International Review of Cytology: A Survey of Cell Biology, Volume 104

INTERNATIONAL REVIEW OF CYTOLOGY A SURVEYOF CELLBIOLOGY VOLUME104 ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G...

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INTERNATIONAL

REVIEW OF CYTOLOGY A SURVEYOF CELLBIOLOGY VOLUME104

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO Y UKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS DONALD G. MURPHY

ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER YUDIN

INTERNATIONAL

Review of Cytology A SURVEY OF CELLBIOLOGY

Editor-in-Chief

G. H. BOURNE St. George's University School of Medicine St. George's, Grenada

West Indies

Associate Editors

K. W. JEON

M. FRIEDLANDER

Department of Zoology University of Tennessee Knoxville, Tennessee

The Rockefeller University New York, New York and Hackensack Medical Center Hackensack, New Jersqy

VOLUME104

1986

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

COPYRIGHT @ 1986 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. Orlando. Florida 32887

United Kingdom Edition published by

ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London NW I 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 52-5203 ISBN 0-12-364504-2 PRINTED IN THE UNITED STATES OF AMERICA

86878889

9 8 7 6 5 4 3 2 1

Contents

Plasmids of Rhizobium and Their Role in Symbiotic Nitrogen Fixation R . K . PRAKASHAND ALANG . ATHERLY

I. I1. 111. IV .

V.

VI . VII .

VIII .

Introduction . . . . . . . . . ......... ............. ........ Physical Studies on Pla ......................................... Strategies and Genetic Methods for a Functional Analysis of Plasmids . . . . . . . . Functions Controlled by Plasmid Genes . . . . Plasmid-Genome Rearrangements ..................................... Relationship between Rhizobium and Agrobucrerium Plasmids . . . . . . . Restriction Endonuclease Maps ................................ Perspectives ............................................. References . . ..................................................

1 2 4 9 16 16 17 18 19

Mouse Mutants: Model Systems to Study Congenital Cataract AUDREYL . MUGGLETON-HARRIS

I. I1. 111.

IV . V.

VI .

Introduction ........................................................ The Search for Chromosomes Associated with the Mutants Cataract . . . . . . . . . . Lens Crystallins .................................................... Cellular Studies on Lens Epithelial Cells ................................ Manipulations of the Cataractous Phenotype ............................. Possible Areas of Research for the Future ............................... References .........................................................

25 27 27 30 32 33 35

Cell Wall Synthesis in Apical Hyphal Growth J . G . H . WESSELS

I. 11. 111.

IV .

Introduction ........................................................ Observations on Living Hyphae ....................................... The Cytoplasmic Components of Apical Wall Growth ..................... Structure of the Fungal Cell Wall ...................................... V

37 38 46 56

vi

CONTENTS

V . Wall Polymer Assembly at the Hyphal Apex .......................

VI .

Summary .................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 72 73

Connectin. an Elastic Filamentous Protein of Striated Muscle KOSCAKMARUKAMA

............................. I . Introduction . . I1 . ....................... I11. Iv . Native Connectin ................................................... V . Interaction with Myosin and Actin VI . Location in Myofibrils . . . . . . . . . . VII . Connectin as an Elastic Component .......... VIII . Connectin Transformation during Differentiation .......................... IX . Comparative Biochemistry ............................................ X . Perspectives ............. .... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81 81 83

86 96 99 103 105

107 110 112

Cell Interactions during the Seminiferous Epithelial Cycle MARTTIPARVINEN. KlMMO K . VIHKO.

AND

JORMATOPPARI

I . Introduction . . . . . . . . ............................................ I1 . Spermatogenic Cell Ty and Their Metabolism ......................... 111. Cycle. Wave. and Transillumination of the Seminiferous Epithelium . . . . . . . . .

IV . Transcriptional Activity during Spermatogenesis and the Function of the Chromatoid Body

V . Cyclic Interaction between Sertoli Cells and Spermatogenic Cells . . . . . . . . . . . .

VI .

VII . VIII . IX .

Localization and Function of Plasminogen Activators in the Seminiferous Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spermatogenesis in Vitro ......................... . . . . . . . . . . . . . . . . . . . . Interaction between Seminiferous Tubules and the Leydig Cells . . . . . . . . . . . . . Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .........................................................

115 117 121 125 129 138 142 144

146 147

The Cytoskeleton in Protists: Nature. Structure. and Functions JEANGRAIN

I. I1 . Ill . IV . V.

Introduction ........................................................ Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Actin and Myosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tubulins and Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153

154 172 176 I85

VI. VII. VIII. IX.

X. XI.

CONTENTS

vii

Periodic Fibers . . . , . . . , . . . . , . . , . . . . , . . , . . . . . , . , . . , . . . . . . . . . . . , . . . . . . Intermediate Filaments Epiplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonactin Microfilaments Filament Systems of Unkn .................................. . . . . . .. . . Conclusion . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

217 224 228 232 238 24 1 242

The Electrical Dimension of Cells: The Cell as a Miniature Electrophoresis Chamber ARNOLDDE LOOF

I. 11. 111. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of the Plasma Membrane and the Generation of the Transmembrane Potential and Transcellular Currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selected Functions of Ionic Currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Imposed Electrical Fields on Cellular Activities . . . . . . . . . . . . . . . . . . Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ............................

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25 I 252 294 328 333 335 353

This Page Intentionally Left Blank

rNTERNATIONAL REVIEW OF CYTOLOGY. VOL. 104

Plasmids of Rhizobium and Their Role in Symbiotic Nitrogen Fixation R. K. PRAKASH* AND ALANG. ATHERLYT *Native Plants, lnc. Salt Lake City, Utah 84108 and fDepartment of Genetics, Iowa State University, Ames, Iowa 50011 I

I. Introduction Rhizobia are gram-negative soil bacteria which fix nitrogen in a symbiotic association with plants of the family Leguminosae; however, this classical definition must be extended now to include nodulation and nitrogen fixation on a nonlegume, Purusponiu (Trinick, 1973). Rhizobium purusponiu can infect this tropical tree and fix nitrogen in a manner very similar to that observed in legumes. In both cases, establishment of the symbiosis starts with invasion of plant root or stem by free-living rhizobia followed by a series of steps that result in the formation of a nodule. It is in these nodules that nitrogen fixation takes place. Both the plant and the bacteria undergo differentiation that is regulated by gene expression. The details of recognition and nodule formation are only beginning to be understood. It is the purpose of this review to discuss recent literature and the current state of knowledge concerning these genes, their location, methods of study, and organization. Members of the genus Rhizobium are of great economic importance because of their ability to fix nitrogen. The genus has somewhat informally been separated into those species that are fast growers and those that are slow growers. This separation has recently become more formal (Jordan, 1982) by the creation of a new genus, Brudyrhizobium, which includes all of the slow growing species while the fast-growing species associating with root soybean have now been reclassified as Rhizobium fredii (Scholla and Elkan, 1984). The classification was done mainly because of many clearly distinct differences between slow- and fast-growing species. Some of these differences are summarized in Table I. Recently the clear distinctions between fast- and slow-growing strains of rhizobia have been flurred by the discovery of strains that seemingly have some characteristics of both groups (Stowers and Eaglesham, 1983; see Broughton er ul., 1984, for a listing of numerous references). Thus a third class of rhizobia exist that nodulates many of the same plant species as the slow-growing and fastgrowing strains. An understanding of the genes of Rhizobium involved in plant symbiosis and 1 Copyright D 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

R. K. PRAKASH AND ALAN G. ATHERLY TABLE I SOMEPHENOTYPIC CHARACTERISTICS OF Rhizobium Phenotype

Fast growers

Slow growers

Doubling time Mannitol medium Disaccharide utilization

2-4 hours Acid production

7-20 hours Alkaline production

(+)

(-)

6-Phosphogluconate dehydrogenase Antibiotic sensitivity Free-living acetylene reduction

(+)

(-)

5-50 pg/ml

100-1OOO pg/ml

(-)

(+)

Peritrichous 50.6-63.1%

Subpolar 62.8-65.5%

Deley and Russel (1965)

0.20-0.40 M

0.10 M

Yelton ef a/. (1983)

Flagellation Guanine and cytosine Salt tolerance

Reference

Sadowsky ef a / . (1983); Glenn and Dilworth (1981) Martinez-DeDrets and Arias (1972)

McComb ef a / . (1975) Kurz and LaRue (1975) Pagan ef a/. ( 1975)

nitrogen fixation has moved very rapidly with the fast-growing strains (Rhizobium leguminosarum, Rhizobium trifolii, Rhizobium meliloti, and more recently, R. parasponia and R.fredii). This is largely due to their ease of handling, growth rate and lysis, as well as the ability to apply traditional techniques of genetic analysis. On the other hand progress has been hindered in the genetic analysis of the slow-growing strains [Bradyrhizobium japonicum, Bradyrhizobium sp. (Vigna), Bradyrhizobium sp. (Lupinus), as well as species that nodulate Ornithopus, Cicer, Sesbania, Leucaena, Mimosa, Lablab, Acacia, and Parasponia] due to the difficulty of application of the same techniques.

11. Physical Studies on Plasmids A. DETECTION The techniques initially used to isolate plasmid DNA involved alkaline denaturation, renaturation, and removal of single-stranded chromosomal DNA from the preparation (Currier and Nester, 1976; Nuti et al., 1977; Ledeboer er al., 1976; Casse et a f . , 1979; Prakash et al., 1980;'Hirsch et al., 1982). By analysis on gel electrophoresis, the plasmid profiles of particular strains have been determined. Large plasmids with molecular weights ranging from 90,000,000 to 300,000,000 have been identified in most of the fast-growing strains of rhizobia. For literature before 1980, several recent reviews should be consulted (Denarie er al., 1981; Nuti et al., 1982). Recently, using a modified Eckhardt (1978) procedure, a megaplasmid not observed earlier was detected in several fast-growing rhizobia

PLASMIDS OF RHlZOBlUM

3

(Rosenberg er u l . , 1981; Masterson er al., 1985; Heron and Pueppke, 1984). An R. meliloti megaplasmid has been estimated to be 1000 MDa using electron microscopy (Burkhardt and Burkhardt, 1984). Recent studies indicated the presence of at least two megaplasmids, of the same size in R. meliloti (Banfalvi et al., 1985). Very rarely are plasmids of less than 50 MDa found in rhizobia and usually the number of different plasmids within a strain varies from one to six. When the combined size of plasmids and chromosome are totaled the genome of Rhizobium species becomes very large (5.4-7.6 X lo9 Da; Chakrabarti et al., 1983) which is severalfold larger than the Escherichia coli genome (2.2 X lo9 Da), thus plasmid DNA may represent as much as 25% of the total DNA of some strains. Rhizobium plasmids have been given designations as proposed by Prakash er al. (1980). The indigenous plasmids of Rhizobium are named as pRtr, pRle, pRme, pRph, pRja, etc. (for plasmids of R. trifolii, R. leguminosarum, R . meliloti, Rhizobium phaseoli, B. japonicum, respectively) followed by the number of the strain in which the plasmid was found and serial letter a, b, c, etc. in case of multiple plasmids where a designates the largest plasmid in a series. Plasmids containing symbiotic genes are also sometimes referred to as pSym. Slow-growing strains of Rhizobium have only been recently examined for the presence of plasmids because gentle lysis procedures did not readily adapt to the slow-growing strains. An exception was the analysis of a number of slowgrowing strains isolated from alkaline soils (Gross er al., 1979). More recently, Masterson et (11. (1982) and Cantrell et al. (1982) developed a plasmid isolation procedure that is useful with all Bradyrhizobiurn strains. Using this procedure, several B. japonicum strains (USDA 110, 122DES, 143, 136, 142, 6, and SR) have been shown not to contain any plasmids. Very large plasmids are present in most B. japonicum strains including 61A76, 61A24, USDA strains 31, 71a, 74, 94, and 143 (Haugland and Verma, 1981; Masterson et al., 1985).

B. NUCLEICACIDHYBRIDIZATION DNA hybridization studies have provided information about the relatedness of plasmids derived from the same or different species of bacteria. Jarvis et al. (1980) compared the DNA sequences from total DNA of 35 different fastgrowing strains of Rhizobium by DNA:DNA reassociation kinetics and found 40-80% sequence similarities. On the other hand, Hollis et al. (1981) compared a wide variety of slow-growing species for DNA sequence homology and found that they could be grouped into three homology groups: high homology, moderate homology, and very little sequence homology (10% or less). Haugland and Verma (1981) examined the intraspecific homology of total DNA sequences in three strains of B . japonicum. Strains 61A76 and I10 showed very little sequence homology with eachrother (24%), while a third strain 61A24, showed 50% homology with strain 110. Although strain 61A76 was found to have one large plasmid and 61A24 was found to have two plasmids, no discern-

4

R. K. PRAKASH AND ALAN G. ATHERLY

ible plasmid could be detected in strain 110. Heterologous hybridizations between plasmid and total DNAs from these strains indicated that sequences which were plasmid-borne in one strain were located on the chromosomal DNA in other strains. Jouanin et al. (1981) analyzed the sequence homology of the plasmids of symbiotically effective strains of R. meliloti. chosen because of their wide geographical origin. They used restriction endonuclese patterns and Southern DNA:DNA hybridization to reveal sequence homologies of the purified plasmid DNA. Sequence homology was present between all plasmids tested, irrespective of geographical origin. The homology was concentrated in a few restriction fragments, but because restriction maps are not available they could not provide evidence for any degree of clustering of these sequences. Likewise, they did not indicate any biological function that might be associated with these highly conserved sequences. Prakash et al. (1981) found that plasmids from different fast-growing Rhizobium species share extensive homology. Organization on the restriction endonuclease map indicated that these homologous sequences are clustered within a region that contained sequences for symbiotic nitrogen fixation (Prakash et al., 1982a,b). Masterson et al. (1985) examined the homology of a R. fredii Sym plasmid with the plasmid DNA and total DNA from various other R. fredii strains and 8 . japonicum strains. They found extensive homologous sequences in plasmid DNAs as well as in chromosomal DNA of different R. fredii strains. However, in one R . fredii strain USDA194 and in B. japonicum strains the homology was observed only with total DNA. A possible explanation for highly conserved sequences in plasmid DNA was obtained by Watson and Scholfield (1985). In R. trifolii, they found a repeated sequence that is a reiteration of the nijHDK promoter. This sequence is highly conserved within all geographically distinct isolates and located exclusively in the Sym plasmid. The sequences were specific for R. trijolii. They propose a model in which the expression of symbiotic genes is host-specifically activated via their species-specific sequences. 111. Strategies and Genetic Methods for a Functional Analysis of Plasmids

A. TRANSPONSONS, GENEFUSIONS,AND MARKER EXCHANGE A number of symbiotically important functions are coded by the plasmids of fast-growing strains, but no such functions can be assigned to the plasmids of the slow-growing rhizobia. A wide variety of approaches have been used to identify

PLASMIDS OF RHIZOBIUM

5

and characterize plasmid-borne genes and gene functions. These includes plasmid transfer, plasmid curing, mutant analysis, expression in minicells, fusion of genes with facZ, and restriction endonuclease mapping. Some of these procedures apply to both plasmid genetic analysis as well as to genome analysis. An extremely useful approach for genetic analysis of Rhizobium and other gram-negative bacteria is transposon mutagenesis. Insertion of a transposon results in a relatively stable mutation (insertional inactivation). One disadvantage is that transposon insertion usually results in polar mutations (Berg et a f . , 1980). A variety of suicide vehicles to deliver transposons into Agrobucrerium and Rhizobium are available. The first of these vectors was developed by Van Vliet et al. (1978) and Beringer e t a f .(1978) and has been effectively used to genetically tag plasmids for plasmid transfer (Johnston et a f . , 1978; Hooykaas et a f . , 1981). The vectors were designed to replicate only in E. cofibut not in other nonenteric, gram-negative bacteria. When replication is not possible, the transposon present in the plasmid is rescued by insertion into the host DNA. For unknown reasons the presence of Mu phage DNA sequences in RP4 does not allow replication of RP4 in Rhizobium or Agrobacterium. Unfortunately, the Mu-induced failure of RP4 to replicate can occasionally be overcome, thus complicating interpretation of results (Simon et a f . , 1983). Another complicating event is the frequent simultaneous transposition of both Mu and Tn5 sequences which then makes genetic analysis difficult (Banfalvi et a f . , 1981b; Meade et a f . , 1982; Forrai et al., 1983). To overcome these problems, Simon et al. (1983), Selvaraj and Iyer (1983), and Yakobson and Guiney (1984) have developed broad-host-range mobilization systems for transposon mutagenesis. Simon et uf . (1983) constructed a family of vector plasmids containing the P-type recognition site for mobilization (mob site) which can be mobilized with high frequency from a donor strain carrying the transfer genes (oriT)of Incp-type plasmid RP4 integrated into the chromosome. The mobilization vectors were derived from pACYC184, pACYC177, and pBR325 which are able to replicate in E. cofi and its close relatives but not in Rhizobium and Agrobacterium. Vectors were prepared that include Tn5 (Nm, Km; pSUPlOl1) Tn7 (Sp/Sm; pSUP2017), and Tcr genes integrated into a nonfunctional area of Tn5 (pSUP10141) which then gives Tc‘ and Nmr or Kmr simultaneously. In a similar system developed by Yakobson and Guiney (1984), the 760-base pair oriT region of broad-host-range plasmid RK2 was cloned into Tn5 residing on a derivative of pBR322. The resulting “suicide” plasmid, pEYDGl , can be transferred to a variety of gram-negative bacteria in the presence of RK2 helper plasmid pRK2073. Once transposon-generated mutants are isolated, replicons which have acquired Tn5-oriT can be mobilized to other gram-negative hosts following the introduction of helper plasmid pRK212.1 into the mutants. Selvaraj and Iyer (1983) constructed suicide plasmids containing Tnl , Tn5, or Tn9 in a pl5A-type of replicon with an N-type of bacterial mating system. These

6

R . K . PRAKASH AND ALAN G . ATHERLY

vectors also replicate in E . coli but not in Rhizobium and because of the N-type mating system they are very efficient in matings with E . coli and Rhizobium. These vectors are useful for random mutagenesis as well as for site-specific mutagenesis. Tn5-induced mutant clones of genes can be reinserted into a genome by homologous recombination, selecting for kanamycin. For example, a clone of the recA gene containing Tn5 (pRMB 1002) has been isolated by Better and Helinski (1983) from R . melifoti genome bank. By reinserting this cloned gene into a suicide vector (pSUP205; Simon et al., 1983) the Tn5 in the recA gene can be rescued creating a recA mutant. Such a procedure has been used by Shantharam and Iyer (1986) to construct a double mutant affected in recombination in R. meliloti. Simon et af. (1983) and Hahn and Hennecke (1984) have used this approach to create site-specific mutants of R . melifoti and B . japonicum, respectively. The procedure initially developed for site-directed mutagenesis (Ruvkun and Ausubel, 1981) involved the cloning of restriction fragment containing the mutagenized gene of interest on a P-group broad-host-range vector. This is followed by replacement of the wild-type parental DNA sequence with the mutant sequence by conjugation with a second P-group plasmid, with simultaneous selection for Tn5 and a marker on the second plasmid. R. K. Prakash and A. G . Atherly (unpublished) further simplied the site-specific mutagenesis technique by cloning the Tn5-mutagenized gene in vector pBR322, which can replicate in E . coli but not in Rhizobium. This vector has been found to be mobiliziable with the plasmid DNA that mobilize the P-group broad-host-range vector. Recently lac gene fusions have been used to study a wide variety of genetic functions both at the transcriptional and translational levels (Weinstock et al., 1983). lac fusions are especially useful in analyzing gene functions and regulation where the gene product is difficult, if not impossible, to assay or when the gene in question is turned on/off under undefined conditions. Techniques for fusing genes with lac have been very well developed (Silhavy et al., 1984) and a large number of convenient cloning vectors have been constructed to facilitate genetic analysis (Casadaban et al., 1980; Kahn and Timblin, 1984). These have been applied to the analysis of regulation of nifgene activity in rhizobia. The technique of lac gene fusion has been extremely useful for analyzing trans-acting regulatory functions of the ntrA, n t E , and nifA genes on nijKDH promoters (Sundaresan et al., 1983a,b; Szeto et af., 1984). One criterion for studying lacZ gene fusions to heterologous promoters is for the host cell to have a low level of endogenous P-glactosidase activity. This is true in most fast-growing strains of Rhizobium, and, hence, fur mutants are desirable but not necessary if Rhizobium is used as the host cell (Szeto et al., 1984). A potentially very useful technique of facZ gene fusion makes use of the combination of suicide plasmid and lacZ in a transposable element (Simon et a f . , 1983; Selvaraj and lyer, 1983) and Mu-d lac. Castillo et af. (1984) have constructed mini-Mu transposable elements containing lac operon structural genes.


7

These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form transcriptional and translational lac gene fusions. Olsen et al. (1985) developed a procedure for selecting mini-Mu insertions by inserting Mu-d lac (kan) into the suicide vector pGS6 (Selvaraj and Iyer, 1983). They mated this vector to R . fredii strain USDA201. Kanamycinresistant transconjugants arose at a frequency of lop4 and appear to insert randomly into the genome. Several transconjugants exhibited increased lac expression when grown in the presence of Glycine max root exudates and nodule extracts, suggesting fusion to nod (nodulation) andfir (fixation) gene promoters. Another potentially useful promoter probe for Rhizobium has been recently developed for Caulobacter crescentus. Bellofatto et al. (1984) removed the promoter of Tn5 while still retaining its translational start site and inserted it into a suicide plasmid containing Mu phage sequences. Consequently, neomycin phosphotransferase is expressed only when fused to the functional promoter in the genome. B. CLONING VECTORSAND

THE

USE OF PHAGE

A considerable number of DNA cloning vectors have become available over the years; however, the vast majority of them are restricted for use in E . coli. Since 1980, two groups of broad-host-range DNA cloning vectors have been developed for use in specialized functions. The first of these vectors (pRK290) was developed by Ditta et al. (1980) from RK2, a large (56 kb) P-1 incompatibility group plasmid. RK2 and its derivative plasmids have the ability to transfer at a high frequency via conjugation into a wide variety of gram-negative bacteria, including species of Rhizobium, Klebsiella, Serratia, Pseudomonas, Acinetobacter, and Agrobacterium. pRK290 is very stable in R . meliloti, with only a 0.2% loss per generation (Ditta et al., 1980). The 20 kb plasmid confers tetracycline resistance, is not mobilizable (mob ,tra-), and contains a single recognition site for EcoR1 and BglII. However, a second derivative of RK2 (Figureski and tielinski, 1979) contains the tra functions and the neomycin resistance gene ligated to a ColE1 replicon and will mobilize pRK290, respectively. To further extend the use of pRK290, Friedman et al. (1982) and Knauf and Nester (1982) added a cos fragment into the unique Bglll site creating the cosmid pLAFRl , and pVKlOO and pVK102. The plasmid pRK290 has now been reduced in size to 10 kb and has been modified for gene expression studies for a wide variety of gram-negative bacteria (Ditta et al., 1985). A second group of broad-host-range cloning vectors has been developed from the 8.9 kb IncQ/P4 plasmid RSFlOlO (Bagdasarian et al., 1981; Guerry et al., 1974). Simon et al. ( 1983) removed the replication and mobilization functions of pKT210, a chloramphenicol-resistant derivative of RSF1010, and inserted them into pACYC184, pBR325, orpACYC177 which resulted inpSUP104, pSUP204, and pSUP304, respectively. By inserting a 0.40 kb cos fragment into the single +

8

R. K. PRAKASH AND ALAN G . ATHERLY

PsrI site of pSUP104, a cosmid pSUP106 was developed. However, fragments greater than 10 kb cloned into pSUP106 are very unstable and cannot be easily maintained (R. K. Prakash and A. G. Atherly, unpublished). RSFlOlO and its derivatives are nontransmissible plasmids but are efficiently moblized by Inc- 1 plasmids or the mob site of IncP-1 plasmids. David et al. (1983) using derivatives of RSF1010, (pKT248. pKT210; Bagdasarian eral., 1981)demonstratedthat they were transferred at a high rate from E. coli to R. melilori in the presence of RP4 and were stably maintained. A valuable adjunct of plasmid cloning vectors for genetic analysis is the discovery of generalized transducing phage in R . melilori (Casadesus and Olivaries, 1979; Sik e f al., 1980; Martin and Long, 1984; Finan er al., 1984), R . leguminosarum and R. trifolii (Buchanan-Wollasten, 1979), and R. japonicum (Shah et al., 1981, 1983). Not only can phage be used for fine structure mapping and strain construction of Rhizobium but also for transfer of large plasmids from one strain to another. The molecular size of the phage N3 (Martin and Long, 1984) is very large (approximately 195 kb) thus allowing transfer of large plasmids intact. Unfortunately, rhizophage are restricted to only a few species and are usually strain specific. C.

PLASMID

TRANSFER AND R-PRIMES

Plasmids present in Rhizobium species are frequently of very high molecular weight (150,000,000 to more than 500,000,000) and therefore biochemical and genetic analysis is extremely difficult. Procedures that reduce the working size of a DNA segment or plasmid are therefore very helpful. Identification of symbiotic functions can be accomplished by the transfer of whole or partial plasmids from Rhizobium to E. coli or to plasmidless Rhizobium and Agrobacrerium strains. Plasmids coding for symbiotic functions in some strains of R . leguminosarum, R. trifolii, and R. phaseoli are self-transmissible (Higashi, 1967; Johnston er al., 1978; Beynon er al., 1980; Rolfe et al., 1981; Hooykaas er al., 1981, 1982a; Lamb er al., 1982; Scott and Ronson, 1982), but self-transmissibility has not been observed in symbiotic (Sym) plasmids from R. melilori or R. fredii (Kondorosi er al., 1982; Atherly et al., 1985; Appelbaum et al., 1985). In the case of nontransmissible plasmids, several approaches are available to obtain transfer to new hosts. Using R plasmid, Hooykaas et al. (1982a,b) developed a helper plasmid PRL180 to transfer nontransmissible plasmid. Kondorosi er al. (1982) inserted the mob region of the P-1 type plasmid RP4 into the megaplasmid pRme4lb of R. melilori and subsequently obtained pRme4lb transfer at a frequency of 1 X Kanamycin on Tn5 was used as a selective marker but transconjugants were also tested for nod (nodulation) and f i x (fixation). mobRP4 was inserted into the megaplasmid by cloning a fragment of pRme4lb and the mob region into pBR322. This hybrid plasmid was mobilized into R. melilori but will not replicate; however, recombination occurs due to homology between the


9

resident plasmid and the hybrid pBR322. A similar approach was used by Julliot et al. (1984) for creating R’ plasmids of R. melifoti pSym megaplasmid of strain 201 1. After mobilization of the cointegrate RP4:pSym into E. coli, they obtained a set of RP4-primes that contained large fragments of the pSym megaplasmid. One of them has been extensively characterized (285 kb) and contains the symbiotic genes. Simon (1984) constructed a convenient suicide vector by inserting mob into Tn5 which, in turn, was present in a suicide vector. Thus, by inserting this mob:Tn5 into a Rhizobium plasmid, mobilization could be effected. Appelbaum et al. (1985) used this approach to mobilize (with RP4 in trans) the Syrn plasmid of R. fredii. D. PLASMID DELETION AND CURING P-1 group plasmids will occasionally form cointegrates with resident Rhizobium plasmids if naturally occurring plasmid-plasmid homology sites exist. The P-1 group plasmids apparently can be maintained as integrated units only if a recA or low recA gene activity is present. Ronson and Scott (1983) obtained a cointegrate of pRtr514 and R68.45, which totaled about 770 MDa and transferred it to a variety of different bacterial strains at frequencies as high as Similarly, cointegrates of Sym plasmid from R. fredii strain USDA191 and PRL180 (Hooykaas er al., 1982a,b) were transferred to E. coli and Agrobacreriurn tumefaciens (Engwall et al., 1986). But, cointegrates were very unstable in recA+ E. coli strains yielding only pRLl8O upon conjugation. In recA- E. coli hosts the Sym plasmids suffered random deletions and it was thus possible to create a family of deletions encompassing the entire plasmid. Useful information on plasmid function can also be obtained from plasmidcured strains, such as lost phenotypic characteristics and reintroduction of plasmid fragments to regain only specific plasmid functions. Plasmid curing is most easily accomplished by prolonged exposure to high temperatures (Zurkowski and Lorkiewicz, 1978), but this procedure may also result in internal deletions within plasmids (Banfalvi et al., 1981b). Heat treatment of slow-growing strains of R. japonicum also resulted in deletions with the majority of them occurring in the nif (nitrogen fixation) or nod (nodulation) region (Skogen-Hagenson and Atherly, 1983).

IV. Functions Controlled by Plasmid Genes A. GENETIC EVIDENCE FOR SYMBIOTIC NITROGEN FIXATION GENES The study of symbiotic nitrogen fixation in Rhizobium has been mainly limited to Rhizobium plasmids. A great percentage of DNA in Rhizobium is in the form of large plasmids (up to 25%) and it was of interest to know what genes are

10

R . K . PRAKASH AND ALAN G. ATHERLY

carried on these large plasmids. Some early genetic evidence indicated that in Rhizobium the symbiotic nitrogen fixation genes might be on the plasmid DNA (Higashi, 1967; Dunican and Cannon, 1971). Several genetic and physical studies have now clearly established that, at least in fast-growing Rhizobium species, the genes for symbiotic nitrogen are usually on a large plasmid. Zurkowski and Lorkiewicz (1976, 1978, 1979) and Hooykaas et al. (1981) showed that nonnodulating mutants of R. trifolii, resulting from treatment at high temperature, were due to loss of plasmid DNA. This nod- mutant also lost its ability to attach to the root hair surface and that property was also restored upon the reintroduction of the plasmid. These data strongly suggested the involvement of plasmid DNA in nodulation. Furthermore, Johnston et al. (1978) demonstrated that transfer of the Tn5 marked plasmid into a j r strain of R . leguminosarum restored its normal symbiotic functions, strongly implying that the symbiotic genes were located on plasmids. Brewin et al. (1980a) showed that transfer of R. leguminosarum bacteriocinogenic plasmid pRLl JI into four symbiotic mutants restored them to normal symbiotic function. In R. meliloti, genes controlling the early and late function in symbiosis were reported to be on a megaplasmid (Rosenberg et a f . , 1981). Further Palomares et al. (1978, 1979) showed that in R . meliloti, extrachromosomal DNA was responsible for polygalacturonase, a key enzyme responsible for the early infection process (Ljunggren and Fahraeus, 1961). Thus, it seems clear that early functions in the infection process are plasmid controlled in R . meliloti. Using the genetic strategy described in Sections III,C and D the presence of both nodulation and nitrogen fixation genes on a fastgrowing Rhizobium plasmid has been established by other authors (Banfalvi et al., 1981b, 1983; Kondorosi et al., 1982, 1983; Julliot et al., 1984; Rolfe e t a l . , 1983; Appelbaum et al., 1985; Hombrecher et al., 1981a; Kowalczuk et a / . , 1981; Sadowsky and Bohlool, 1983). In addition to nifand nod genes the Sym plasmid seem to confer host specificity to Rhizobium. Recently Brewin et al. (1981) reviewed the role of Rhizobium plasmids in host specificity. Plasmid controlled host-range specificity in R . leguminosarum was first described by Johnston et al. (1978). In this study a plasmid (pJB5JI) bearing Tn5 was transferred to R . trifolii and R . phaseoli. Transconjugants arose at a frequency of lo-* and all were capable of forming nodules on peas, in addition to retaining their normal host nodulation properties. Later, several other reports provided evidence for the presence of host-range determinants on other Sym plasmid DNA (Brewin et al., 1980c; Hooykaas et al., 1981, 1982a,b; Djordjevic ef al., 1983; Morrison er al., 1983; Lamb et al., 1982; Appelbaum et al., 1985). Besides Rhizobium species, the Sym plasmid has also been transferred to closely related Agrobacterium. Hooykaas et al. (1981, 1982a) transferred the Sym plasmid from R. trifolii and R. leguminosarum to A . tumefaciens LBA288 (a nonvirulent strain cured of its tumorigenic plasmid) and observed that the trans-

11


conjugants were able to form nodules on clover. Electron microscopy of the nodules showed root hair curling and infection threads filled with bacteria. Furthermore, the bacteria were surrounded by a periplasmic membrane indicating that the agrobacteria had been released from the infection thread into the plant cell. But there were no typical bacteroid-like structures and no nitrogen fixation was observed. Similar findings are obtained with other Rhizobium plasmids when transferred to Agrobacterium (Kondorosi et a / . , 1982, 1983; Broughton et al., 1984; Djordjevic et al., 1983). In most cases the agrobacteria were confined to the infection threads and the nodule was essentially devoid of bacteria (Hirsch e t a / . , 1985; Schofield et al., 1984; Truchet et al., 1984; Wong et a/., 1983).

B. EXPRESSION OF Sym PLASMID

IN

RHIZOBIACEAE

Transfer of Syrn plasmids within Rhizobiaceae often results in variable expression. Hooykaas e f al. (1981, 1982a,b) observed that the Syrn plasmids of R . leguminosarum and R. rrifolii expressed symbiotic nitrogen fixation properties completely when transferred between these two strains. However when these Sym plasmids were transferred to A . tumefaciens or R . meliloti they induced root nodules but did not fix nitrogen. Djordjevic et al. (1983) and Rolfe et al. (1983) found that transfer of plasmid pJB5J1 or pBR IAN, which encodes pea and clover specificity, respectively, to various R . meliloti plasmid-cured strains did not result in the ability of R. mefifoti to nodulate peas and clover. The transfer of pBRl AN to an A . tumefaciens strains conferred clover nodulation to this strain, whereas pJB5JI could not induce this Agrobacrerium strain to nodulate peas. The transfer of pBR 1 AN to various Syrn plasmid-cured or deleted R. leguminosarum or R . rrifolii strains resulted in the ability of these strains to nodulate clover, whereas the reverse was true when pJB5JI was transferred to these strains. When plasmid pJB5JI and pBRlAN were introduced into a fast-growing R. parasponium strain, the resulting transconjugants showed a change in the spectrum of plants that could be nodulated. The plasmids have also been transferred to slowgrowing Rhizobium species, but the plants had an ineffective phenotype. The transfer of a host-range plasmid pJB5J1 from R . leguminosarum to R. fredii elicited only early stages of nodule development on peas (Rviz-Sainz et al., 1984). On the other hand R. fredii Syrn plasmid from strain USDA191 is Fix in ANU265 genetic background (Appelbaum et a/., 1985). ANU265 is a pSymcured derivative of NGR234, a broad-host-range fast-growing strain. This implies that the chromosomal genetic background of NGR234 is very similar to R . fredii. Incompatibility and instability of the Syrn plasmids could also be attributed to variable expression of the Syrn plasmid in different hosts (Djordjevic et al., 1982; Christensen and Schubert, 1983). +

12

R. K. PRAKASH AND ALAN G.ATHERLY

C. PHYSICAL EVIDENCE FOR NITROGEN FIXATION GENES The first physical evidence for the presence of nifgenes on a plasmid was obtained by hybridizing R. leguminosarum plasmid with the cloned nif structural genes KDH of Klebsieflapneumoniae (Nuti et al., 1979). The nifstructural genes of K . pneumoniae are found to be highly conserved in all nitrogen-fixing bacteria (Ruvkun and Ausubel, 1980). With the exception of R. melilori, in most of the fast-growingRhizobium species the nifgenes are located on medium size isolatableplasmids (Prakashetal., 1981; Broughtonetal., 1984;Mastersonet u f . ,1982, 1985).Rhizobium meliloti nifgenes are present on the megaplasmid (Rosenberg et al., 1981; Banfalvi et al., 198lb). Slow-growing strains of Rhizobium. including B. japonicum, have not been shown to carry nif sequences on plasmid DNA (Mastersonet al., 1982; Haugland and Verma, 1981). Likewise in a fast-growing R. fredii strain (USDAl94) homologous sequences for nif structural genes were identified only when total DNA was used (Masterson e t a f . , 1985). DNA hybridization studies indicated that in R. phuseoli (Quinto et al., 1982) and R. fredii (Prakash and Atherly, 1984) the nif structural genes are reiterated. The nif structural genes have been cloned and characterized from several Rhizobium species (Ruvkun and Ausubel, 1980; Schetgens et af., 1984; Hennecke, 1981; Schofield et al., 1983; Quinto et al., 1985). Site-directed mutagenesis and complementation studies verified that the region homologous to K . pneumoniae nif structural genes is involved in nitrogen fixation (Schetgens er al., 1984; Ruvkun and Ausubel, 1981; Ruvkun et al., 1982a). Further, RNA isolated from nitrogen-fixing nodules hybridizes strongly to the nifregion on the plasmid DNA (Prakash et al., 1982a; Krol et al., 1980, 1982; Corbin et af., 1982, 1983). Apart from nifstructuralgene KDH, the nifregulatory gene A of K . pneumoniae, has also been localized on plasmid DNA of R. leguminosarum (Downie et al., 1983a),R . meliloti (Szetoer al., 1984), and in chromosomal DNA of B. japonicum (Fuhrman et af., 1985). In most of the fast-growing Rhizobium species the nifand nod genes have been found to be located on one plasmid except in few strains of R . fredii. In some R . fredii strains the nif and nod genes are not only present on different plasmid but also specified on the chromosome (Masterson et al., 1985; Scholla et al., 1984; Mathis et al., 1985).

D. IDENTIFICATION AND ISOLATION OF NODULATIONGENES In addition to nif structural genes, other genes involved in effective nitrogen fixation and nodulation have been identified. Using both nitrosoguanidine and transposon mutagenesis, Forrai er al. (1983) identified 5 nod- (noduation) and 57 Fix- (fixation) symbiotic mutants of R . meliloti. Of these mutants 1 nodand 11 Fix- mutants were localized on the chromosome while 5 Tn5-induced


13

Fix- mutants and 1 Tn5 nod- mutants were localized on the megaplasmid. Long et al. (1981) and Meade et al. (1982) also found several nod- and Fixmutants of R. meliloti using Tn5 mutagenesis. Among 19 symbiotic mutants characterized, at least 6 were shown to reside on the megaplasmid (Buikema et al., 1983). Microscopic examination of four nodulation-defective ( n o d - ) mutants (Hirsch et a f . , 1982) showed that some did not induce root hair curling and entered root epidermal cells even though no infection threads were formed. In R. phaseoli, 10 symbiotic Tn5-induced mutants were isolated (Noel et a f . , 1984). Of three that were located on the Sym plasmid, one mutation led to abnormal nodule development and two of the mutations, which eliminated nitrogenase activity, were located outside the immediate region of the nif structural genes. The remaining seven mutants were seemingly on the chromosome. They resulted in slow nodule development and nodule dispersement on the root system. Three of the chromosomally located mutants (Vandenbosch et al., 1985) induced the formation of uninfected root nodule-like swelling on bean. The technique which utilized transponon Tn5 mutagenesis has facilitated the isolation of nodulation genes from several Rhizobium species. In R. trifolii the nodulation region, identified by Tn5 insertion, was cloned and used to isolate the wild-type sequence. Reintroduction of the wild-type sequence, present on a 14 kb Hind111 restriction fragment, into Sym-plasmid-cured R. trifolii strain and A. tumefaciens resulted in the restoration of nodulation on clover (Schofield et a l . , 1984). This 14 kb nod fragment of R. trifolii also formed nodules on clover plants when present in Lignobacter and Pseudomonas strains (Plazinski and Rolfe, 1985). All nodules formed by Lignobacter transconjugants showed bacterial release from the infection threads into host cytoplasm. Pseudomonas transconjugants formed pseudonodule without bacterium, except within the cellular species of the outermost cells. Similarly a 10 kb region of DNA-encoding nodulation functions was cloned from R. leguminosarum (Downie et al., 1983b). Long et al. (1982) cloned the nodulation gene from R. melilori strain 1021 by complementation studies. In this procedure, a clone bank of wild-type R. meliloti sequences was conjugated into each of two nod- R. meliloti mutants. Plants were inoculated with the transconjugants. The bacteria isolated from the nodules contained a plasmid pRmSL26, bearing nodulation genes. Further it was found (Hirsch et al., 1985; Jacobs et al., 1985) that two subclones of pRmSL26, one an 8.7 kb EcoRI fragment and a 5.5 kb PstI fragment elicited the same response in A. tumefaciens on roots of alfalfa as did pRmSL26. In addition, Hirsch et al. (1985) found that 8.7 kb nod fragment alone is sufficient for nodule morphogenesis. In R. meliloti strain 41, the essential nod genes were localized in 8.5 and 6.8 kb EcoRI restriction fragment, respectively. In nod- deletion mutant lacking the 8.5 kb fragment the nodulation ability on alfalfa was restored upon the introduction of Sym plasmid of R. leguminosarum or R. trifolii (Banfalvi et al., 1981a; Djordjevic et al., 1983; Kondorosi er a l . , 1984). Similarly the nod-

14

R. K. PRAKASH AND ALAN G . ATHERLY

mutant of Sym plasmid from R. meliloti, R . trifolii, and broad-host-range Rhizobium strain can be complemented with the 8.7 kb nod fragment of R . meliloti strain 1021 or 14 kb nod fragment of R. trifolii (Fisher et a l . , 1985; Djordjevic et al., 1985). Moreover, the nod region of the 8.5 and 8.7 kb EcoRI nod fragment of R . meliloti strain 1021 and 41, respectively, hybridized with nod genes from other rhizobia (Prakash and Atherly, 1984; Masterson et al., 1985; Broughton et al., 1984). Therefore, these nod gene clusters are called as “common” nod genes. By complementation and sequence analysis, four genes were identified in the common nod region, which were designated as nod A , B, C, and D ,respectively (Torok et a l . , 1984; Fisher et al., 1985; Jacobs et al., 1985). Comparison of nod A, B, C nucleotide and amino acid sequences of R. meliloti and R . leguminosarum (Torok et al., 1984; Rossen et al., 1984) showed that the organization of these three genes is fairly similar and the nucleotide sequence of nod A, B, and C genes shared 72, 69, and 71.4% homology, respectively. Using directed Tn5 mutagenesis, a nod gene cluster of about 3.5 kb was found within the 8.7 kb EcoRI fragment (Jacobs et al., 1985; Kondorosi et al., 1984). The other 6.8 kb region contains two nod gene regions separated by a 1 kb region nonessential for nodulation (Kondorosi et al., 1984). With the use of 3.5 kb nod sequence as a probe, the nod genes from R. fredii were also cloned (Prakash et al., 1986). As in R. meliloti, the nod genes of R. fredii are not clustered, but instead are present at different regions on the plasmid DNA. Further, in R.fredii, the nod genes are reiterated (Prakash and Atherly, 1984).

E. OTHERGENESINVOLVED IN SYMBIOTIC NITROGEN FIXATION 1. Polysaccharide Synthesis In 1974 an attractive theory involving legume seed lectins was proposed for the specific attachment of Rhizobium to their host plant (Bohlool and Schmidt, 1974). This theory has subsequently been pursued and discussed by numerous investigators (Bauer, 1981; Graham, 1981; Dazzo and Truchet, 1983; Dazzo and Gardiol, 1984). The biochemical basis for this theory is that the seed glycoproteins known as “lectins” bind to specific sugar haptens on rhizobial cell-surface polysaccharides, thus aiding in attachment and recognition. Rhizobium cellsurface polysaccharides have been shown to carry the receptor sites of lectin molecules (Bal et al., 1978; Shantharam et al., 1980). Suggestions for the involvement of Rhizobium plasmid genes in the recognition system stem mainly from the properties of plasmid-free symbiotically defective mutants of Rhizobium. A nod- derivative of R. leguminosarum, obtained by culturing at elevated temperatures, lost pea lectin-binding properties because of the loss of its smallest plasmid (Prakash er al., 1980). Also, a strain of R. trijolii cured of a large plasmid failed to nodulate its host. In addition, it lost lectin-binding capaci-


15

ty and the ability to attach to the root hair surface. When the Sym plasmid was restored from the wild-type strain, the bacteria regained all of the above properties (Zurkowski and Lorkiewicz, 1979; Zurkowski, 1980). Mutants that are incapable of producing exopolysaccharide (EPS) and nitrogen-fixing root nodules have been isolated from Rhizobium (Sanders et a l . , 1978; Chakravorty et al., 1982) suggesting again that polysaccharides may be involved in the recognition process. From a Tn5-derived mutant of R. trifolii the wild-type DNA sequence was cloned which was capable of restoring the mutant strain to synthesize normal levels of EPS and simultaneously restored its ability to nodulate clover (Chakravorty et a l . , 1982). On the other hand, in Azotobacter, independent transfer of R. trifolii lectin-binding property has been shown without the transfer of nodulating ability (Bishop e f a l . , 1977). Evidence that extracellular polysaccharide is involved in the nodulation of R . meliloti comes from the work of Leigh et al. (1985) and Finan et al. (1985b). They obtained mutants in polysaccharide synthesis by screening with the fluorescent strain calcafluor and also by insensitivity to monoclonal antibodies to the rhizobial surface (Johansen et al., 1984). These two independently isolated sets of mutants (Exo-) formed ineffective nodules on alfalfa and fell into six distinct genetic groups as determined by complementation analysis. Apparently, the exopolysaccharide, although not required for nodule formation, is involved in nodule invasion. One of these mutants has been mapped to a second megaplasmid (pRme-SU47b) which is slightly larger than the symbiotic plasmid (pRme-SU47a) (Finan et al., 1985a). The overwhelming evidence supports the suggestion that polysaccharides are involved in the nodulation process; however, the exact mechanism is not known. It is clear that the genes involved in polysaccharide synthesis are located on both the plasmid and the chromosome.

2 . Hydrogen Uptake Genes Large quantities of energy are lost during the nitrogenase-catalyzed reaction as hydrogen gas. Many Rhizobium species possess an active hydrogen uptake system (Hup) permitting hydrogen to be recycled. Thus, Hup+ Rhizobium strains generally are more effective symbionts than those with Hup- phenotypes (Albrecht e f a l . , 1979). Evidence for the presence of hydrogen uptake genes on plasmid DNA comes from the work of Brewin et al. (1980b) who cotransferred the genetic determinants for hydrogenase activity and nodulation ability between R. leguminosarum strains. DeJong et al. (1982) found that transfer of such plasmids to different R . leguminosarum strains improved symbiotic nitrogen fixation. However, the Hup plasmid from R . meliloti expressed low levels of Hup activity in alfalfa (Bedmar et al., 1984; Behki et a l . , 1985). This observation may be related to observed variability in expression of Hup genes in relation to the host plant (Bedmar et al., 1984).

16


V. Plasmid-Genome Rearrangements The relationship between the chromosome and plasmids present in Rhizobium strains is of interest because of the large size of the plasmids. The DNA-DNA homology studies from different Rhizobium species (see Section I1,B) and nifand nod DNA hybridization (see Sections IV,A and B) indicated that in some strains the DNA sequences are conserved on a plasmid and in other strains it is on the chromosomal DNA. Studies on the organization of nifand nod sequences in R. fredii (Masterson et al., 1985) showed that although the nif and nod hybridization patterns to restriction endonuclease cut DNA were identical within these species, in one strain (USDA194) nifand nod sequences are on the chromosomal DNA. Further, in strain USDA194, the Sym plasmid DNA sequences present in other R. fledii strains are conserved in the chromosomal DNA, suggesting a plasmid-chromosome DNA rearrangement in R. fredii strains. Recently, Cantrell et al. (1982) examined several Hup+ and Hup- strains of B. japonicum for plasmid content. They discovered two plasmids in three spontaneous, nonrevertable hydrogenase mutants (Hup- ) plasmids. The parent strain SR did not contain isolatable plasmids. The authors concluded from these findings that either a large, Hup- -encoding megaplasmid has rearranged to give rise to two smaller plasmids or the two plasmids were generated from the bacterial chromosome. In either event, a structural rearrangement of the Rhizobium genome is very likely. Further evidence that the plasmid and chromosome of B. japonicum strains can undergo rearrangements was provided by Berry and Atherly (1984). They observed that after introduction of a P-group plasmid (RP1) by spheroplast transformation, the RPl DNA was found to be integrated into the chromosome, but, simultaneously an equal-sized piece of chromosomal DNA transposed into a large indigenous plasmid, producing an even larger plasmid. Thus, the very large plasmid of Rhizobium may possess episome-like behavior. This conclusion seems even more likely in light of the finding that B. japonicum possesses at least 18 copies of two insertion-like sequences (Kaluza et al., 1985). Recently, rearrangement of nif genes during heterocyst differentiation in the Cyanobacterium anabaenu has been reported (Golden et al., 1985). But no such DNA rearrangement was observed during transition of Rhizobium to nitrogenfixing bacteriods (Scott et al., 1984; R. K. Prakash and A. G. Atherly, unpublished).

VI. Relationship between Rhizobium and Agrobacterium Plasmids Agrobucterium and Rhizobium both belong to the family Rhizobiaceae. Agrobacteria are closely related to fast-growing rhizobia but not to slow-growing


17

rhizobia (DeLey, 1968; Gibbins and Gregory, 1972; Heberlein et al., 1967). As in fast-growingRhizobium species, where the Syrn plasmid controls the symbiotic functions, the tumor-inducing ability of A . tumefaciens is controlled by a large plasmid designated as the Ti plasmid (see review by Nester and Kosuge, 1981). The high level of genetic relatedness between Rhizobium and Agrobacterium has been shown in several reports. Hooykaas et al. (1982~)compared chromosomal linkage maps of Rhizobium and Agrobacterium and found a high degree of similarity. Prakash and Schilperoort (1982) demonstrated homologous regions in Rhizobium Syrn plasmids and the Agrobacterium Ti plasmid. Hadley and Szalay ( 1982) showed that T-DNA sequences of Agrobacterium are present in diverse Rhizobium species. The implications of these observations are not clear at present. But, studies with R. fredii plasmid DNA indicate that the observed homology of the Syrn plasmid with 7'-DNA and vir-DNA region of A. tumefaciens is due to the presence of bacterial insertion IS-66-like sequences (Ramakrishnan et al., 1986). Insertion elements have already been described in Rhizobium lupini (Preifer et al., 1981), R . meliloti (Ruvkun et al., 1982b), andB. japonicum (Kaluza et al., 1985).

VII. Restriction Endonuclease Maps Restriction endonuclease maps of plasmids facilitate the identification and genetic characterization of plasmid-borne genes and consequently aids in the genetic manipulation of plasmid genes. Strategies for constructing a physical map of a large plasmid are varied but usually depend upon construction of a plasmid DNA clone bank using a vector that will yield large DNA fragments so as to facilitate construction of overlapping sequences. Prakash et al. (1982b) were the first to establish a restriction fragment map of the symbiotic plasmid (pRle1001a) from R. leguminosarum strain 1001. This 150 MDa plasmid was mapped by hybridization of individual HpaI restriction fragments to blotted SmaI and KpnI digestions of the plasmid. Regions homologous to nifstructural genes, Syrn plasmid DNA of R. trifolii, and Ti plasmid DNA of A . tumefaciens were mapped. A large region around the nifstructural genes was found to be highly conserved in these Rhizobium species. Other regions that are common to both the Syrn plasmid of R. leguminosarum and R . rrifolii were also conserved in octopine and nopaline Ti plasmids of A . tumefaciens. The regions that are transcribed in free-living bacteria and bacteriods were also localized on a restriction endonuclease map of the plasmid pR le lo0 la (Prakash et al., 1982a). One region that is actively transcribed in nitrogen fixation bacteroids includes the regions homologous to nifstructural genes and the region that has homology to the Syrn plasmid of R. trifolii. Similarly Huguet et al. (1983) prepared a physical map of a 150 MDa nonsymbiotic plasmid (pRme4la) from R. meliloti and found extensive regions of homology with both octopine and nopaline Ti plasmids and, surpris-

18

R . K. PRAKASH AND ALAN G. ATHERLY

ingly, less homology with R . meliloti plasmid DNA from strains of various geographical origins. Because of the great difficulty in constructing physical maps of very large plasmids (greater than 150 MDa) no megaplasmid has been totally mapped. However, partial maps have been constructed using R' plasmids derived from the megaplasmid of R . meliloti 41 (pRme4lb) (Kondorosi et a f . , 1983, 1984; Banfalvi et al., 1981b; Rosenberg er al., 1981) and strain 201 (Batut e? al., 1984; David el al., 1984). David et al. (1984) found extensive regions of plasmid gene expression (about 100 kb) from a 285 kb R' fragment and as well two regions (about 20 kb) that are expressed only in vegetatively growing cells. Using restriction endonuclease mapping of segments of plasmid DNA, several laboratories found a close linkage of nod and nif genes in several fast-growing Rhizobium species. In R. meliloti, mutations in nod regions mapped withir. about 25 and 13 kb downstream from the nif KDH operon (Kondorosi et a l . , 1984; Buikema er a f . , 1983). In R. trifolii, nod genes are located some 16 kb from the nifstructural genes KDH (Schofield et al., 1983). In R. leguminosarurn a 45 kb region of DNA has been shown to carry two clusters of genes encoding nifgenes separated by a cluster of nod genes (Downie er al., 1983b).

VIII. Perspectives The amount of genetic information now known about plasmid DNA is incredibly small in relation to the immense information present. An estimated molecular weight of 1 X lo9 for one megaplasmid (Burkhardt and Burkhardt, 1984) and the observation that two such megaplasmids exist in R . meliloti (Kondorosi er af., 1984) indicate that, at least in some Rhizobium strains, the amount of plasmid DNA may be equivalent to the total chromosomal DNA of E. cofi (2.2 X lo9 Da). Very useful genes for the survival of bacteria are frequently present on plasmids; thus it is not surprising that symbiotic functions and nitrogen fixation genes are present on the plasmids of a great many Rhizobium strains. But, by no means have all these genes been identified and their functions determined. Prospects for the future are bright in identifying symbiotic and nitrogen fixation genes due to the advent of many very useful techniques in molecular genetics. In this respect two very useful procedures must include lacZ fusions, including the use of Mu-d lac, and mRNA hybridization to cloned fragments to identify functional genes. Tn5 insertional inactivation of tentatively identified genes, as well as random mutagenesis with Tn5, is extremely helpful in identifying gene functions. At present, the genes involved in competitive ability are of great interest and preliminary evidence suggests they may be on plasmids (DeJong er al., 1982; Olson et al., 1985). Also, the involvement of carbohydrates is strongly implicated in some steps of the symbiotic process, but few genes or mutants have

PLASMIDS OF RHlZOBIUM

19

been identified. Sequences are found on plasmids that are highly conserved (Prakash et al., 1981 ; Masterson et al., 1985; Watson and Scholfield, 1985) and there is little evidence to indicate what functions these sequences play in the symbiotic process, or other necessary functions. The hope for a complete understanding of the symbiotic process is that it will eventually lead to intelligent strain construction. Changes in Rhizobium strains that may be useful include altering the host-specificity genes to broaden the host range, increasing the competitive ability, and optimizing nitrogen fixation efficiency or creating Rhizobium strains specific for particular cultivars, soils, or climatic conditions. It also seems very likely that knowledge of the regulation of genes in the nodule tissue will aid in the construction of strains that export new products to the plant, other than nitrogen. Genes for the synthesis of insect hormones, plant hormones, fungicides, or other chemicals can be engineered into Rhizobium strains with expression regulated during the bacteroid form and the products exported to the plant. In this respect Rhizobium may become more useful to the farmer than just a nitrogen source.

ACKNOWLEDGMENTS We wish to thank Dr. S. Shantharam for his critical reading of this manuscript and Miss Ruth Richman for typing the manuscript.

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Ruvkun, G. B., and Ausubel, F. M. (1980). Proc. Narl. Acad. Sci. U.S.A. 11, 191. Ruvkun, G. B., Sundaresan, V., and Ausubel, F. M. (1982a). Cell. Ruvkun, G. B., Long, S. R., Meade, H. M., Van Den Bos, R. C., and Ausubel, F. M. (1982b). J . Mol. Appl. Genef. 1, 405. Sadowsky, H. J., and Bohlool, B. B. (1983). Appl. Environ. Microbiol. 46, 906. Sadowsky, M. J., Keyser, H. H., and Bohlool, B. B. (1983). Inr. J . Sysr. Bacreriol. 33, 716. Sanders, R. E., Carlson, R. W., and Albersheim, P. (1978). Nature (London) 271, 240. Schetgens, T. M. P., Bakkeren, G., Van Dun, C., Hontelez, J. G. J., Van Den Bos, R. C., and Van Kammen, A. (1984). J. Mol. Appl. Genet. 2, 406. Schofield, P. R., Djordjevic, M. A.. Rolfe, B. G., Shine, J., and Watson, J. M. (1983). Mol. Gen. Genet. 192, 459. Schofield, P. R., Ridge, R. W., Rolfe, B. G., Shine, J., and Watson, J. M. (1984). PlantMol. Biol. 3, 3. Scholla, M. H., and Elkan, G. H. (1984). Inr. J. Sysr. Bacreriol. 34, 484. Scholla, M. H., Moorefield, J. H., and Elkan, G. H. (1984). Inr. J . Sysr. Eacreriol. 34, 382. Scott, D. B., and Ronson, C. W. (1982). J . Bacreriol. 151, 36. Scott, D. B., Court, C. B., Ronson, C. W., Scott, K. F., Watson, J. M., Schofield, P. R., and Shine, J. (1984). Arch. Microbiol. 139, 151. Selvaraj, G., and Iyer, V. N. (1983). J. Bacreriol. 156, 1292. Shah, K., Sousa, S., and Modi, V. V. (1981). Arch. Microbiol. 130, 262. Shah, K., Patel, C., and Modi, V. V. (1983). Can. J . Microbiol. 29, 33. Shantharam, S., and Iyer, V. N. (1986). In preparation. Shantharam, S., Gow, J. A., and Bal, A. K. (1980). Can. J . Microbiol. 26, 107. Sik, T., Hovath, J., and Chatterjee, S. (1980). Mol. Gen. Genet. 178, 51 1 . Silhavy, T. J . , Berman, M. L., and Enquist, L. W. (1984). I n “Experiments with Gene Fusions.” Cold Spring Harbor Press, Cold Spring Harbor, New York. Simon, R. (1984). Mol. Gen. Genet. 196, 413. Simon, R., Priefer, U., and Puhler, A. (1983). Biorechnology 1, 784. Skogen-Hagenson, M. J . , and Atherly, A. G. (1983). J. Bacreriol. 156, 937. Stowers, M. D., and Eaglesham, A. R. J. (1983). J. Gen. Microbiol. 129, 3651. Sundaresan, V., Ow, D. W., and Ausubel, F. M. (1983a). Proc. Narl. Acad. Sci. U.S.A. 80,4030. Sundaresan, V., Jones, J. D. G., Ow, D. W., and Ausubel, F. M. (1983b). Nature (London) 301, 728. Szeto, W. W., Zimmerman, J. L., Sundaresan, V., and Ausubel, F. M. (1984). Cell 36, 1035. Torok, I., Kondorosi, E., Stepkowski, T., and Konodorosi, A. (1984). NucleicAcids Res. 12,9509. Trinick, M. J. (1973). Nature (London) 244, 459. Truchet, G., Rosenberg, C., Vasse, J., Julliot, J. S., Camut, S., and Denarie, J. (1984). J . Bacreriol. 157, 134. Vandenbosch, K. A., Noel, K. D., Kaneko, Y.,and Newcomb, E. (1985). J . Bacreriol. 162,950. Van Vliet, F., Silva, B., Van Montagu, M., and Schell, J. (1978). Phsmid 1, 446. Watson, J. M., and Scholfield, P. R. (1985). Mol. Gen. Genet. 199, 279. Weinstock, G. M., Berman, M. L.. and Silhavy, T. J. (1983). I n “Expression of Cloned Genes in Prokaryotic and Eukaryotic Vectors” (T. S . Papas er al., eds.), p. 27. Elsevier, Amsterdam. Wong, C. H., Panhurst, C. E., Kondorosi, A., and Broughton, W. J. (1983). J. CeNEiol. 97,787. Yakobson, E. A., and Guiney, D. G. (1984). J. Bacreriol. 160, 451. Yelton, M. M.. Yang, S. S., Edie, S. A., and Lim, S. T. (1983). J. Gen. Microbiol. 129, 1537. Zurkowski, W. (1980). Microbios 7, 27. Zurkowski, W., and Lorkiewicz, Z. (1976). J. Bacreriol. 128, 481. Zurkowski, W., and Lorkiewicz, Z. (1978). Genet. Res. 32, 311. Zurkowski, W., and Lorkiewicz, Z. (1979). J . Bacferiol. 128, 481.

INTERNATIONAL KEVIEW OF CYTOLOGY. VOL I W

Mouse Mutants: Model Systems to Study Congenital Cataract AUDREYL. MUGGLETON-HARRIS MRC Experiment01 Embryology cind Teratology Unit, Medicul Research Council Laboratories. Carshalton, Surrey SM5 4EF, England

I. Introduction Congenital and early developmental cataracts are common ocular abnormalities and represent an important visual impairment in childhood; 10-38% of all blindness in children is caused by developmental cataracts. One of every 250 newborns (0.4%) has some form of congenital cataract; 26% of those children operated on for congenital cataract are able to attend school, and the majority of those have impaired vision. In the light of the statement that “In view of the inevitable operative risk, in other words, of anesthetic accident, post operative infection and surgical complications, congenital cataract surgery should be undertaken only after the most careful consideration” (Nelson, 1984), it would appear that an analysis of the genetic and phenotypic mechanisms underlying congenital and developmental cataractogenesis is a necessity. In a child with cataracts who is otherwise healthy, between 8.3 and 23% of cataracts are familial, autosomal dominant hereditary being the most frequent mode of inheritance (Nelson, 1984). The limited data on the causative mechanisms of human cataract are related to the fact that man is such a slow breeder, and detailed in vivo morphological classification of inherited cataract and family histories are not readily available. Also the surgical procedure for cataract removal involves fragmentation of the lens, and thus valuable material for histological, cellular, and biochemical analysis is destroyed. Therefore animal models are especially suited for a genetic analysis of cataractogenesis, because the litter size is large and they have a relatively short life-span. Animal Models. There are various animal models with which to study congenital cataract; the advantages of using mouse mutants are obvious: the genetics of the mouse are well researched and the breeding of large litters is comparatively easy. As stated earlier, autosomal dominant hereditary is the most frequent mode of inheritance of congenital cataract, therefore mutants which inherit their cataracts in this manner are specifically interesting. One such mutant is the Cuturuct Fruser (CutFr)mouse. The CatFr mouse develops a cataract prenatally and was first described as a dominant autosomal mutation known as 25 Cupyripht 0 1986 by Academic Prcm Inc. All right? uC reproduction in any t u r n reserved.

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AUDREY L. MUGGLETON-HARRIS

“shriveled” (Fraser and Schabtach, 1962). Mice begin to show lens deterioration between 10 and 14 days of intrauterine life. Initially the cell nuclei in the deep cortex become abnormally pyknotic; degeneration of cytoplasm and destruction of the lenticular nucleus follow. Two weeks postnatally, the anterior epithelia show unusual mitotic activity with the formation of multiple cell layers that infiltrate into the abnormal fibers of the anterior cortex. Cells at the equatorial region retain their ability to differentiate until complete hydration of the lens occurs after 1 year of age. lnhibition of elongated older cells takes place by the breakdown of the nuclear membrane (Gelatt and Das, 1984). The abnormalities associated with this congenital cataract can be appreciated by observing the normal structure of the lens and its development (McAvoy, 1981). The pathogenesis of another genetically malformed lens mutant, Elo, has indicated that the eye lens obsolescence is independent of the extraocular environment and that the y-crystallin synthesis in the lens of the Elo mutant and the normal lens was similar, thereby suggesting that the necrotic process in the lens fibers of the Elo is not due to defects in y-crystallin synthesis. Initial cytological changes in this mutant were the appearance of numerous lysosomal bodies, the destruction of mitochondria at the basal cytoplasm of the lens fibers, and nuclei with elevated perinuclear cistema (Oda et al., 1980; Watanabe et al., 1980). A systematic search for heritable electrophoretic variation among the lens crystallins to date yielded no useful markers for the mammalian crystallin genes. However, isoelectrofocusing of soluble lens proteins from 29 strains of mice revealed 3 electrophoretic phenotypes. Genetic experiments and molecular sieving studies demonstrated that the variation was encoded by three alleles at a locus, designated Len-Z, and that the affected protein was a y-crystallin. Len-/ was determined to be located on mouse chromosome 1. Based upon the nature of the genetic variant, it is plausible to consider Len-1 as the structural locus for one of several mouse y-crystallins. Two mutations affecting the mouse lens, vacuolated lens (VL), and eye lens obsolescence (Elo) have also been mapped to chromosome 1 (Skow, 1982). Certainly in the light of the y-crystallin genes being closely linked to cataractous mutants (Skow, 1982), there is the possibility of some regulation mutation affecting gene expression of the lens crystallins in the C U P mouse. The fact that the present C U P mouse is not on the A I J background as first reported a number of years ago and appears not to be on several alternative albino inbred strains which we have studied could indicate that it is now a “unique” strain of cataractous mouse (Muggleton-Harris et a l . , 1986). A mutation which arose spontaneously in a stock of mice homozygous for the Robertsonian translocation (Rb) Ald is an albino, whose pink eyes are opaque. Examination of the eyes revealed the opacity in the lens, and it was present when the eyes first opened. Breeding showed the character to be inherited as an autosomal dominant, and it was given the name and symbol lens opacity, Lop. Linkage tests of Lop with markers on various chromosomes, to map the locus

MOUSE MUTANTS: MODEL SYSTEMS FOR CATARACTS

27

and to exclude allelism with other genes for cataract known to map in other positions, confirm that there is no allelism with several already mapped genes affecting the eye and that there is a locus for a dominant cataract gene of chromosome 10 for Lop. The Lop occupies almost an end position on the chromosome and thus provides a useful marker in linkage studies (Lyon et al., 1981; West and Fisher, 1985).

11. The Search for Chromosomes Associated with the Mutants Cataract

Using available genetic markers, various loci on the chromosomes of the mouse may be eliminated as being associated with the CutFrcongenital cataract. By breeding and cross-breeding the inbred CarFr mouse with the appropriate mice which carry specific genetic markers, the chromosome carrying the C U P gene can be determined by a process of elimination. Once a specific marker is linked to the CatFrmouse, and if the chromosome on which the marker is carried can be identified, then the chromosome with the CatFrgene will also be known. Already a number of chromosomes (specific) are known not to carry the CatFr mutation. Details of the previous work undertaken by Fraser and Schabtach (1962) and the chromosomes mapped by ourselves are given in Table I. The markers that are available for mapping the loci for the CarFrmouse are indicated in Table I. We have crossed inbred and cataractous strains to establish dominance and then backcrossed to establish genetic segregation. The backcross progeny are then typed for all relevant marks (i.e., those at which the parental strains differ). Fifty animals are studied for initial matings, and once provisional evidence indicates a positive, then the numbers will be increased to 200-300 animals. The CutFrmutant has been partially mapped. Previous studies by Fraser and Schabtach (1962) used the genes a, b, c, N, and Sex; we have covered those with an X on the marker list. A map of the chromosomes already mapped at specific loci is now available (Fig. 1). Kosambi’s function was used to convert the frequency of crossing over to physical chromosomal distance (Robinson, 1971). There are some obvious chromosomes yet to cover: chromosome 1 (with which other lens abnormalities are associated) and chromosome 10 (Lop lens defect). 111. Lens Crystallins Specialized proteins called crystallins are associated with the lens structure (Papaconstantinou, 1967, 1967). The synthesis of these proteins in the lens is ‘The CarFr and Lop lens abnormalities are linked on chromosome 10 and are probably allelic. This gene location has been recently confirmed by Muggleton-Hams er al. (1986).

28

AUDREY L. MUGGLETON-HARRIS TABLE I MARKERS FOR MAPPING THE LOCI Locus Idh-1 A0x-1

Marker

I I

Biochemical Biochemical Biochemical Coat color Biochemical Biochemical Biochemical Biochemical Biochemical Coat color Biochemical Biochemical Coat color Biochemical Biochemical Coat color Biochemical Biochemical Biochemical Coat color Biochemical Coat texture Limb mutant Biochemical Biochemical Coat color Biochemical Biochemical Limb mutant

Pep-3

1

2 3 3 3 4 4

Gpd-1 Pgm-2

B Pgm-1

Ldr-1 Miwh

Gpi-I Hbb C

Es-1 Mol-1 Tcf d Es-3 Re Xt

Es-10 Gpr-I

N Ce-2 PgK-2 bm Sex

CUf“ MOUSE

Chromosome

A Adh-3 Cur-2 Sep-1

FOR THE

4 5 6 6 7 7 7 8 9 9

9 I1 II 13 14

15 15 17 17 19 20

Sex

Mapped

X X X

X X X X

well documented for development, differentiation, aging, and cataract (Bloemendal, 1977, 1981; Hoenders and Bloemendal, 1983; Harding and Dilley, 1976; Piatigorsky, 1981). The initiation of crystallin synthesis in the mouse does not require proper development of the lens (Zwaan and Williams, 1968; Zwaan, 1975). Altered patterns of crystallin synthesis and aggregation of lens proteins have been reported for aging and cataractous lenses (Mostafapor and Reddy, 1980; Genis-Galvez et al., 1968; Harding, 1981). The eye lens of the CatFr mouse contains reduced amounts of y-crystallins (Garber et al., 1984), and, in initial studies, the presence of “abnormal” proteins was found in CatFrlenses, but using more sensitive methods, the authors found that traces of these “abnormal” proteins are in fact present in normal lenses (Garber et al., 1985). The

1

2

FIG.I .

3

4

5

6

7

8

9

10

11

12

13

Map of chromosomes covered for specific loci for the Curfr mutant. (---)

cr a / . ( 1986).

14

15

16

17

18

19

X

Fraser and Schabtach (1962): (- - - -) Muggleton-Harris

30


murine a-crystallin-related proteins were not present in cultures of normal or mutant CatFr lenses, nor were they present in the in vitro translation products directed by RNA extracted from such lenses (Mostafapor and Reddy, 1980; Harding, 198 1). Column chromatography of total soluble lens proteins from various cataractous mutants has shown increased amounts of a-crystallin aggregated in the lenses. However, each of these mutations has a distinct etiology, making it likely that the formation of a-crystallin derivatives and high-molecular-weight aggregates is an indirect effect of the mutations. In each case, some primary cataractogenic defect presumably initiates a sequence of events leading to the development of the cataract (Genis-Galvez et al., 1968). One or more of these events could be responsible for the changes noted in crystallin synthesis, structure, or protein aggregation. Therefore the primary genetic mutation which results in the initiation of the cataract is an area of study which needs clarification. It is known that the mRNA for a-crystallin synthesis is present in nearly normal amounts and in translatable form in lenses of CatFrmice at birth (Garbor et al., 1985); and yet, as stated previously, the lens deterioration associated with the CutFr mouse is present between 10 and 14 days of the embryo’s intrauterine life. The observation that the loss of the y-crystallin mRNA correlates in time with the destruction of the lens fiber cells in the CatFr mouse demonstrates the need to study the primary genetic defect.

IV. Cellular Studies on Lens Epithelial Cells The lens epithelial cells from normal and cataractous ( C U P )mice have been cloned and characterized for morphology, replication, and growth patterns. The C U P cells undergo fewer replications than the normal cells and the growth pattern is also different (Muggleton-Harris et al., 1981). One observation made on the cultured lens epithelial cells which was in conflict with a number of previous studies (Eguchi and Kodarna, 1979; Russell et al., 1977; Okada et af., 1971) was that the mouse lens epithelial cells did not undergo differentiation to fibers and form lentoid bodies in vitro. Only if the cells were left undisturbed with cellular or lenticular membrane/capsule present did those alterations take place. Cells in close proximity to one another will form plaques of cells (Muggleton-Harris et al., 198 1). The stages of differentiation in lens epithelial cells in culture has been well documented (Creighton et al., 1981). The cytoskeleton of cultured cells has been linked with elongation (Piatigorsky er al., 1972). Cell volume and nuclear size are altered with elongation in vivo (Hendrix and Zwaan, 1974) and in v i m ; the cytoskeletal structures generating intracellular forces and/or membrane interactions may also be important factors for cell elongation (Piatigorsky, 198 1; Ramaekers and Bloemendal, 1981). Lens epithelial cell elongation has been reported in the absence of microtubules (Beebe et al., 1979),


31

and in rat lens epithelial cell cultures, the presence of neural retina-conditioned medium precipitated cell fiber differentiation and synthesis of y-crystallin (Campbell and McAvoy, 1984). The loss of regulation of cell replication and growth which is a major factor of congenital cataractogenesis may be reflected in a distortion of the microtubulemicrofilament connections to the cell membrane. Transformed fibroblasts show a different distribution of actin-containing material to their normal counterparts (Puck, 1977). Anti-actin serum stains stress fibers in the cultured cells; the morphological appearance of this major component of the cellular microfilament network has been shown to depend upon the cell’s physiological state and upon the presence of pathological changes (Ramaekers and Bloemendal, 1981; Puck, 1977). In cultured bovine MLE the intermediate-sized filaments of the vimentin type are associated with certain regions of the cytoplasm, and the number of bundles of intermediate filaments, with and without microtubules, is arranged almost perpendicular to the microfilament bundles (Ramaekers and Bloemendal, 1981). The epitheloid lens-forming cells grown in vitro as monolayers are connected by gap junctions, and the intermediate filaments occur in close proximity to the intercellular boundary. At the cell-to-cell boundary, terminal anchorage sites of microfilament bundles and a densely stained web of fine intermediate filamentous material appear at the site of membrane anchorage (Ramaekers and Bloemendal, 1981). Actin and tubulin immunofluorescence and y-crystallin synthesis have been reported to be related to stages of differentiation of lens epithelial cells in culture (Creighton et al., 1981). A tabulation of the various observations of this correlation was made with specific stages associated with the cells as they replicate and grow in virro. At stage 1 when the cells are rounded or cuboidal the actin immunofluorescence was diffuse and in globules (Creighton et al., 1976; Hamada and Okada, 1977; Mousa and Trevithick, 1977). When the cells are elongated (fibroblastic polygonal stellate), the actin immunofluorescence is associated with the microfilament fibers (Tamura, 1965; Okada et al., 1971; McDevitt and Yamada, 1969; Piatigorsky and Rothschild, 1972; Creighton et al., 1976; Hamada and Okada, 1977). Observations on cultured lens epithelial cells by Creighton et al. (1977) indicate that the nucleus of the cell moves to one side and the cytoplasm is filmy or lace-like, the actin immunofluorescence appeared fibrous and associated with the microfilaments at this stage (Van der Veen and Heyen, 1959; Tamura, 1965; Friedrich and Glaesser, 1971; Creighton et al., 1976; Hamada and Okada, 1977). At stage 4 the production of y-crystallin has been observed (Papaconstantinou, 1967; McDevitt and Yamada, 1969; Creighton et al., 1976) and there was no alteration in the distribution of actin observed. Stage 5 brought about the disintegration of the nucleus in the cells (Papaconstantinou, 1967; Mamo and Leinfelder, 1958), and at stage 6 the cells became a fibrous mass with globules and/or “lentoid bodies” forming (Mamo and Lein-

32


felder, 1958; Okada et al., 1971; Creighton et al., 1976; Hamada and Okada, 1977; Russell et al., 1977). In cloned mouse lens epithelial cells which are kept isolated from degenerating cells and lens capsule material, cell elongation and the formation of “lentoid bodies” have not been seen (Muggleton-Harris et al., 1981). Conflicting reports on the presence of lens proteins in cultured lens epithelial cells appear to be related to species and culturing conditions. Cloned cells of chicken lens epithelium have formed differentiated colonies when conditioned medium was used, a gelatin layer encouraged clonal growth, and “lentoid bodies” were observed at high cell density (Okada et al., 197 1 , 1973). Growth control of lens epithelium cells by cell substratum and interactions of the cell with the substratum to induce cell shape and alterations in morphology highlight the extracellular influences on the cell’s morphology and behavior (Iwig and Glaesser, 1979a,b). The replication, growth, and morphology of the CatFr mouse lens epithelial cells in vitro have been well characterized (Muggleton-Harris et al., 1981). The distribution of lens proteins and actin microfilaments in these cells is not known, but preliminary studies appear to indicate that the CarFrcells do not synthesize the lens y-crystallins in v i m , whereas the noncataractous mouse lens epithelial cells do (unpublished results).

V. Manipulations of the Cataractous Phenotype Somatic cell hybrids and manipulative studies have been undertaken on the cultured CatFrmouse lens epithelial cells. The micromanipulation of the cells is achieved with the aid of deFonbrane micromanipulators (Lipman and Muggleton-Harris, 1982). The results from these experiments demonstrated that the two parental genotypes which constituted the hybrid had been retained by the clone of cells derived from that hybrid. The parental cells from a noncataractous mouse and the C U P mouse display a finite life-span. The cells of cataractous origin have a decreased number of population doubling levels compared to the capacity of the normal cells when replicated in v i m . Hybrid cells derived from individual cell fusions of these two types have a mode of replication similar to that of normal cells, thus indicating that the cataractous mouse lens epithelial cells have been modified by the addition of noncataractous lens epithelial cell components. Which components of the noncataractous lens epithelial cells had this effect is not known, but the system is available to analyze with the technique of nuclear transfer. This technique was developed and used to identify the components controlling the cellular aspects of replication in vitro (Muggleton-Harris and Hayflick, 1976; Muggleton-Harris and Palumbo, 1979; Muggleton-Harris and DeSimmone, 1980).


33

VI. Possible Areas of Research for the Future If we are to move toward the eventual prevention of cataract, the initiating point in pathogenesis becomes critical. During early morphogenesis, organ or tissue specific “stem” cell lines are established. Initially multipotent, the progeny become committed to the expression of an increasingly restricted number of phenotypes. This process leads to acquisition by cells of specialized structures, e.g., cytoskeletal filaments, altered nuclear-cytoplasmic ratio, and specific functions, e.g., synthesis of lens proteins. Implicit in this process is the idea of a progressive loss of the cell genome, for reprogramming from one type of behavior to another. In vivo the CatF’-differentiated central lens epithelium cells continue to replicate for at least 3 weeks following birth. The noncataractous mouse lens cells cease replicating once the animal is born. This demonstrates a lack of cell regulation during embryogenesis. At which point was this initiated and how can this problem be studied? One approach is chimera studies; chimeric or allophenoic mice (Tarkowski, 1961, 1963; Mintz, 1969, 1971) can readily be made by aggregating 4-8 cell embryos. The embryos are flushed from the oviduct of the mouse, and after removal of the zonae pellucida with acid tyrodes, the embryos are pushed together and held for a few minutes until they adhere. These manipulations are carried out at 37°C. To enable these early embryos to develop they are inserted into the uterus of a pseudopregnant mouse after cultivation to the late morula/early blastocyst stage of development. Differing coat colors, or other genetic variants such as glucose-phosphate isomerase (GPI; EC 5.3. I .9) isozymes will act as genetic markers. The growth and development of the lens can be studied histologically on embryos before birth. Slit lamp observations on a day-to-day basis can monitor the lens in vivo after birth. One area of research would involve the removal of the epithelium and a GPI analysis of the different populations of cells; this will provide an estimate of the contribution of cells from both donor embryos. Strain-specific allelic variants of GPI are analyzed electrophoretically for parental cell lines and the experimental hybrid cells and their resultant clones. The isozyrnes are separated using a Titan I1 ZipZone cellulose acetate plate (Helena Labs, Beaumont, Texas) with 0.025 M Tris base, 0.192 M glycine for 1 hour at 200 V . Following the application of a 2% agar overlay containing 0.3 M Tris-HC1 at pH 8.0,0.3 1 mM NADP, 15 mM fructose 6-phosphate, 20 mM Mgacetate, 2.4 pM dimethylthiazolyldiphenyltetrazolium bromide, 1.3 pM phenazine methosulfate, and 10 IU of glucose-phosphate dehydrogenase, the GPI could be detected. Since such analyses can be accomplished on blood samples from mice, lenses from embryos or chimeras, or manipulated mouse blastocysts, very little material is needed. Thirty cells derived from one hybrid MLE gave

34


clear results that the strain-specific allelic variants of both parental cells were present in the cells (Lipman and Muggleton-Harris, 1982). Rescue of an abnormal developmental defect by embryo aggregation or manipulation of early mouse embryos has been achieved. In a study of inherited photoreceptor cell degeneration (ru/ru), the interaction of mutant and normal pigment epithelium was analyzed in experimental chimeric mice. The eyes of the resulting chimeric mice had particles of normal retina interspersed with particles lacking photoreceptors. The synthesis of rod outer segment disks proceeded normally in photoreceptor cells underlying mutant pigment epithelial cells. The results indicated the site of the mutant gene action to the neural retina (LaVail and Mullen, 1976). Experimental chimerism has also been used as a genetic tool to study the X-linked lethal mutation jimpy up) (Eicher and Hoppe, 1973). Aggregates of the early mouse embryo were made between Tajp/ + x + + / Y crosses and wild-type embryos, the viable X-linked tabby gene serving as a marker to identify chimeras. The experimental chimera was shown to transmit the TajpX chromosome demonstrating that sufficient amounts of the normal cell components of the embryo were present to counteract the lethality of the jp/Y genotype and allow jp/Y cells to differentiate in the testis and form functional jp sperm. When trisomic and diploid (Ts-2n) mouse embryos were aggregated, the resultant chimeras can have Ts and 2n cells in all embryos. This approach has been used for trisomes 15, 16, 17, and 19 and the results indicate that significant numbers of trisomic cells are found in brain, heart, liver, and kidney; the mean proportion of Ts cells was between 40 and 55%. Trisomic cells contribute in a normal manner to development in those chimeras which came to term (Cox et al., 1984). Possible levels of interaction between genotype and environment can be studied at both the cell and embryonic stages. Mouse blastocysts can be injected with genetically distinct embryonic cells, then develop normally and tissues of the resulting offspring are often chimeric consisting of cells from both donor and host origin (Gardner, 1968, 1972). The developmental potential for normal development of embryonal carcinoma cells has been tested in this manner, and in the experimental chimeras, it was found that these “malignant” abnormal cells participated in normal development (Papaioannou et af., 1973). Lack of material with which to study the human congenital cataract makes it necessary to fully utilize available animal models. Mouse mutants enable baseline data to be obtained in sufficient numbers as to be statistically significant. Such data on the embryonic and cellular aspects of congenital cataractogenesis may be used to further our knowledge of this abnormal birth defect. The fields of recombinant DNA and related technologies have revealed much about the structure of the genes associated with lens crystallin synthesis (Piatigorsky, 1981; Garber et af., 1985; Skow, 1982). Both the in vitro and in vivo approaches compliment one another, and eventually advantage will be taken of the rapid

+


35

advances in the gene technology field to challenge the genes directly by incorporating them into the embryonic genome.

REFERENCES Beebe, D. C., Feagans, D. W., Blanchette-Mackie, E. I., and Nau, M. E. (1979). Science 206, 836-838. Bloemendal, H. (1977). Science 197, 127-138. Bloemendal, H. (1981). In “Molecular and Cellular Biology of the Eye Lens” (H. Bloemendal, ed.), pp. 1-47. Wiley, New York. Campbell, M. T., and McAvoy, J. (1984). Exp. Eye Res. 39, 83-94. Cox, R. D., Smith, S. A., Epstein, L. B., and Epstein, C. J. (1984). Dev. Biol. 101, 416-425. Creighton, M. O., Mousa, G. Y., and Trevithick, J. R. (1976). Diflerenfiafion 6, 155-167. Creighton, M. 0.. Mousa, G. Y., and Trevithick, J. R. (1981). Visual Res. 21, 25-35. Eguchi, E., and Kodama. R. (1979). Ophthalmic Res. 11, 308-315. Eicher, E. M., and Hoppe, P. C. (1973). J. Exp. 2001.183, 181-184. Fraser, F. C., and Schabtach. (1962). Gener. Res. 3, 383-387. Friedrich, E., and Glaesser, D. (1971). Acra. Biof. Med. Germ. 27, 41-43. Garber, A. T., Goring, D., and Gold, R. J. M. (1984). J . Biol. Chem. 259, 16, 10376-10379. Garber, A. T., Winkler, C., Shinohara, T.. King, C. R., Inana, G., Piatigorsky. J., and Gold, R. I. M. (1985). Science 227, 74-77. Gardner, R. L. (1968). Nature (London) 220, 596-597. Gardner, R. L. (1972). In “Reproduction of Mammals” (R. V. Short and C. R. Austin, eds.). Cambridge Univ. Press, London and New York. Gellatt, K. M., and Das, N. D. (1984). Curr. Eye Res. 3, 765-778. Genis-Galvez, J.. Maisal, H., and Castro, J. (1968). Exp. Eye Res. 71, 593-605. Hamada, Y., and Okada, T. S. (1977). Dev. Growrh Direr. 19, 265-273. Harding, J. J. (1981). In “Molecular and Cellular Biology of the Eye Lens” (H.Bloemendal, ed.). Wiley, New York. Harding, J. J., and Dilley, K. J. (1976). Exp. Eye Res. 22, 1-73. Hendrix, R. W., and Zwaan, J. (1974). Nature (London) 247, 145-147. Hoenders, H. J., and Bloemendal, H. (1983). J . Geronrol. 38, 278-286. Iwig, M., and Glaesser, D. (1979a). Ophthalmic Res. 11, 293-298. Iwig, M., and Glaesser, D. (1979b). Ophthalmic Res. 11, 298-301. LaVail, M. M., and Mullen, R. M. (1976). Exp. Eye Res. 23, 227-245. Lipman, R. D., and Muggleton-Harris, A. L. (1982). Somatic Cell Genef. 8, 791-800. Lyon, M. F., Jarvis, S. E., Sayers, I., and Holmes, R. S. (1981). Genet. Res. Cambr. 38,337-341. MacAvoy, J. W. (1981). In “Mechanisms of Cataract Formation in the Human Lens” (G. Duncan, ed.). pp. 7-47. Academic Press, New York. McDevitt, D. S., and Yamada, T. (1969). Am. Zool. 9, 1130-1 131. Mamo, J. G., and Leinfelder, P. J. (1958). AMA Arch. Ophthalmol. 59, 417-419. Mintz, B. (1969). Orig. Article Ser. 5, 11-22. Mintz, B. (1971). In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., ed.). Freeman, San Francisco, California. Mostafapor, M. K., and Reddy, V. N. (1980). Invest. Ophthalmol. Visual Sci. 19, 206. Mousa, G. Y., and Trevithick, J. R. (1977). Dev. Growth Difler. 19, 265-273.

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Muggleton-Harris. A. L., and DeSimmone, D. W. (1980). Somatic Cell Genet. 6, 689-698. Muggleton-Harris. A. L., and Hayflick, L. (1976). Exp. CeNRes. 103, 321-330. Muggleton-Harris. A. L., and Palumbo, M. (1979). Somatic Cell Genet. 5 , 397-407. Muggleton-Harris, A. L., Lipman, R. D., and Kearns, J. (1981). Exp. Eye Res. 32, 563-573. Muggleton-Harris, A. L., Festing, M. F. W., and Hall, M. (1986). Genet. Res., in press. Oda, S., Watanabe, K., Fujisawa, H., and Kamagama, Y. (1980). Exp. Eye Res. 31, 673-681. Okada, T. S., Eguchi, G., and Takeichi, M. (1971). Dev. Growth Difler. 13, 323-335. Okada, T. S., Eguchi, G., and Takeichi, M. (1973). Dev.Biol. 34, 321-333. Nelson, L. B. (1984). Ophthalmol. Surg. 8, 697-688. Papaconstantinou, J. (1965). Biochim. Biophys. Acta 107, 81-90. Papaconstantinou, J. (1967). Science 156, 338-346. Papaioannou, V., BcBurney, M. W., and Gardner, R. L. (1973). Nature (London) 258, 70-73. Piatigorsky, J. (1975). Ann. N.Y. Acad. Sci. 253, 333-347. Piatigorsky, J. (1981). Differentiation 19, 134-153. Piatigorsky, J., and Rothschild, S. S. (1972). Dev. Biol. 28, 382-389. Piatigorsky, J., Webster, H. de F., and Wollberg, M. (1972). J . Cell Biol. 55, 82-92. Puck, T. T. (1977). Proc. Narl. Acad. Sci. U.S.A. 74, 4491-4495. Ramaekers, F. C. S., and Bloemendal, H. (1981). In ‘‘Molecular and Cellular Biology of the Eye Lens” (H. Bloemendal, ed.), pp. 85-135. Wiley, New York. Robinson, R. (1971). “Gene Mapping in Laboratory Animals.” (Part A). Plenum, New York. Russell, P., Fukui, H. N., Tsunemetsu, Y.,Huang, F. L., and Kinoshita, J. H. (1977). Invest. Ophthalmol. Visual Sci. 16, 243-246. Skow, L. C. (1982). Exp. EyeRes. 34, 509-516. Tamura, S. (1965). Jpn. J. Ophthalmol. 9, 1130-1131. Tarkowski, A. K. (1961). Nature (London) 190, 857-860. Tarkowski, A. K. (1963). Narl. CancerInst. Monogr. 11, 51-67. Van der Veen, J., and Heyen, C. F. A. (1959). Nature (London) 183, 1137-1138. Watanabe, K., Fujiosawa, H., Oda, S., and Kameyama, Y. (1980). Jpn. J. Ophthalmol. 31, 683689. West, J. D., and Fisher, G. (1985). Genet. Res. Cambr. 46, 45-56. Zwaan, J. (1975). Dev. Biol. 44, 306-312. Zwaan, J., and Williams, R. M. (1968). J . Exp. Zool. 169, 407-422. Zwaan, J., and Williams, R. M. (1969). Exp. Eye Res. 8, 161-167.

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. I W

Cell Wall Synthesis in Apical Hyphal Growth J. G . H. WESSELS Department of Plant Physiology, Biological Centre, University of Groningen, 9751 N N Hnren, The Netherkinds

I. Introduction Fungi, with the exception of the unicellular yeasts, typically grow by means of hyphae which elongate at their apices. The mycelial colony, consisting of a system of branched hyphae, may thus grow over and through substrates. Consequently, the actively growing part of the organism constantly moves away from its original position while colonizing dead organic substrata (saprotrophs) or other living organisms (biotrophs) as in parasitic and symbiontic associations. Particularly with plants, such associations have developed as manifested by the frequent occurrence of fungi as plant pathogens (Dickinson and Lucas, 1982; Misaghi, 1982) and their association with the roots of nearly all plants known as mycorrhizae (Harley and Smith, 1983). Indeed, there is evidence of a longstanding mutual dependence of plants and fungi. They are the main decomposers of the lignocellulose wall of plants (Crawford, 1981) and they have been observed in a vesicular-arbuscular mycorrhizal association with the roots of primitive Devonian land plants (Nicholson, 1975). While the possession of cell walls makes plants immobile and the loss of cell walls was a factor in the aquisition of mobility in animals, the fungi have evolved as organisms with cell walls which nevertheless have a certain mobility due to apical growth. The typical way cell wall growth occurs in plants, namely, diffuse extension growth, can also be found among fungi but never during the invasive growth of the mycelium. Examples of diffuse wall growth are found in a certain stage of elongation of sporangiophores of zygomycetes such as Phycomyces or in the hyphae of elongating stipes of fruit bodies of the Agaricales (basidiomycetes). In fact, one of the hypotheses to explain microfibril orientation in elongating plant cells (Lloyd, 1984), namely, the multinet-growth hypothesis, was formulated on the basis of observations on fibril orientation during intercalary growth of P hycomyces sporangiphores (Roelofsen, 1959). On the other hand, apical growth of tubular cells also occurs in plants, notably during growth of root hairs and pollen tubes (Sievers and Schnepf, 1981). Cytologically as well as functionally these cells resemble fungal hyphae and therefore reference to research on apical wall synthesis in these systems will be made when appropriate. 31 Copyright 0 1986 by Acadumic Prms. Inc. All rights of rcproduction in any form rc\crvcd.

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The problem of apical hyphal growth does not only concern the morphogenesis of a tubular cell. It is also through the growing hyphal tips that the fungus communicates with its biotic and abiotic environment and translates environmental signals in terms of changes in rate or direction of growth or the formation of adaptive apical structures. The direction of growth is regulated in negative autotropism, i.e., the tendency of the hyphae to grow away from each other (Robinson, 1973) and in chemotropism and positive autotropism as in sexual interactions (Gooday, 1975). Recognition between fungal hyphae and plant-host tissues probably also involves hyphal tips with strong evidence for hyphal wall polymers as elicitors of some of the ensuing reactions (Kosuge, 1981; Albersheim er al., 1983). Acceptance or rejection of the fungus by the host tissue is most probably mediated by mechanisms which either allow or check apical growth of the fungus. In the case of biotrophic fungi, acceptance often leads to morphogenetic changes at hyphal apices resulting in typical intracellular infection structures, such as haustoria in parasitic associations and vesicular arbuscular structures in mycorrhizas. Finally, it should be realized that fungi can only display their invasive growth in solid dead substrata or living organisms by virtue of the production of an array of hydrolytic enzymes which hydrolyze polymers in the milieu. On the one hand, the breakdown products can be taken up and used to sustain growth of the fungus. On the other hand, hydrolysis of the polymers clears the way for penetration of the hyphae into the solid substrata. Although little direct evidence is available, it is likely that these enzymes reach the milieu by secretion at the growing hyphal apices (Sentandreu et al., 1981) where the structure of the wall must allow for the passage of these proteins (Chang and Trevithick, 1974). This brief overview of biological activities associated with apical growth in fungi serves to illustrate the importance of understanding how wall synthesis and expansion at the hyphal apex takes place. This is not an easy problem because the apex represents but a tiny part of a hypha. Also, while in growing hyphae wall synthesis per unit area is maximal at the tip, the total amount of wall material synthesized subapically at the same time is appreciable and may qualitatively differ from wall material synthesized at the apex (Sietsma et al., 1985). Therefore, conventional techniques of biochemistry must be combined with a variety of other approaches, including cytochemical and autoradiographic methods, to begin to understand the process of apical wall growth. 11. Observations on Living Hyphae A. THEPHENOMENON OF APICALGROWTH The phenomenon of apical cellular growth received much attention in the nineteenth century and hypotheses generated at that time to explain polarized growth of tubular cells surrounded by rigid walls survive in modem concepts on

CELL WALL SYNTHESIS IN APICAL HYPHAL GROWTH

39

apical growth. Most notable is a publication of Reinhardt (1892) which reviews concepts prevailing at that time. This work added a wealth of new observations interpreted within the framework of a model which still contains elements worth consideration in the light of modem knowledge. Reinhardt worked with the wide hyphae (16- 18 pm) of Peziza species, particularly Peziza sclerotiorum, which displayed a growth rate of 14-23 p,m min- with a maximum of 34 pm min- I . These values are similar to those recorded for the fast-growing leading hyphae of Neurosporu crussa (Steele and Trici, 1975), another ascomycete which has become the subject of many related observations. The most relevant observations made by Reinhardt (1892) can be summarized as follows. 1. There is a relationship between the shape of the apex and the growth rate of the hypha. Slowly growing hyphae have half-spherical tips; with increasing growth rates the tips assume a more tapered appearance going to half-ellipsoids of revolution. 2. When hyphal growth is temporarily stopped, e.g., by applying water to the cultures, the apices swell. With minor disturbances growth resumes after some time from these apices but the emerging hyphae have a smaller diameter. If growth is checked for a longer time, then the swollen tips flatten and hyphal growth from these tips is irreversibly blocked. Resumption of growth takes place by branches arising just under the modified tip. 3. A positive correlation exists between growth rate of hyphae and their diameters. 4. Flooding of hyphae with water often causes bursting of tips. However, bursting never occurred at the extreme apex but always at the boundary of the apical dome and the cylindrical part of the hypha. 5. The three first observations and observations concerning the the site of Occurrence of curvature in growing hyphae were considered as evidence for growth at the tips. Reinhardt concedes that the observation of subapical bursting of tips could be construed as evidence for growth just under the tip because there the wall is apparently weakest. However, he considers that just in this area the tangential stress in the wall, due to turgor, becomes maximal and turgor pressure could thus cause rupture of the young wall in this area. Direct demonstration of apical growth by observing the displacement of particles applied to the apex was possible only with root hairs of Lepidium sativum.

With regard to the mechanisms involved in apical extension growth Reinhardt ( 1 892) considered the then prevailing theory of leading botanists who regarded enlargement of wall area at the apex as a process in which an elastic wall expands under turgor pressure while new wall material is being added by apposition or intussusception. However, he regarded this theory as inadequate because it would require an increase in mechanical strength of the wall going from the very apex to the base of the extension zone. As he puts it, such an increase in strength could be achieved by a proportional increase in wall thickness or by a change in

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the quality of the molecules that make up the wall. He found no evidence for a change in wall thickness. A change in the quality of the wall molecules-which is precisely the kind of change which recent work in our laboratory suggests (see Section V,B)-was considered unlikely. In both cases one would expect that an increase in hydrostatic pressure as caused by flooding with water would result in swelling and bursting at the extreme tip which he did not observe. Reinhardt (1 892) concluded that the wall must have uniform strength over the whole apex and that the wall grows by intussusception of wall material, maximally at the extreme tip and declining to zero at the base of the extension zone. In his view turgor pressure does not cause plastic expansion of the wall at the apex which is rigid enough to withstand this pressure. The fact that relief of turgor pressure by applying solutions of low osmotic potential stopped growth was interpreted as being due to detachment of cytoplasm from the apical wall, thus interrupting the organized delivery of new wall materials by the cytoplasm to the wall. The basic observations of Reinhardt have been confirmed and extended by later workers. Using artificial markers, direct evidence was obtained that growth of hyphae is confined to the apex (Burgeff, 1915; Smith, 1923; Stadler, 1952) and, moreover, that there exists a gradient in expansion rate of the surface declining from the extreme tip to the base of the extension zone (Castle, 1958). Trinci and Saunders (1977) have confirmed that apices of fast-growing hyphae are not hemispherical but more closely approximate half-ellipsoids of revolution. The many measurements made by Trinci and co-workers (Trinci, 1973; Steele and Trinci, 1975) have also revealed that there is indeed a rather strict positive correlation between the growth rates of individual hyphae, their diameters, and lengths of the extension zones. The uniformity of wall thickness over the apex was confirmed by electron microscopy of sections (Girbardt, 1969; Grove and Bracker, 1970; Trinci and Collinge, 1975). In agreement with Reinhardt’s observations, experiments of Robertson (1958) with Fusarium oxysporum showed that after flooding of hyphae with solutions of high- or low-osmolarity arrestment of growth was accompanied by swelling just under the apex. Continuation of hyphal growth occurred either from the swollen apex, after a brief arrestment, or by subapical branching when arrestment was longer than 40 seconds. The phenomenon that hyphal apices when flooded with solutions of low osmolarity tend to swell and burst in a region just under the very tip has also been noted by others (Robertson, 1958, 1965; Park and Robinson, 1966; Bartnicki-Garcia and Lippman, 1972b; J. H. Sietsma and J . G . H. Wessels, unpublished). Although these independent studies largely confirm the basic observations made by Reinhardt (1892), his model of apical growth has generally been rejected in favor of models featuring expansion of the wall by turgor pressure. It can be surmised that this was partly due to d’Arcey Thompson’s (1918, 1942) considerations on the origin of cell form. Yet, as will be discussed later, modern research on the cytoskeleton in the hyphal apex may support some of the ideas of Reinhardt on the formative capacity of the cytoplasm in apical growth.


41

B. TURGOR A N D WALLEXPANSION To what extent the wall at the apex is under turgor pressure is not entirely clear. Relatively few studies have addressed this question. Robertson (1958) flooded colonies of F . oxysporurn, growing on agar, with sucrose solutions and examined the effect on the growth of leading hyphae. When flooded with solutions between 0.125 and 0.25 M sucrose growth of the hyphae was unaffected. Solutions of lower or higher osmotic potential resulted in temporary arrestment of apical extension followed by subterminal branching. Robertson concluded that the internal osmotic potential is equivalent to 0.125-0.25 M sucrose. Park and Robinson (1966) found rapid bursting of apices and extrusion of cytoplasm when hyphae of Aspergillus niger were flooded with 0.5% acetic acid. By combining solutions with varying concentrations of sucrose with 0.5% acetic acid they determined the osmotic equivalent between 0.1 and 0.15 M sucrose. Like Robertson they showed that hyper- or hypotonic solutions caused arrestment of apical growth but the hyphae quickly equilibrated apparently by reestablishing a normal internal hydrostatic pressure after which growth resumed. Using the wide hyphae of N . crassa, Robertson and Rizvi (1968) have made measurements of the water potential by determining swelling and shrinking in diameter of hyphae in various sucrose concentrations, and of the osmotic potential by determining incipient plasmolysis. They arrived at a turgor pressure of 1240 and I750 kPa for apical and basal hyphal compartments, respectively. As pointed out by the authors this pressure difference may be a factor in forcing cytoplasm toward the apex. All these observations and measurements attest to the fact that growing hyphae are turgescent and that internal hydrostatic pressure is a component of the apical growth process. However, to what extent the extending apical wall is subjected to internal hydrostatic pressure is not immediately evident because the underlying cytoplasm represents a highly structured entity (see Section HI). Vacuoles responsible for building up high internal pressures are present only in the basal part of the hypha. In addition, there seem to be no reports establishing a quantitative relationship between the growth rate of hyphae and the magnitude of turgor pressure. It could be argued that bursting of the tip and the violent extrusion of cytoplasm after applying various chemicals represent proof that the growing wall at the apex is under high turgor pressure. Chemicals, such as polyoxin D, acetic acid, chelating substances, salts, alcohols detergents, etc., have all been thought to act by weakening the cell wall at the apical dome (Park and Robinson, 1966; Bartnicki-Garcia and Lipprnan, 1972a,b). Although some of these substances without doubt act in this way, e.g., polyoxin D, some of them may also have acted primarily on the structured cytoplasm underlying the apical wall (see Section 111). However, the strength of the argument that bursting indicates full turgor pressure in the apex is particularly weakened by the fact that swelling and bursting are often localized just under the apex where extension has

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ceased. In fact, often the whole apical dome is blown off under these circumstances (J. H. Sietsma and J. G. H. Wessels, unpublished observation). It thus appears possible that bursting occurs when the apical cytoplasmic organization is destroyed or when a weakened cell wall synthesized at the apex is displaced by growth toward the subapical region where turgor pressure becomes higher. Picton and Steer (1982) have also argued that it seems very unlikely that the thin wall covering the apices of extending pollen tubes has sufficient mechanical strength to contain the osmotic pressure of the cytoplasm. They propose that the pollen tube apex is stabilized by a fibrillar cytoplasmic network similar to that found in amoebae and slime molds. Although there is no proof for the existence of a cytoplasmic component which protects the expanding wall at the hyphal apex against the turgor pressure prevailing elsewhere in the hypha, it seems appropriate to keep this possibility in mind when discussing biophysical models of hyphal growth. In these models the hypha is treated as a tubular wall expanding at one end under uniform turgor pressure, with the role of the cytoplasm reduced to the delivery of wall precursors and enzymes to the growing wall.

C. BIOPHYSICAL MODELSOF APICALGROWTH Inspired by considerations of d'Arcy Thompson (1917) on the origin of cell form, several mathematical models have been put forward to describe hyphal morphogenesis (de Wolff and Houwink, 1954; Da Riva Ricci and Kendrick, 1972; Green, 1974; Trinci and Saunders, 1977; Koch, 1982). These models describe the gradients in expansion rate of wall areas in the apical dome in order to generate a tubular structure. Depending on the shape of the tip, hemispherical or half-ellipsoid, the rate of expansion of any point on the apex is proportional to the cosine or the cotangent of the angle between this point and the longitudinal axis of the hypha (Green, 1974; Trinci and Saunders, 1977; see Fig. 1). In all I

I

I

I

I

I I

I

--+-nsion zone

I,

I

I

FIG. 1. The rate of expansion of any point at the apex is proportional to the cosine of the angle a when the shape of the tip is hemispherical (A) or to the cotangent of the angle a when the shape of the tip is half-ellipsoid (B).


43

cases expansion is maximal at the very tip and declines to zero at the base of the apical dome. In these models turgor is considered as the force that drives expansion. For a hemispherical tip, Green has argued that the stress in the wall due to turgor is uniform over the apex and that the decline in expansion must thus be caused by a decreasing tendency of the wall to yield to the internal pressure. Qualitatively this also holds for tips with deviating shapes. Because expansion does not lead to thinning of the wall there must be a gradient in the addition of wall materials to the wall which matches the gradient in expansion of the wall. Indeed, in N. crussu hyphae it was found (Trinci and Saunders, 1977) that the cotangent function describing wall expansion was matched by similar gradients in chitin synthesis, as determined by autoradiography , and in the concentration of cytoplasmic vesicles thought to deliver wall materials to the growing wall. Such correlates, however, do not reveal causal mechanisms and the mathematical models are probably carried too far if they are used to prove or disprove mechanisms as attempted by Saunders and Trinci (1979). Their mathematical model could not have excluded a mechanism of growth as envisaged by Reinhardt (1 892) in the absence of preconceived mechanistic concepts. Nevertheless, growth by expansion of a plastic wall is a much more appealing mechanism than the mechanism proposed by Reinhardt because it allows for the addition of wall materials by apposition and because stretching of the wall is a well-known phenomenon in the nonapical diffuse extension growth of cell walls of plants and some fungal systems, e.g., stage IV sporangiophores of Phycomyces. By different means Roelofsen (1950) and Ahlquist and Gamov (1973) artificially stretched these sporangiophore walls and showed that the growing zone, an area just under the developing sporangium, was the most extensible part of the wall exhibiting permanent nonelastic deformation. Nongrowing parts of the wall displayed only small reversible elastic deformations. In Phycomyces stage IV sporangiophores the extensible wall area clearly arises from a formerly nonextensible wall area and there is little doubt that extension occurs under considerable turgor pressure. The growing apical wall, however, has no history of stiffness, its viscoelastic properties have not been determined, and thus the internal pressure necessary to deform this wall is unknown. Assuming that the wall deposited at the apex has plastic properties, then some mechanism must exist that gradually stiffens this wall so that eventually a tubular wall shape can be maintained even under the highest turgor pressure. Green (1974) has advanced the interesting idea that the wall could automatically acquire this stiffness if the increase in resistance to stretch were a function of previous stretch. Whether this is a realistic proposition is hard to tell on the basis of present knowledge of hyphal wall structure. On the basis of what is known about the structure of the wall, two apparently opposing views have been presented regarding the maintenance of a gradient in

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plasticity of the wall at the growing tip. One view, explicitly formulated by Bartnicki-Garcia (1973), holds that the newly deposited wall is inherently rigid and that lytic enzymes permanently loosen this wall and create gaps for the insertion of new wall material. The wall thus grows by intussusception and the theory is mostly referred to as implying “a delicate balance between lysis and synthesis.” Another view, more recently emanating from work in the reviewer’s laboratory (Wessels et al., 1983; Wessels, 1984), holds that the wall material deposited at the growing apex by apposition is inherently plastic in nature but gradually develops stiffness due to secondary processes occurring in the wall. Both theories will be more fully discussed in Section V.

D. ELECTRICAL CURRENTS AND ION GRADIENTS Elongation of fungal hyphae is a clear example of polarized growth. With the advent of the vibrating electrode (Jaffe and Nuccitelli, 1974) it has been become clear that all tip-growing systems generate endogenous electrical currents such that positive ions enter the tip and leave the system subapically (Jaffe and Nuccitelli, 1977; Quatrano, 1978; Nuccitelli, 1983). The first indications for the occurrence of such currents in fungi stem from Slayman and Slayman (1962) who used intracellular electrodes with the wide hyphae of N. crassa. The first measurements with the vibrating probe with fungi were by Stump et al. (1980) in the water mold Blastocladiella emersonii in which they showed a current of positive ions, presumably protons, entering the tips of the rhizoids and leaving over the surface of the thallus. Since then transhyphal currents have been demonstrated in a variety of filamentous fungi, always showing entry of positive currents at a growing hyphal apex: the ,oomycetes Achlya debaryanum (Armbruster and Weisenseel, 1983) and Achlya bisexualis (Kropf et al., 1983), the ascomycetes N. crassa and A. niger and the basidiomycetes Schizophyllum commune and Coprinus cinereus (Gow, 1984), and the deuteromycete Trichoderma harzianum (Horwitz et al., 1984). Not only do these endogenous currents accompany apical growth, they also precede the emergence of an apical growth center as shown for pollen germination (Weisenseel et al., 1975) and branching in fungal hyphae (Kropf et al., 1983). In addition, the property of applied constant electrical fields to determine the site of organization of an apical growth center, as shown for rhizoid outgrowth in fucoid eggs (Peng and Jaffe, 1976), has also been demonstrated in a fungal system by de Vries and Wessels ( 1982). They showed that at an applied field of 25 mV/cell, 75% polarization of the outgrowth of hyphae from regenerating protoplasts of S. commune occurred toward the anode. It appears that the endogenous currents in fungi are largely carried by protons (Gow et al., 1984), possibly by a spatial difference in the location or activity of an electrogenic proton-translocating ATPase in the plasma membrane (Goffeau


45

and Slayman, 1981). Jennings (1979) has suggested a role for a subapically active K + ,Na+ -ATPase for generating an ionic current in the marine fungus Dendryphiella salina. What is the significance, if any, of these self-maintained transhyphal currents? At the moment only speculations are possible.

I . Self-electrophoresis of vesicles toward the growing wall area. This mechanism, originally proposed by Jaffe et al. (1974), has been surmised as operative in fungal hyphae (Bartnicki-Garcia, 1973; Harold, 1977). Alternatively, the vesicles could be carried by water flow caused by electroosmosis (Jennings, 1979). 2 . Polarization of the plasma membrane by lateral movement of charged particles in the lipid bilayer under the influence of the electricalfield (Jaffe, 1977). Once initiated, the electrical current could sustain itself by positioning the pumps generating the current in the proper location along the membrane. Also, lateral diffusion of wall synthetic enzymes in the membrane by the electrical field could determine their proper distribution in the apex. The low strength of the fields sufficient to effect polarization in S. commune protoplasts (10% polarization at 0.7 mV/cell) has suggested such a mechanism to deVries and Wessels (1982) but they were unable to show dislocation of chitin synthesis under influence of the applied electrical field. 3. Direct influence of the membrane potential on the activity of wall synthetic enzymes. Such a mechanism has been suggested on the basis of experiments on cellulose biosynthesis in bacteria (Delmer et a l . , 1982) and plants (Bacic and Delmer, 1981). A disturbing fact in applying any of these theories is that apical hyphal growth is apparently possible without a net positive current traversing the hypha, as shown by Kropf e f al. (1983). In Ac. bisexualis they showed that a maximum inward current developed at a site along the longitudinal wall before the emergence of a branch at that site. During this process, the inward current at the tip of the main hypha decreased and even turned outward before slowly reverting to an inward current. During the period of outward current, lasting about 1 hour, the main hypha continued to elongate at normal speed. However, by using pHsensitive microelectrodes Cow et af. (1984) have shown that during the period of reversed current protons continue to flow into the apex. They concluded that tip growth is always accompanied by an influx of protons but that reversal of the electrical current must have been caused by the flux of other ions. The influx of protons in growing hyphal tips may be related to the reported acidity of the apical cytoplasm as detected with pH indicator dyes (Turian, 1978). The apparent importance of the flux of specific ions makes if attractive to consider a role for cytoplasmic gradients for such ions. 4. Muintenunce qf a polarized cytoskeleton organization in the apex by ion

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gradients. As discussed in Section 111, disturbance of the cytoskeleton with antitubulins and cytochalasins can dramatically influence wall deposition. In addition, evidence is accumulating for a general role of the cytoskeleton in the movement of vesicles (Schroer and Kelly, 1985). The electrical transhyphal currents may be related to the proper distribution in the hyphae of, e.g., H + and Ca2+ necessary for the proper functioning of processes based on this cytoskeleton. Most information relates to the distribution of Ca2+ in apices. X-ray microanalysis by Galpin et al. (1978) showed that in Chaeromium Ca2+ is the only cation with a significant spatial gradient. X-ray microanalysis also showed a Ca2+ gradient in pollen tubes (Reiss et al., 1983). Using chlorotetracycline fluorescence Reiss and Herth (1979a) showed indirectly a tip-to-base gradient in Ca2+ concentration in Achlya hyphae and in various other tip-growing systems. Growing pollen tubes require an optimal external Ca2 concentration and disturbance of the Ca2+ gradient with ionophores and chelaters leads to cessation of growth and a collapse of the ultrastructural polarity of the cytoplasm (Herth, 1978; Reiss and Herth, 1979b, 1980, 1982). Picton and Steer (1982, 1983) have developed interesting ideas about the role of Ca2 in the dynamics of the cytoskeleton in the tips of pollen tubes. Such studies are not yet available for fungal hyphae but Reissig and Kinney (1983) have shown that a Ca2 ionophore induces branching in N . crassa. Also the presence of calmodulin in fungi (Gomes et al., 1979; Grand et al., 1980; Ortega-Perez er al., 1981; Muthukumar et al., 1985), a protein known to regulate many Ca2+-mediated processes (Cheung, 1982), should be considered when searching for possible important roles of Ca2+ in hyphal elongation. +

+

+

111. The Cytoplasmic Components of Apical Wall Growth A. CYTOPLASMIC VESICLES

In an excellent review Grove (1978) has surveyed the apical cytoplasmic organization of the various groups of fungi, as seen after conventional chemical fixation. Emphasis was placed on the occurrence of a large number of vesicles apparently en route for fusion with the plasma membrane. This appears to be an attribute of all tip-growing cells and can be regarded as a highly polarized system of exocytosis. In conventially fixed preparations fusion profiles are frequently seen (Girbardt, 1969; Grove and Bracker, 1970) but they are extremely rare in preparations fixed by freeze substitution (Howard and Aist, 1979; Hoch and Howard, 1980; see Fig. 2). This may be due to the rapid fixation achieved by this procedure and the extreme velocity at which fusion occurs (Hoch and Howard, 1980). Historically, the assemblage of vesicles at the hyphal apex relates to the so-


47

called Spitzenkorper (apical body). In phase optics this is a dark area seen in the apex of elongating hyphae of ascomycetes and basidiomycetes but not of zygomycetes and oomycetes (Girbardt, 1957, 1969). Girbardt ( 1969) and McClure et al. (1968) have equated the Spitzenkorper with the whole concentrated assemblage of vesicles while Grove and Bracker (1970) have argued its equivalence with a central vesicle-free zone in the apices of septate fungi (Fig. 2 and see below). Howard (1981) has discussed this controversy and concluded that Girbardt’s view is probably correct. Girbardt (1957, 1969) found that growth is closely associated with the presence of the Spitzenkorper. When growth is arrested the Spitzenkorper vanishes and reappears just before growth resumes and its position is related to the direction of growth. A central position precedes straight growth while an eccentric location is followed by a change in the direction of growth. In the latter case thickening of the wall, and presumably retardation of wall expansion, occurs in the wall area closest to the Spitzenkorper resulting in curvature of the hypha. The vesicles in the hyphal apex vary in size and contrasting contents. For a description of the vesicles after conventional fixation the reader is referred to the review of Grove (1978). Because the details observed after fixation by freeze substitution are superior, the following summary is from the work of Howard and Aist (1979) on the ascomycete Fusarium acuminatum and Hoch and Howard (1980) on the basidiomycete Laetisaria arvafis (Fig. 2). Vesicles of 70-120 nm diameter occur nearly exclusively at the apices and at the sites of branch formation. They are therefore referred to as apical vesicles and two types of these can be distinguished on the basis of staining of the contents with osmium tetroxide. Microvesicles, 20-50 nm diameter and often characterized by an hexagonal outline, were seen throughout the apex (in the ascomycete, also in the central zone free of apical vesicles). In addition, these microvesicles and a special type surrounded by fibrous material (called filasomes) were found along lateral walls and at the sites of septum formation. Apical vesicles and microvesicles were also seen in association with fenestrated sheets of smooth endoplasmic reticulum located subapically in tip cells presumably representing the Golgi equivalent in these septate fungi. These Golgi equivalents occurred in close association with mitochondria and probably are the source of both apical vesicles and microvesicles. No direct information is available on the rate at which vesicles are produced and fuse with the plasma membrane. On the assumption that membrane material for extending the plasma membrane is delivered by fusion of the apical vesicles, Collinge and Trinci (1974) calculated a fusion rate of 38,000 vesicles per minute for fast-growing hyphae of N . crassa. This rate may even be an underestimate because recycling of membrane material was not considered. Using a method in which a short exposure to cytochalasin D only interfered with vesicle transport, Steer and Picton (1984) estimated the rate of production of apical vesicles in

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FIG.2. Apex of Fusarium acuminatum fixed by freeze substitution (A) and conventional technique (B). (A) Apex of a freeze-substituted hyphal tip cell. Apical vesicles of two electron densities partially surround a cluster of ribosome-like particles. Note the presence of a microtubule (MT) within the apical dome and the close association between mitochondria (M) and smooth cistemae (SC). Expansion of the outer cell wall layer can be surmised at the upper right (arrows). Bar, 1.O km. Inset: Enlargement of the boxed area. The apical cell wall is distinctly four layered. Note the apical vesicles (V) and smooth contour of the plasmalemma (P).Bar, 100 nm. (B)Apex of a conventionally fixed hyphal tip cell. The mitochondria (M) have been so grossly distorted that enclaves of cytoplasm (*) appear as though part of the mitochondria. Electron-lucent vesicles and ribosome-like particles


49

pollen tubes growing at different rates (28 and 7 pm min-I). There was little difference in the vesicle production rate and the calculations indicated 8 0 4 7 % recycling of membrane material delivered to the plasma membrane at the lower growth rate. What is the role of the vesicles in wall formation at the hyphal apex? In many publications (cf. Grove, 1978) they are loosely regarded as being involved in making new wall. One possibility would be that they carry wall polymers ready for insertion in the growing wall. lntracellular synthesis of wall polymers delivered to the wall by vesicles applies to pectin, hemicellulose, and hydroxyprolinerich glycoprotein in plants (Willison, 1981; Northcote, 1984) and to wail mannoproteins in yeast (Sentandreu et al., 1981). For filamentous fungi there is no good evidence for this. Cytochemical staining does detect polysaccharide material in the apical vesicles (Grove, 1978), but this material may represent glycoprotein enzymes destined for export. Electron microscopic autoradiography of hyphal apices of S . commune, labeled to detect chitin synthesis (van der Valk and Wessels, 1977), and Saprolegnia monocica, labeled to detect glucan synthesis (Fkvre and Rougier, 1982), has failed to detect intracellular synthesis of wall polymers. Girbardt (1969) has expressed doubts that wall constituents could be carried by apical vesicles because they are not seen near septa where active wall synthesis is taking place. However, because lateral walls may contain polymers not present in the septa (Hunsley and Gooday, 1974; van der Valk er al., 1977; Wessels and Sietsma, 1979) it is still possible that the apical vesicles carry such polymers to the growing wall. Apart from their possible role in carrying some wall polymers to the surface, there is little doubt that the vesicles are part of the general secretory pathway studied much better in yeasts (Sentandreu et al., 1981; Scheckman, 1982). Presumably they contain the whole array of hydrolytic enzymes so abundantly excreted by fungi while their membranes contain plasma membrane proteins among which are those functioning in wall synthesis. Again, there is little experimental evidence to substantiate this point. Cytochemistry has revealed the presence of cellulase in apical vesicles of Achlya ambisexualis (Nolan and Bal, 1974). In the same fungus Hill and Mullins (1980) demonstrated the vesicular location of a nucleoside diphosphatase and acid phosphatase while Dargent (1975) detected alkaline phosphatase in apical vesicles and Golgi cisternae of Ac. bisexualis. Various studies have attempted to assign polysaccharide synthase and hydrolase activities to cellular structures by differential centrifugation of homogenates (e.g., see Fkvre, 1979). However, vesiculation of the plasma membrane

(arrows) appear at the cell apex. Bar, 1.0 pm. Inset: Enlargement of the boxed area. The cell wall is seen as two layers (i, ii) and is irregular in outline on the inside. The contents of apical vesicles (V) appear coarsely fibrous. Bar, 100 nm. (With permission from Howard and Aist, 1979.)

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and cross-contamination of membranes make it very difficult to draw conclusions, except for the chitin synthase particles to be discussed below. Microvesicles (Fig. 2) are not confined to hyphal apices but are also abundantly present along lateral walls and developing septa (Girbardt, 1969; Hoch and Howard, 1980; Howard, 1981). Howard (1981) noted a hexagonal outline in sections of these microvesicles but dismissed the possibility that they represented mycovirus on the basis of absence of dsRNA, the usual nucleic acid component of these viruses. Yet, the general Occurrence of mycovirus without any apparent disease symptoms (Lemke, 1979) should be kept in mind. Like Bracker er al. (1976) and Girbardt (1979), Howard (1981) suggested that at least some of the microvesicles could be identical to chitosomes which look similar when sectioned after isolation (Bracker er al., 1976). Chitosomes can be isolated from a variety of chitin-containing fungi (RuizHerrera et al., 1975; Bartnicki-Garcia et al., 1978; review by Bartnicki-Garcia and Bracker, 1984). They measure 40-70 nm, have a membrane-like shell rich in sterols and no glycoprotein, and apparently contain exclusively chitin synthase in an inactive form. By proteolytic digestion chitosomes are activated in virro and then synthesize crystalline chitin from uridinediphospho-N-acetylglucosamine, apparently one microfibril per chitosome. Toluene-permealized germlings of Mucor rouxii after treatment with trypsin-synthesized chitin from added widine diphospho-N-acetylglucosaminewithin the cytoplasm without an apparent preferential localization, as shown by electron microscopic autoradiography (Sentandreu et al., 1984). This may reflect the observation that the microvesicles are not strictly localized at the apex. Bartnicki-Garcia and Bracker (1984) concluded that chitisomes convey a package of chitin synthase zymogen to the cell surface, probably to the plasma membrane. Although there is now good evidence for the operation of chitin synthase in the plasma membrane (see below), they still considered both intracellular synthesis of chitin and synthesis of chitin by chitosomes extruded into the periplasmic space as possibilities. Previous claims as to the presence of inactive chitin synthase in the so-called gamma particles of zoospores of B. emersonii (Mills and Cantino, 1981) have been refuted (Dalley and Sonnebom, 1982; Hohn et al., 1984). B. CYTOSKELETON Microtubules in filamentous fungi have been extensively described in relation to mitosis (Heath, 1978) and nuclear migration (Girbardt, 1968; Raudaskoski and Koltin, 1973) but their abundance in apices only became evident after rapid fixation of hyphae by freeze substitution (Howard and Aist, 1979; Hoch and Howard, 1980; Howard, 1981; see Fig. 2). As in subapical areas the microtubules in the apex run parallel to the hyphal axis and some are seen to end at the apical plasma membrane. The abundance of microtubules at the tiyphal apex has


51

now also been demonstrated by immunological techniques in Uromyces phaseofi (Hoch and Staples, 1985). This became possible by using monoclonal antibody against yeast tubulin (Kilmartin and Fogg, 1982) and permeabilization of the fungal wall by lytic enzymes to allow the antibody to gain access to the cytoplasm. Hoch and Staples (1985) even obtained evidence for the presence of microtubule nucleating regions in the hyphal apex. In S . commune microtubules, extending into the apex, have been visualized using antibodies against mammalian a- and p-subunits of tubulin (Runeberg and Raudaskoski, 1986). Fungal microtubules, except those of the oomycetes, are relatively insensitive to standard antimicrotubule agents, such as colchicine, colcimid, and Vinca alkaloids. However, certain systemic fungitoxic benzimidazole derivatives, such as benomyl and its hydrolysis product methyl benzimidazole-Zyl carbamate (MBC), have similar effects on fungal microtubules as colchicine on those of plants and animals (Davidse, 1975; Davidse and Flach, 1977). Morris and coworkers (see Gambino et al., 1984, and lit. cit.) have indicated the p-tubulin subunit as the target for these antitubulins and have examined a number of benomyl resistance mutations of Aspergiffusnidufans with respect to effects on mitosis and nuclear migration. Howard and Aist (1977, 1980) have investigated the effects of MBC on hyphal tip elongation and ultrastructure of F. acuminatum. At a concentration of MBC permitting reduced growth (65-30% of control) light microscopy showed the formation of a wavy hyphal tube, rounding off of the tips, disappearance of the Spitzenkorper, and an increasing distance between the mitochondria-rich area and the tip. The ultrastructural study (Howard and Aist, 1980) showed the extreme susceptibility of apical microtubules to MBC and a dramatic effect on the distribution of the 70-90 nm diameter vesicles. In control cells 50% of all the vesicles in the apical 30 pm of the hypha were found to lie within 2 pm of the apex. While the total number of vesicles did not change much, MBC caused this vesicle distribution to become uniform resulting in a substantial increase in the number of vesicles in subapical regions. Howard and Aist (1980) proposed this to be due to continued fusion of vesicles with the plasma membrane together with a reduction in the rate of transport of the vesicles. Consequently they implied a role for apical microtubules in the transport of apical vesicles. Such a role would seem plausible because of the general implication of microtubules in vesicle transport in animals (Dustin, 1978) and the recent in vitro demonstrations of movement of axoplasmic vesicles along single microtubules in the presence of ATP (reviewed by Schroer and Kelly, 1985). In pollen tubes of Lilium and Clivia, colchicine, at concentrations which led to complete disappearance of subapical microtubules, failed to inhibit growth (Franke et al., 1972). Also in secretory root cells, colchicine apparently did not affect migration of secretory vesicles (Mollenhauer and Morrk, 1976). On the other hand, colchicine has been shown to inhibit apical growth in pollen tubes of

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Nicotiana tabacum (Derksen and Traas, 1985) and in tip-growing moss and algae cells (Schmiedel and Schnepf, 1980; Mizukami and Wada, 1983). In none of these tip-growing plant systems were axially running microtubules observed in the tips (for root hairs, see Traas et a f . , 1985) but, as in fungi, this may reflect the use of conventional fixation procedures. For the moment, the plant systems thus offer no support for the purported role of microtubules in vesicle transport. Microfilaments, 4-7 nm in diameter, have been found in hyphae of many fungi. They are particularly clear in a circumferential filamentous belt in the area of septum formation (Patton and Marchant, 1978; Girbardt, 1979). Girbardt ( 1979) has expressed doubt about contractile properties of this filamentous septal belt because of its position relative to the invaginating plasma membrane and its apparent insensitivity to cytochalasin B. However, cytochalasin B is not usually effective against fungi while cytochalasin A, C, D, and E have been more useful (Betina e t a f . , 1972; Allen e t a f . , 1980; Hoch and Staples, 1983). Again, fixation by freeze substitution has also enabled the visualization of microfilaments in the apical region of fungi, particularly in the central vesicle-free area of septate fungi (Howard and Aist, 1979; Hoch and Howard, 1980; Howard, 1981; Fig. 2), an area which looks finely granular after conventional fixation (Grove and Bracker, 1970). Evidence for equating the microfilaments with actin and myosin is accumulating. Allen and Sussman (1978) have extracted material from N . crassa hyphae which in the presence of ATP precipitates to form actin-like filaments which could be decorated with heavy meromyosin. Cytochalasins which interfere with the polymerization of actin (MacLean-Fletcher and Pollard, 1980; Flanagan and Lin, 1980) inhibit hyphal tip growth and cause irregular deposition of wall materials in N . crassa (Allen et a f . , 1980), Gifbertellapersicaria(Grove and Sweigard, 1980), and U. phaseoli (Herr and Heath, 1982). Rhodamineconjugated phalloin, a derivative of phalloidin which binds to F-actin, has been used to visualize actin in situ in U.phaseoli (Hoch and Staples, 1983, 1985). The actin filaments were present throughout the germ tube but especially in more basipetal regions whereas fluorescent plaques, possibly equivalent to filasomes, occurred near the periphery of the cell’s cytoplasm most abundantly in the hyphal tip region. Using the fluorescent probe 7-nitrobenz-2 oxa- 1,3-diazole-phallacidin (NBD-Ph) or anti-actin antibodies, distinct fluorescence was also noted in apices of S . commune (Runeberg and Raudaskoski, 1986). In addition, intense fluorescence was seen in the septal belt area before the formation of a septum, suggesting the equivalence of the constituting filaments with F-actin. The recent discovery of heavy myosin in Saccharomyces cerevisiae (Watts et a f . , 1985) probably forecasts the demonstration of this cytoskeletal element in the filamentous fungi. Cytochalasins also inhibit tip growth and transport of vesicles in pollen tubes (Herth et a f . , 1972; Franke et a f . , 1972). This inhibition of vesicle transport has been used (Picton and Steer, 1981) to measure the rate of production of vesictes by dictyosomes, although prolonged exposure to cytochalasin also seems to in-


53

hibit the latter process (Shannon et a f . , 1984). A possible role for Ca2+ in the proper functioning of the cytoskeleton has already been mentioned (Section 11,D).According to Picton and Steer (1982, 1983) rather high concentration of Ca2+ at the extreme apex of the pollen tube are necessary for the fusion of the vesicles with the plasma membrane while a decreasing Ca2+ concentration in a subapical direction is necessary to maintain the microfilament network at the tip sufficiently relaxed. In summary, the fragmentary information that exists does suggest that the growing fungal hypha contains in its apex a cytoskeleton of microfilaments and longitudinally running microtubules. There are also indications for connections of cytoskeletal elements with the plasma membrane. As in nonwalled systems (animals, amoebae, slime molds) the cytoskeleton could thus have form-determining properties. This is particularly important if it is assumed that the new wall deposited over the apex does not as yet have enough strength to maintain form. Although the bursting of hyphal tips by chemical agents (Park and Robinson, 1966; Bartnicki-Garcia and Lippman, 1972b) or cold (Robinson and Morris, 1984) has been attributed to changes in wall metabolism, some of these influences may actually work via disturbance of the apical cytoskeleton. Most probably the cytoskeleton at the apex is involved in the polar transport of vesicles for fusion with the plasma membrane. In addition, by maintaining connections with plasma membrane proteins the cytoskeleton could be involved in regulating the distribution of certain proteins, e.g., transport proteins or polysaccharide synthases, in the plasma membrane. C. PLASMAMEMBRANE With respect to wall synthesis many autoradiographic studies have shown that wall components, such as chitin and glucans, are most actively synthesized in the apical portion of the growing hypha (e.g., Bartnicki-Garcia and Lipmann, 1969; Gooday, 197 I ; Katz and Rosenberger, 197 1 ; Wessels et al. , 1983). In N . crassa it was shown (Trinci and Saunders, 1977; Gooday and Trinci, 1980) that the gradient in chitin synthesis at the apex closely follows the theoretical gradient to maintain uniform wall thickness, maximal at the extreme tip and declining to a minimal value at the base of the extension zone (Section 11,C). Autoradiography also shows heavy chitin synthesis at the site of forming septa while a low but significant degree of synthesis, particularly of glucan (Gooday, 1971 ; Sietsma el al., 1985), may occur along the whole length of the longitudinal wall. For chitin there is good evidence that its synthesis occurs only on the plasma membrane. Electron microscopic autoradiography of chitin deposition in regenerating protoplasts and hyphal apices of S . commune has shown that chitin is synthesized only at the plasma membrane/wall interface (van der Valk and Wessels, 1977). Similar observations have been made for chitin synthesis along

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longitudinal walls in stipes of C. cinereus fruitbodies (Gooday, 1982). The presence of chitin synthase in the plasma membrane has been ascertained by isolating pure plasma membranes using the concanavalin A method developed by Scarborough (1975). Thus Durin et al. (1975) showed the association of chitin synthase with the plasma membrane of protoplasts from the yeast Sac. cerevisiae. The chitin synthase was largely in an inactive form and could be activated by limited proteolysis. Similar results were reported for protoplasts from yeast and mycelial phases of Candida albicans (Braun and Calderone, 1978) and the mycelial fungus S . commune (Vermeulen et al., 1979) in which the plasma membrane-associated chitin synthase was largely in an active state. Cabib et al. (1983) have provided good evidence that the enzyme accepts N-acetylglucosamine residues from uridinediphospho-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded at the outside of the membrane. The association of chitin synthase with the plasma membrane is compatible with the presence of chitin synthase in the previously discussed chitosomes if the latter are considered as the transport vehicles for inactive chitin synthase to convey the enzyme to the plasma membrane. A clear distinction between two chitin synthase fractions could be made after subcellular fractionation of concanavalin A-coated protoplasts of S . commune (Vermeulen et al., 1979). About 90% of the active chitin synthase was found associated with the plasma membrane while proteolytic treatment of this fraction resulted in a stimulation of only a factor of 1.7. But these protoplasts also contained a chitin synthase fraction, sedimentable at high centrifugal speed and in an inactive form like chitosomes, which accounted for about 50% of the total chitin synthase activity after proteolytic stimulation. Although this study does not prove that the cytoplasmic inactive form is the precursor for the active plasma membrane-bound chitin synthase, this is a suggestive pathway. How the enzyme becomes active in the plasma membrane is not known. There is no direct evidence for proteolytic activation of chitin synthase in vivo although such a scheme has been proposed for activation of chitin synthase at the site of septum formation in yeast with a zymogenic form of the enzyme uniformly present in the plasma membrane (Cabib et al., 1982, for review). Being an integral membrane protein the influence of the lipid environment can be surmised as important for regulating activity. Arrhenius plots show clear transition points in the activity of the enzyme, delipification inactivates the enzyme, and phospholipids activate or restore the activity of partially delipified enzyme (Durin and Cabib, 1978; Vermeulen and Wessels, 1983; Montgomery and Gooday, 1985). Mutations which alter the lipid composition of membranes have an effect on chitin synthesis (Hanson and Brody, 1979; Pesti et al., 1981). Fungicides which interfere with normal ergosterol biosynthesis cause irregular deposition of chitin as inferred from hyphae stained with the optical brightener diethanol


55

(Kerkenaar and Barug, 1984; Marichal et al., 1984). Polyene antibiotics, known to interact with sterols, inhibit chitosomal chitin synthesis (Rast and BartnickiGarcia, 1980). Since the chitosomal lipids differ from those present in total membrane preparations (Hernandez et al., 198I), and thus probably also from the lipids in the plasma membrane, it seems possible that the mere transfer of chitin synthase from the chitosomes to the plasma membrane results in activation of the enzyme. There is a clear need for in vitro reconstitution experiments attempting incorporation of chitosomes into plasma membrane vesicles or liposomes. If it is accepted that chitin synthase acts as a vectorial integral plasma membrane enzyme, how then is the polarized pattern of chitin synthesis in the fungal hypha achieved? On the basis of the available information a few possibilities can be considered. Chitin synthase may be continuously inserted into the plasma membrane at the growing apex while its polarized activity could be regulated by local perturbations of the lipid environment in the membrane or by some other reversible mechanism such as phosphorylation/dephosphorylation.Assuming a long halflife for the enzyme this would imply the persistence of a latent chitin synthase in the subapical plasma membrane. Some evidence for this scheme comes from experiments of Issac et al. (1978): “early” protoplasts, formed preferentially from hyphal tips, had chitin synthase with a much higher specific activity and only slight proteolytic activatability in comparison to “late” protoplasts formed preferentially from older areas of the hyphae. One may ask, however, whether the protoplast membrane faithfully reflects the original hyphal plasma membrane in composition. During protoplasting the membrane may grow and new lipids and proteins may be inserted. This uncertainty even applies to the assumed uniform distribution of chitin synthase in the plasma membrane of intact yeast which was inferred from the distribution of chitin synthase in the protoplast membranes (DurBn et al., 1975; Cabib et al., 1983). There is a clear need for the development of immunological methods to detect chitin synthase in situ. Contrary to a reversible activation/deactivation mechanism, Ruiz-Herrera (1984) has proposed an irreversible mechanism for creating a gradient of activity of chitin synthase at the hyphal apex. The proposed mechanism is based on the property of chitosomes to synthesize in vitro a single chitin microfibril upon proteolytic activation after which activity is irreversibly lost. In his words: ‘‘Chitosomes are directed towards the apical pole, they become activated, synthesize a single chitin microfibril, and are deactivated in a matter of minutes to guarantee the polarized growth of fungi.” This mechanism would imply an extremely high turnover rate of chitin synthase for which no evidence is available at the moment. On the contrary, cycloheximide blocking the synthesis of chitin synthase has little effect on the chitin-synthesizing ability of regenerating protoplasts (de Vries and Wessels, 1975) and the drug causes a displacement rather

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than a decrease of chitin synthesis in growing hyphae (Katz and Rosenberger, 1971). This apparent stability of chitin synthase can be reconciled with a rapid turnover of the enzyme at its site of activity only if there were an enormous reserve of latent chitin synthase molecules in the cytoplasm. The autoradiographic study of Sentandreu et al. (1984) indeed indicates that large amounts of chitin synthase may be present in the cytoplasm. A completely different view of localized chitin synthesis, so far not considered in the literature, could be based on the newly discovered cytoskeletal elements in the apex and septa1 region and the possible connections between these elements and proteins in the plasma membrane. A polarized assemblage of stable chitin synthase molecules in the plasma membrane highest in density at the extreme apex could be just another manifestation of the polarized activity of the apical cytoskeleton. The whole assemblage of chitin synthase molecules, stabilized by connections to the cytoskeleton, could be pushed through the membrane while this membrane extends. A disturbance of the cytoskeleton, e.g., by osmotic shock, could then easily lead to a displacement of chitin synthesis as observed (Katz and Rosenberger, 1971). Preparation of protoplasts would then probably result in a completely new pattern of chitin synthase insertion and stabilization in the plasma membrane. Numerous studies have demonstrated the synthesis from uridinediphosphoglucose of a (1-.3)-P-glucan by particular or vesicular preparations from fungi (ref. cit. in Quigley and Selitrennikoff, 1984; Sonnenberg et al., 1985). Control mechanisms involving nucleotides have been proposed to explain localized activity (Ftvre, 1983; Szaniszlo et al., 1985). However, the evidence that the glucan synthase is another plasma membrane-bound enzyme is scarce. An autoradiographic study (Ftvre and Rougier, 1982) suggests that all wall glucans in Saprolegniu monoica are synthesized at the plasma mernbrane/wall interface while a (1-.3)-P-glucan synthase has been found to occur in the plasma membrane of S. cerevisiae protoplasts (Shematek et al., 1980).

IV. Structure of the Fungal Cell Wall A. WALLPOLYMERS

Cell wall analyses of different taxonomic groups of fungi have revealed a remarkable heterogeneity with respect to polymers or combinations of polymers present, as first pointed out clearly by Bartnicki-Garcia (1968). On the other hand, ultrastructural studies (Bracker, 1967; Troy and Koffler, 1969; Hunsley and Burnett, 1970; van der Valk et al., 1977; Burnett, 1979; Wessels and Sietsma, 1979) have shown a general similarity in construction of the wall, i.e., an inner layer containing chitin or cellulose microfibrils apparently embedded in


57

TABLE I VAKIOUS POLYM~K OCCUKRING S I N THE CELLWALLw- FUNGI" Cell wall polymers ~~

Taxonomic groups Basidioniycotina Ascomycotina Zygomycotina Mastigomycotina Chytridiomycetes Hyphochytridiomycetes

Alkali soluble Xy lo-manno-protein ( 1+3)-u-u-glucanh (Galacto)-manno-protein ( 1+3)-a-u-glucanh Glucuronomanno-protein pol yphosphate Glucanc Not determined

Alkali insoluble

(1+3)-P/( I+6)-P-~-Glucan Chitin ( l+3)+3/( l+6)-P-~-Glucan Chitin Polyglucuronic acid Chitosan Chitin Glucan' Chitin Chitin Cellulose (l+3)-P/( 1+6)-P-o-Glucan Cellulose

"From Wessels and Sietsma (1981a) with permission. "In a number of cases the alkali-soluble fraction also contains part of the (l+3)-P/( 1+6)-P-uglucan. Incompletely characterized; probably (1+3)-P and ( 1+6)-P-linked.

other polymers and one or more outer layers. As a rule the outer layers are soluble in dilute alkali leaving the inner layer as an insoluble residue. Table I lists various types of alkali-soluble and alkali-insoluble polymers as they occur among the fungi. It appears that within the fungi convergent evolution has provided for the occurrence of a variety of different polymers fulfilling similar functional requirements of the walls. Chitin is the most characteristic component of fungal walls. It is the common feature of the walls of fungi belonging to the Basidiomycotina, Ascomycotina, and Zygomycotina, which according to Whittaker and Margulis ( 1978) constitute the Kingdom Fungi. Chitin has also been found in two classes of the Mastigomycotina, the Chitridiomycetes and the Hyphochitridiomycetes (BartnickiGarcia, 1968) and recent work even suggests the presence of chitin in oomycetes (Compos-Takaki rt a / ., 1982) which typically have cellulose in their walls. Wall composition in the Mastigomycotina will not be considered further. With regard to the "true fungi," chitin and associated polymers such as P-glucans and glycuronans will be discussed because they are likely to play an important morphogenetic role. (For a more comprehensive treatment of fungal wall polymers, see Bartnicki-Garcia, 1968; Wessels and Sietsma, 198 1 a; Bartnicki-Garcia and Lippnian, 1982.) Chitin is usually considered as a homopolymer of N-acetylglucosamine but

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even in crystalline chitin a number of nonacetylated glucosamine residues may occur. X-ray diffraction of fungal chitin shows a microcrystalline conformation known as cx-chitin (which also occurs after spontaneous crystallization of chitin from solution) in which chains presumably run antiparallel. In addition, two other polymorphs, p and y, occur in nature (cf. Muzzarelli, 1977). Evidence for the Occurrence of chitin in walls is mostly obtained by chemical analysis, infrared spectroscopy, and X-ray diffraction analysis of wall residues obtained after consecutive extractions with alkali and acid. After such treatments electron microscopic observations mostly reveal long interweaving microfibrils of varying width in random orientation (Troy and Koffler, 1969; Hunsley and Burnett, 1970; Burnett, 1979; van der Valk er al., 1977), sometimes short rodlets (Kitazima er al., 1972) or granules (Houwink and Kreger, 1953; Pollack er al., 1983). Usually nontreated walls do not show X-ray diffraction lines of chitin (Houwink and Kreger, 1953; Kreger, 1954; Reid and Bartnicki-Garcia, 1976; Sietsma and Wessels, 1977). For the walls of S. commune it was concluded that in native and in alkali-extracted walls most of the chitin is poorly crystallized. Only after treatments which removed the P-glucan did sharp X-ray reflections of chitin (Sietsma and Wessels, 1977) and abundant microfibrils (van der Valk er al., 1977) appear. On the other hand, microfibrils were seen on the inner surface of the native walls but the relationship between crystallinity and microfibrillar structure is not straightforward (Gow and Gooday, 1983). In contrast, on regenerated protoplasts where initially chitin is deposited without alkali-insoluble pglucans the chitin in the native wall is microfibrillar and highly crystalline (van der Valk and Wessels, 1976). Also when chitin is synthesized in virro with chitosomes (Ruiz-Herrera and Bartnicki-Garcia, 1974) or with membrane preparations (Vermeulen et al., 1979) the chitin is microfibrillar and highly crystalline. In the hyphal wall secondary modifications of chitin and interactions between the chitin chains and P-glucan chains (see below) probably prevent the formation of perfect crystallites of chitin. Chitosan is a homopolymer of glucosamine (deacetylated chitin). As mentioned above even in chitin a number of residues may be deacetylated and thus there may be a continous range of polymers from chitin to chitosan varying in the degree of acetylation. This is in line with the observation that chitosan is synthesized from chitin by a deacetylase (Araki and Ito, 1975; Davis and BartnickiGarcia, 1984). Chitosan and partially deacetylated chitin may occur in the walls of chitin-glucan fungi (e.g., Novaes-Ledieu and Garcia Mendoza, 1981) but these polymers are particularly abundant in the walls of zygomycetes where their cationic nature is balanced by the presence of anionic polymers such as inorganic polyphosphate and glycuronans (Bartnicki-Garcia and Reyes, 1968a,b; Datema er al., 1977a,b). Using nitrous acid which specifically attacks nonacetylated glucosamine residues three fractions could be distinguished in the wall of Mucor mucedo (Datema


59

et al., 1977b). One fraction which was solubilized by nitrous acid contained N-acetylglucosamine interspersed with glucosamine. Another fraction became nitrous acid soluble after treatment of the walls with pronase or alkali, indicating a polymer containing (N-acety1)glucosamine to which peptides were linked. The remaining fraction appeared to consist of a homopolymer of N-acetylglucosamine with an X-ray diffraction pattern of a-chitin. In contrast, other zygomycetes, e.g., Phycomyces blakesleeanus (Kreger, 1954) and Mucor rouxii (Bartnicki-Garcia and Nickerson, 1962; Bartnicki-Garcia and Reyes, 1968a), apparently contain the homopolymer chitosan. How this chitosan exists in the native wall is unknown but a significant observation is that the crystalline conformation of chitosan is detected only after extraction of the substance from the wall with dilute acid, a treatment likely to break covalent bonds. With the exception of the hyphal walls of zygomycetes, glucans with (1+3)-p and (1+6)-p linkages occur in the walls of all fungi (Bartnicki-Garcia, 1968; Gorin and Spencer, 1968; Rosenberger, 1976; Fleet and Phaff, 1981; Wessels and Sietsma, 1981a). They may occur as more or less water-soluble glucans forming a gel-like layer around hyphae (Gorin and Spencer, 1968; Buck et al., 1968; Wessels et al., 1972) or as a genuine wall component often existing in an alkali-insoluble complex together with chitin. The extreme insolubility of these glucans makes their analysis extremely difficult. Their extraction often involves the use of strong alkali and acid with the risk of breaking covalent bonds and modifying the glucans. For instance, heating with dilute mineral acid may render the glucans soluble in alkali with simultaneous hydrolysis of ( 1+6)-P-linked residues and crystallization of the remaining ( 1-3)-P-linked chains to form “hydroglucan” (Kreger, 1954). The finding that acid-treated pieces of rhizomorphs of Armillaria mellea showed oriented X-ray reflections of hydroglucan enabled Jelsma and Kreger (1975) to show that hydroglucan has a conformation of three intertwining helices each containing six glucose residues per turn of the helix, in accordance with Marchessault er al. (1977). In native walls or alkaliextracted residues, which only give very diffuse hydroglucan reflections, such ordered structures are probably frequently disrupted by side branches giving the glucan gel-like properties (Sato et al., 1981). The reasons for insolubility of a considerable portion of the (1+3)+- and (1-6)-P-linked glucans in the wall are not completely clear. Since the alkaliinsoluble fraction of the wall always contains (N-acety1)glucosamine it is possible that the glucans are actually part of a glucosaminoglucan. In many fungi a large proportion of the alkali-insoluble glucan can be extracted by dimethyl sulfoxide (Sietsma and Wessels, 1981) but the conclusion that this glucan fraction is apparently unbound to an (N-acety1)glucosamine polymer was not substantiated by showing the absence of amino sugars in the extracted material. In all fungi investigated there was a fraction of glucan which resisted extraction with dimethyl sulfoxide and even extraction with 10 M NaOH at 100°C. In S.

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commune walls this fraction represented nearly all of the glucans insoluble in I M KOH at 60°C. However, after specific hydrolysis of the chitin chains present in these alkali-resistant residues, either with purified chitinase or with nitrous acid after deacetylation of chitin, the glucan became soluble partly in water, the rest in dilute alkali (Sietsma and Wessels, 1979, 1981). The released alkali-soluble glucan very much resembled a glucan also found in the culture medium, a (1+3)-P-glucan with single glucose branches attached by (1+6)-P linkages. The released water-soluble glucan was also (1+3)-@-linked but contained longer (1+6)-P-linked branches. These studies thus indicate that the insolubility of these glucan chains of moderate lengths is caused by linkage to the alkaliinsoluble (acety1)glucosamine-containing polymer. Amino acids, particularly lysine, appear to be involved in linking the two polymers together (Sietsma and Wessels, 1979). Apparently this linkage is stable in alkali but it is very labile to acid (Sietsma and Wessels, 1977). Acidic polysaccharides, although sometimes found in the other groups, are major constituents in the walls of zygomycetes (Bartnicki-Garcia and Reyes, 1968a,b; Miyazaki and Irino, 1970, 1971; Ballesta and Alexander, 1971; Datema et al., 1977a). A heteroglucoronan isolated with alkali from walls of M. rouxii (Bartnicki-Garcia and Reyes, 1968a,b) contained fucose, mannose, and glucuronic acid in a 2:3:5 ratio (mucoran). Another acidic polysaccharide resisted both alkali and acid extraction but could be solubilized by alkali after acid treatment. The water-insoluble polysaccharide extracted in this way (mucoric acid) was microcrystalline and contained mainly glucuronic acid. The X-ray diffraction pattern showed this substance to be identical to a substance previously demonstrated in acid-treated walls of P. blakesleeanus (Kreger, 1954, 1970). Although isolated as two distinct polymers the possibility that mucoran and mucoric acid were derived from a single polymer was not excluded (BartnickiGarcia and Reyes, 1968a). By depolymerizing the glucosamine-containing polymers with nitrous acid, Datema et al. (1 977a) found that this procedure solubilized from the wall of M . mucedo a single heteroglucuronan containing all the neutral and acidic sugars of the wall (fucose, mannose, galactose, and glucuronic acid in a molar ratio of 5: 1: 1:6). This water-soluble heteroglucuronan, apparently held insoluble in the wall by ionic bonds to the glucosamine-containing polymers, could also be extracted quantitatively from the wall by salt solutions of high ionic strength and partially by alkali. By treatment of the isolated watersoluble heteroglucuronan with 1 M HCl at 100°C it was partly converted into a water-insoluble crystalline compound containing only glucuronic acid with the properties of mucoric acid. This suggests that, at least in M. mucedo, mucoric acid, which can be extracted from the wall by alkali after acid treatment, is not a genuine wall component but arises by partial acid hydrolysis of a single heteroglucuronan and subsequent crystallization of acid-resistant homopolymeric stretches of glucoronic acid contained in this heteroglucuronan.


61

B. STRUCTURE OF THE HYPHALWALL In order to reveal the molecular architecture of the hyphal wall electron microscopic observations combined with the use of more or less specific enzymic or chemical extractions have been made on the walls of a variety of fungi (lit. cit. in Wessels and Sietsma, 1981a). On the basis of such observations Hunsley and Burnett (1970) have modeled the wall as a coaxially layered structure. For example, going from the outside to the inside, the wall of N . CYUSSU is thought to contain the following layers (also see Burnett, 1979): ( I ) a layer of mixed a-and P-glucans, (2) a glycoprotein reticulum merging into a proteinaceous layer, (3) a distinct layer of protein, and (4)an innermost chitinous region in which chitin microfibrils are embedded in proteinaceous material. As Burnett (1979) has pointed out it should be understood that the coaxially arranged regions are not discrete but grade into each other. As pointed out by Wessels and Sietsma ( 198 la) the techniques used can easily lead to misinterpretations and they considered most published studies in agreement with a model of the wall in which the various wall components are more closely associated with each other forming essentially one layer with some components accumulating at the outside apparently forming extra layers. This simple model applies only to vegetative hyphae and not to the walls of specialized structures, such as spores and aerial hyphae, where genuine outer layers may be present. Figure 3 depicts a model of the hyphal wall of S. commune integrating the results of a number of chemical, enzymatic, and ultrastructural analyses (Wessels et a/., 1972; Sietsma and Wessels, 1977, 1979; van der Valk er al., 1977). In this case a water-soluble gel-like (1+3)-P/(1+6)-P-glucan and an alkali-soluble ( 1+3)-a-glucan (S-glucan) accumulate at the outside of a layer which contains the alkali-insoluble chitin-P-glucan complex. In this complex the glucan chains are ( 1+3)-P-linked with ( 1+6)-P-linked branches attached. In some of the (1+3)-P-linked chains the branches consist of just one glucose residue and these chains thus resemble the gel-like glucan accumulating at the outside of the hyphae. Other (1-3)-P-linked chains carry longer (1+6)-@linked glucan branches. Both types of branched glucans are thought to be attached to “chitin” chains through their reducing ends via amino acids. Probably these substituted chitin chains are hydrogen bonded to microfibrillar chitin. As mentioned earlier this chitin is only weakly crystalline. The presence of hydrogen-bonded triple helices among the glucan chains is inferred from the weak hydroglucan reflections seen in X-ray analyses of the chitin-glucan complex. Treatment of the complex with hot dilute acid breaks the linkages between chitin and glucan and leads to sharper X-ray reflections of hydroglucan and chitin. The model suggests that the excreted gel-like glucan may consist of glucan chains which have either escaped linkage to chitin or have been secondarily split

62

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M EDlUM

L

---G F

I-

plasma membrane2

C Y TO PLASM

FIG.3. Model of the mature hyphal wall of Schizophyllum commune. Partially crystallized chitin chains (A) are hydrogen bonded to chitin chains which carry covalently linked p-glucan chains. The coupling fragment (B) contains amino acids with a high proportion of lysine. The P-glucan chains are (1+3)-/3-linked and carry single (1+6)-P-linked glucose branches (C) or longer (1+6)-@-linked glucan branches (D) or are alternatively (1+3)-p- and (I+6)-P-linked (E). Some unsubstituted or sparsely branched (1+3)-P-glucan segments may form triple helices (H) which add to the strength of the glucan network. Crystalline (1+3)-a-glucan fibrils (alkali soluble) (F) occur throughout the wall and accumulate at the outer surface as a layer. Free water-soluble (1+3)-p-glucan chains with single (1+6)-p-linked glucose branches (G)are also present in the wall and may be excreted into the medium. (Adapted from Wessels and Sietsma, 1981b, with permission.)

off. It was found that the formation of the water-soluble glucan is inversely related to the formation of the alkali-insoluble glucan, depending on genotype and environmental conditions (Sietsma et al., 1977). In addition a small amount of water-insoluble but alkali-soluble p-glucan was found. It should also be noted that in other fungi often a large amount of wall bound p-glucan is found which is alkali insoluble but soluble in dimethyl sulfoxide and therefore possibly not covalently bound to chitin (Sietsma and Wessels, 1981). The most important evidence for postulating linkages between P-glucan chains and chitin is that the glucan chains become soluble in water or alkali after


63

specific depolymerization of (acety1)glucosamine-containing polymers (Sietsma and Wessels, 1979). Both in regenerating protoplasts and growing hyphae watersoluble and alkali-soluble P-glucan chains were shown to be precursors for the alkali-insoluble glucan which could subsequently be released by depolymerization of chitin (Sonnenberg et af., 1982; Wessels er af., 1983). These precursor glucans are primarily pure ( 1+3)-P-glucans; ( 1-6)-P-linked residues seem to be attached at a later stage of generation of the complex (Sonnenberg ef al., 1982, 1985; Sietsrna et al., 1985). Importantly, in regenerating protoplasts in which the synthesis of chitin can be effectively blocked by polyoxin D this inhibition also leads to complete inhibition of the insolubilization of the glucan chains (Sonnenberg et al., 1982). The model given in Fig. 3 is necessarily speculative and may undergo modifications when other fungal walls are similarly analyzed. As it stands its most characteristic feature is that it predicts a rigid structure based on chitin microfibrils bonded together by P-glucan chains. In essence the model is similar to that proposed for primary walls of plants (Keegstra et al., 1973), although the nature of the polymers and the way they interact are very different. In the zygomycetes which do not contain glucan in their hyphal walls little is known about the location of polymers in the wall. One would expect, however, that the cationic polymers containing glucosamine and the anionic glucuronans and polyphosphates which together with chitin make up most of the wall occur in close association. Possibly in this case chitin microfibrils coated with partly deacetylated chitin chains are fixed into a rigid structure by their ionic interactions with the anionic polymers. In that case an enzymatic apparatus responsible for covalently linking P-glucan chains to chitin as possibly operating in basidiomycetes and ascomycetes could be simply replaced by a deacetylase in the zygomycetes (see Section V,B).

C. WALLSTRUCTURE AT

THE

APEX

It is clear that the tiny amoung of wall material present in growing apices does not significantly contribute to the mass of wall material used in chemical analyses. In fact, such apical wall material may be entirely lacking in broken hyphal wall preparations (Wessels et af., 1983). After autoradiography of pulse-labeled hyphae of S . commune labeled wall material over apices could be detected only before but not after breaking the hyphae for preparing wall preparations. After breaking the hyphae labeled glucan did not precipitate with the wall fragments at low-speed centrifugation while labeled chitin did precipitate but as fragments without recognizable form. However, after a chase, allowing for the labeled wall material to move in subapical direction, the labeled wall material stayed with the broken walls. Now also some tips, apparently arrested in growth at the moment of chasing the radioactivity, were labeled. These observations clearly indicate

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that growing and nongrowing hyphae differ in the type of wall material that covers their apices. The absence of alkali-insoluble P-glucan at the very apex of growing S . commune hyphae has been demonstrated by light microscopic autoradiography (Wessels et al., 1983). This same study showed that the chitin synthesized at the apex is insoluble in alkali. A subsequent study using electron microscopic autoradiography on shadowed preparations (Vermeulen and Wessels, 1984) revealed that the chitin in growing apices, though alkali insoluble, must be in a conformational state quite different from that in nongrowing apices and subapical parts. In contrast to the chitin in these older parts the newly synthesized chitin at apices appeared nonfibrillar, very susceptible to chitinase degradation and partly soluble in hot dilute mineral acid. There have been a few earlier observations indicating discontinuities in the presence of microfibrils at hyphal apices (Marchant, 1966; Strunk 1968) but these have been contradicted by many workers showing a continuous network of chitin microfibrils over the apex after chemical treatments that remove “matrix substance” (cf. Hunsley and Burnett, 1970; Bartnicki-Garcia, 1973; Schneider and Wardrop, 1979; Burnett, 1979). However, the study of Vermeulen and Wessels (1984) suggests that the chemical treatments used to visualize microfibrils could have removed chitin from growing apices so that there is the suspicion that images showing apical microfibrils represent nongrowing apices which abundantly occur in growing mycelia. After chemical treatments to visualize chitin microfibrils these were seen to increase in thickness and density from the very tip toward the base of the apex (Hunsley and Burnett, 1968; Burnett, 1979). This was interpreted as an increase in the number of “elementary fibrils” making up the microfibrils (Burnett, 1979). However, any explanation for this remarkable phenomenon should now also take into account the uncertainty of whether the examined tips were actually growing at the time of fixation. There are a number of light microscopic studies employing fluorescent probes which attest to the fact that the wall covering growing apices is different from that covering nongrowing apices and present in subapical regions. In these studies use was made of fluorescently labeled antibodies (Fultz and Sussman, 1966; Marchant and Smith, 1968; Hunsley and Kay, 1976), fluorescent brighteners such as calcofluor (Gull and Trinci, 1974), and fluorescently labeled wheat germ agglutinin (Galun et al., 1976). The two latter probes probably detect chitin. Differential staining at growing tips may be due to the absence of covering wall materials but also to a difference in the conformation of the polymers that bind these probes. For instance, it can be expected that probes for chitin, such as calcofluor or congo red or wheat germ lectin, would bind much better to noncrystalline chitin possibly present at growing apices than to microfibrillarordered chitin chains. In fact, substances, such as calcofluor and congo red, are


65

known to inhibit crystallization of chitin and microfibril formation (Herth, 1980; Elorza et a/., 1983).

V. Wall Polymer Assembly at the Hyphal Apex A. AUTOLYSINS AND APICAL WALLGROWTH Autolysins are considered to be cell envelope-associated wall-lysing enzymes (Farkas, 1979; Rosenberger, 1979). As pointed out by Gooday (1978) a role for such enzymes in keeping the wall at the hyphal apex in a soft and extensible condition has been suggested nearly 100 years ago by Marshall-Ward. In essence, a rigid wall at the hyphal apex would only yield to turgor pressure if locally bonds in the wall polymers are broken. In a wall growth model involving apposition of new wall polymers this would mean that breaks are permanent and that the partially hydrolyzed polymers at the outer surface may even become soluble. Such a model has been of value in explaining wall growth in bacteria (Koch et a/., 1982; Tomasz, 1984) and plant cells (Cleland, 1981). In a wall growth model involving intussusception of new wall materials the breaks in the polymers would be temporary because after extension of the wall the gaps must be filled up with newly synthesized polymer. This is the essence of the unitary model of apical wall growth in fungi as formulated by Bartnicki-Garcia ( 1973). The wall at the hyphal apex is regarded as a rigid entity but growth is possible by the maintenance of a delicate balance of lysis and synthesis of wall polymers. More specifically the model assumes that a high turgor pressure forces broken microfibrils away from each other before synthesizing enzymes can insert new microfibrils or extend the broken ones. Amorphous wall material is thought to be delivered to the wall in vesicles and forced in between the fibrillar network by the turgor pressure. However, concomitant lysis and synthesis of these matrix polymers was not excluded. This concept has gained wide acceptance in the literature, although Bartnicki-Garcia (1973) has indicated that the evidence for participation of wall-lytic enzymes in the apical growth process is circumstantial. Until now no direct evidence has been forwarded and a critical examination of the arguments in favor of a role of lysins in apical growth therefore seems in place. One piece of evidence concerns the presence of autolytic enzymes in many wall preparations. The occurrence of the bulk of these enzymes in older, nongrowing portions of the wall (Polacheck and Rosenberger, 1978; Kritzman et al., 1978; Rosenberger, 1979; Hoch et a/., 1979) would seem to lessen the strength of this argument for a role of these enzymes in apical wall growth. In addition, these wall-bound enzymes [including chitinase, (1+3)-P-glucanase, P-N-acetylglucosaminidase,and P-glucosidase] are subject to catabolite repres-

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sion and their activities rise dramatically during carbon starvation (Polacheck and Rosenberger, 1978; Rosenberger, 1979). This feature is unprobable for enzymes involved in a delicately balanced growth process but more typical for extracellular enzymes involved in degradation of polymers for nutritive purposes (Reese, 1977). The presence of such enzymes tightly bound to older portions of the wall seems strange but could be due to entrapment of export vesicles in the forming wall since the enzymes can be released from the walls by cationic detergents (Polacheck and Rosenberger, 1978). Another piece of evidence forwarded in support of the involvement of lytic enzymes in apical wall growth is the often found correlation between the level of these enzymes and the process of branching or germination. It appears inescapable to infer the action of autolysins whenever a new hyphal apex must arise from a rigid wall area but the increased levels of wall-lytic enzymes detected in the mycelium or in the medium during increased branching can also be regarded as a consequence of the increase in the number of apices. Hydrolytic enzymes are probably excreted at the apices where the vesicles that package these enzymes fuse with the plasma membrane followed by diffusion of the enzymes through the wall into the medium. It has also been pointed out that the increased levels of lytic enzymes found upon branching may result from a lack of nutrients in dense highly branched mycelia leading to relief from catabolite repression (Polacheck and Rosenberger, 1978). It is a generally established fact that some lytic enzymes apparently produced for nutritive purposes are able to attack walls or wall components from the same fungus (Wessels and Sietsma, 1981a). For example, it is possible to liberate protoplasts from T. harziunum by a concentrate of the hydrolytic enzymes excreted into the medium by the organism itself (de Vries and Wessels, 1973). Yet this would not justify implicating these enzymes in normal wall metabolism at the apex. The fact that the first protoplasts are released from apices indicates that the wall is most sensitive in this area. Also by assaying degradation in wall preparations after briefly labeling of wall components it was shown that newly synthesized wall material is most easily attacked by lytic enzymes (Polacheck and Rosenberger, 1975, 1977; Fkvre, 1977; Vermeulen and Wessels, 1984). There thus remains the problem of why potentially lytic enzymes passing the wall at the apex do not destroy the wall in this area. Part of the answer may be that the concentration of the enzymes as they pass the wall may be relatively low and that the susceptible polymers at the apex are quickly integrated in a crosslinked and partially crystalline wall structure where they are much less susceptible to degradation. Moreover, if the wall at the apex were protected against high turgor pressure by a cytoskeleton, as discussed in Section III,B, occasional cuts in the polymers at the apex would also be of little consequence for the maintenance of apical integrity. The bursting tendency of hyphal tips after flooding with water or solutions of


67

acids, chelating substances, salts, etc. was construed as a main argument for the concept of a balance between lysis and synthesis during apical wall growth by Bartnicki-Garcia and Lippman (1972b). They noted that the temperature coefficient of bursting in water was 1.3-2.0 and concluded that bursting thus could not be considered as a purely osmotic phenomenon but that the participation of a biochemical reaction, as a rate-limiting step, was indicated. They also argued that the fact that various substances could trigger bursting was difficult to reconcile with a direct effect of these substances on the apical wall but that bursting was more likely to be associated with interference with wall metabolism. Specific interference with chitin synthesis using polyoxin D also induced bursting (Bartnicki-Garciaand Lippman, 1972a). If lysis and synthesis were an integral part of wall formation at the apex any disturbance tipping the balance toward decreased synthesis or increased lysis would lead to disintegration of the wall fabric at the apex. Recent support for the concept of lysis as an integral part of wall growth at the apex has come from studies showing a close association of chitinase with chitin synthase preparations (Lopex-Romero et al., 1982; Humphreys and Gooday, 1984) and an increase in reducing ends in chitin during light-stimulated growth in stage I sporangiospores of Phycornyces (Herrera-Estrella and Ruiz Herrera, 1983). It must be admitted that all these pieces of evidence are very indirect and alternative explanations are possible. As discussed earlier bursting of hyphae is mostly subapical and some of the substances used to induce bursting may have influenced the integrity of the cytoskeleton underlying the apex. Inhibition of synthesis of one of the principal wall polymers could lead to the assembly of a wall of insufficient strength to withstand turgor pressure in subapical parts and would result in bursting of the wall in this region. Studying the antagonism of Ca2+ and acid in bursting of hyphal tips, Dow and Rubery (1975) came to the conclusion that the assumed role of lysins could not explain their results. Also the apparent absence of significant wall turnover in growing walls (Rosenberger, 1979) does not support the activity of lysins in apical wall growth. In addition, no conceptual framework has been advanced to understand how a balance between lysis and synthesis of the wall at the growing apex can be maintained. First, for lysis and concomitant synthesis to account for intussusception of wall material one must assume that the enzymes for both processes operate within the wall fabric. For lytic enzymes this presents no problem. For the synthetic enzymes, however, most of the evidence points to a cytoplasmic localization, e.g., the plasma membrane in the case of chitin synthase (Section 111,C). Second, the activities of both enzyme systems should be precisely balanced with the magnitude of intussusception which should decrease when a wall segment moves from the extreme apex to subapical parts. For synthesizing enzymes located in the plasma membrane mechanisms originating from the cytoplasm can be envisaged to account for their regulation. This is much more difficult to envisage for stable

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lytic enzymes operating within the wall away from the plasma membrane. Humphreys and Gooday (1984) have suggested that a chitinase and a chitin synthase, both present in the plasma membrane, could perform the delicate balance required. However, growth of a rigid wall cannot proceed unless cuts are produced in at least the outer zone of the wall which would require the lysidsynthase complex to move away from the plasma membrane. Control of the activity of wall-lytic enzymes has also presented a problem in explaining intercalary growth of plant cells and bacteria in which extension of the wall probably does involve the continuous breakage of bonds. However, in these cases it is generally thought that new wall material is formed by apposition and it has been suggested, both for plants (Cleland, 1967) and bacteria (Koch et al., 1982), that the lysins act only in the outer zone of the wall on polymers under stress having an extended conformation. This would allow for breakage of bonds in the stretched older portion of the wall while such bonds would not be subject to hydrolysis in the not yet stretched newly deposited wall polymers. But even if apposition of wall material is also assumed at the hyphal apex, it is still difficult to see how the prevailing stress pattern in the apical wall could lead to proper control of lysis according to this hypothetical mechanisms. It is clear that wall-lytic enzymes play important roles in various aspects of the life of fungi. These included net degradation of wall polymers during starvation, degradation of septa for nuclear migration, hyphal fusions, branch initiation, and germination. As discussed by Wessels and Sietsma (1981a) the key enzymes involved need not be identical with the commonly assayed enzymes using artificial substrates. A few specific breaks in the complicated network of the wall structure by special enzymes may be sufficient to weaken the wall sufficiently or to initiate complete degradation by more general hydrolytic enzymes. As may be evident from the foregoing discussion the participation of lytic enzymes in apical wall growth can at least be doubted. Probably the most fruitful route to clarify this issue would be a search for conditional mutants blocked in apical extension. This route has been taken to assess the role of autolysins in bacterial growth (Tomasz, 1984) and has been started for yeasts (Nombela and Santamaria, 1984). IN B. ASSEMBLY

THE

WALL:A STEADY-STATE MODEL

Only if the wall at the hyphal apex is considered inherently rigid is it necessary to invoke the continuous action of lysins to keep the wall plastic and to allow for the insertion of new wall material. However, the new views on fungal wall structure (Section IV) in which the polymers are cross-linked by various bonds make it highly unlikely that this structure is built in one step. At least two steps can be envisaged. First the individual polymers must be deposited outside the plasma membrane, either by synthesis on the plasma membrane or by extrusion


69

through vesicles that fuse with the plasma membrane. Second, extracellularly the individual polymers may become modified, undergo partial crystallization, and mutual interactions involving covalent or ionogenic bonds. This would imply that a major part of the assembly of the wall structure occurs in the domain of the wall itself. Because of the macromolecular nature of the assembly system it may take considerable time before the structure has reached its final state and it would seem plausible that during this time the mechanical properties of the wall gradually change from a viscoelastic fluid to a rigid composite. The whole process may be likened to that occurring in man-made composites in which by varying the amount of cross-linking between polymers a wide range of plastic and strength properties can be obtained. Cross-linking reduces crystallinity but the additional stiffening effect of an increasing degree of cross-linking between chains more than counterbalances the increasing flexibility due to loss of crystallinity (Kaufman and Falcetta, 1977). Because fungal walls vary widely in the types and amount of the wall polymers they contain it can be expected that there will also be variation in the types of interactions that generate the final rigid wall structure. Because the view on wall biogenesis as outlined above is based mainly on our studies on the structure of the wall of S. commune (Fig. 3 ) and the processes that lead to its construction, this work will be briefly summarized. For the present discussion we ignore the large amount of crystalline (1+3)-aglucan present in the wall of S. commune because this polymer is probably of little morphogenetic significance. Mutations that block the synthesis of this polymer in A . nidulans (Zonneveld, 1974; Polacheck and Rosenberger, 1977) or 2-deoxyglucose that inhibits its synthesis in S . commune (J. H. Sietsma and J. G. H. Wessels, unpublished) do not have a major effect on hyphal morphology. However, this a-glucan may become the major shape-maintaining wall polymer in established hyphae under conditions that most of the P-glucan and chitin have been removed from the walls by autolytic processes (Wessels and Sietsma, 1979). The chitin-P-glucan complex depicted in Fig. 3 is considered to be of major importance in determining the final shape of the hyphae. Interference with its synthesis by congo red or calcofluor white causes the formation of balloonlike structures from growing apices (C. A. Vermeulen and J. G. H. Wessels, unpublished). The proposed structure of the chitin-P-glucan complex immediately suggests that its synthesis must occur in at least two distinct phases. First the individual chitin and P-glucan chains must be polymerized and deposited outside the plasma membrane. Only then can enzymes in the wall proceed to couple the pglucan chains to chitin probably competing with the tendency of the chitin chains to crystallize. The following observations support to occurrence of the suggested sequence of events (Fig. 4). Not only in regenerating protoplasts (Sonnenberg et al., 1982), where initially

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A

B

I

1'

FIG.4. Highly schematic diagram of possible changes in wall structure in the growing hyphal apex of Schizophyllum commune. Increase in stipling in C indicates degree of cross-linking and crystallizationof p-glucan and chitin chains, paralleled by an increase in rigidity. A shows the mature wall structure in which chitin chains probably assembled in microfibrils are cross-linked to partly crystallized (1+3)-p-glucan chains with (1+6)-p-linked branches attached. B shows individual chitin chains (straight lines) and individual (1+3)-p-glucan chains (wavy lines) as present at the extreme apex. (Adapted from Wessels, 1984, with permission.)

no alkali-insoluble P-glucan is formed, but also in growing hyphae (Wessels et al., 1983; Sonnenberg et al., 1985) pulse-chase experiments with [ *4C]glucose have shown that the precursor for the alkali-insoluble P-glucan is a water-soluble/alkali-soluble ( 1+3)+-glucan. The conversion to the alkali-insoluble form is interpreted as being due to linkage of the glucan chains to chitin because they become soluble again after specific depolymerization of chitin. The conversion to the alkali-insoluble form is a relatively slow process, taking several minutes. By light microscopic autoradiography it could be shown (Wessels ef al., 1983) that both the chitin and glucan chains are synthesized at the apex according to the expected gradient but only label from N-a~etyl-[~H]glucosamine immediately appears in an alkali-insoluble form (chitin). Label from [3H]glucoseis primarily incorporated at the apex into water- and alkali-soluble glucans; at the extreme apex alkali-insolubleglucan is completely missing. While the glucans move in a subapical direction during growth in the chase period, alkali-insoluble glucan appears at the expense of the water-soluble glucan. Like the precursor glucan the alkali-insoluble glucan formed at the apex is mainly (1+3)-P-linked with few, if any, (1-6)-P-linked glucan branches attached (Sietsma et al., 1985; Sonnenberg et al., 1985). But in a subapical direction the number of (1+6)-P-linked glucose residues rises rapidly in the alkali-insoluble glucan. Possibly this is another type of modification which occurs within the domain of the wall. Also the conformational state of the chitin changes while moving from its site of synthesis in subapical direction (Vermeulen and Wessels, 1984). Apparently the


71

chitin at the growing apex is not yet crystalline since no microfibrils can be observed and the chitin in this area is extremely sensitive to dissolution by chitinase or hot dilute mineral acid. This contrasts with the conformational state of chitin in subapical parts and indicates a time gap between polymerization of the chitin chains and their final integration into the wall structure. The existence of a gap between polymerization and microfibril formation has been suggested by using congo red or calcofluor white in organisms which form either P-chitin (Herth, 1980) or a-chitin (Elorza et al., 1983). In addition it has been shown that both nascent chitin (Molano et al., 1979; Lopez-Romero et al., 1982) and nascent (1+3)-P-glucan (Pkrez et al., 1984) synthesized in v i m are much more sensitive to enzymic degradation than some time after their formation, again suggesting a conformadonal change with time. When hyphae of S . commune stop growing the alkali-insoluble form of glucan is produced over the whole apex (Wessels er al., 1983) indicating that the process of insolubilization of the glucan (interpreted as linkage of the P-glucan chains to chitin) proceeds with time independent of elongation of the hypha. Also the (1+6)+-linked glucan chains are now formed in the wall at the apex (Sietsma et al., 1985) and chitin microfibrils become visible in this area after treatment with hot dilute mineral acid (Vermeulen and Wessels, 1984). In other words, after elongation stops the wall at the apex assumes the same structural features as observed in the subapical wall. In essence, the scheme suggested above may be applicable to basidiomycetes and ascomycetes in general but details may be different. In particular, the presence of large amounts of alkali-insoluble glucan possibly not linked to chitin in a number of species (Sietsma and Wessels, 1981) has not been accounted for in the above scheme. It is tempting to speculate that in the zygomycetes the role of the chitin-glucan linkages is played by the ionic linkages between partially deacetylated chitin and glucoronans. Similar to the solubilization of P-glucans by depolymerization of chitin (Sietsma and Wessels, 1979), specific depolymerization of the partially deacetylated chitin chains in zygomycetes causes the solubilization of glucuronans (Datema et al., 1977a). Deacetylation of chitin seems to occur by a deacetylase in the wall shortly after synthesis of the chitin chains before crystallization has occurred (Davis and Bartnicki-Garcia, 1984). Assuming hydrogen bonding between partially deacetylated chitin and chitin microfibrils, the generation of the cationic groups by the deacetylase at the apex could lead to cross-linking of the microfibrils by acidic glucuranans which may be hydrogen bonded among themselves. Thus the process of deacetylation of chitin at the apex could also lead to a significant change in the viscoelastic properties of the wall in these fungi. The model of apical wall growth developed above is a steady-state model because the maintenance of elongation depends on the steady delivery to the apex of individual wall components which then interact in the wall to form a rigid

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structure while moving subapically. Any influence stopping elongation would lead to disturbance of the steady-state process and lead to rigidification of the wall over the apex. This would explain why a hyphal apex arrested in growth for a critical time can no longer resume growth (Reinhardt, 1892; Robertson, 1958). Assuming that the time required for rigidification is not dependent on the elongation rate, the model could also easily explain the positive relationship often found between elongation rate and length and width of the extension zone (Reinhardt, 1892; Steele and Trinci, 1975). The observed random orientation of microfibrils in subapical walls and (nongrowing) apices (Burnett, 1979) would present no problem since no realignment of fibrils has to occur. Finally, the deviating wall structure at growing apices may allow for the passage of large-sized proteins intended for export (Chang and Trevithick, 1974).

VI. Summary Although apical growth of hyphae is the most prominent feature of the organisms belonging to the fungal kingdom, we are still largely ignorant of the mechanisms by which it occurs. However, as this review may have shown, many new analyses and observations as well as comparisons with other cellular systems now open new routes for ideas and experimentation. Understanding the mechanisms involved in apical hyphal growth would not only clarify a longstanding morphogenetic problem, namely, the generation of a tubular cell under turgor pressure by elongation at one end. Understanding these mechanisms would also give a better insight in a number of characteristic features which dominate the life of fungi, such as the colonizing ability of the mycelium, the way in which environmental factors influence growth and morphogenesis, the interactions occurring between hyphae, and the interactions between fungi and other organisms in parasitic and mutually beneficial relationships. It appears that the hyphal apex is best viewed as a highly polarized system of exocytosis. Wall materials, extracellular enzymes, and probably other substances are excreted at the growing end of a tubular cell. The most obvious cellular features that accompany this polarized system are the unidirectional flow of vesicles in the cytoplasm fusing with the plasma membrane at the apex, the gradients in wall synthesis at the apex, and the cytoplasmic gradients in ion distribution that are maintained at the apex. New microscopic techniques are also beginning to reveal a cytoskeletal organization of the cytoplasm at the apex which may be crucial to its polarized activity. How these various aspects are causally related can only be a matter of much speculation at the moment. It is clear that once an apical growth center is initiated it is self-maintained. It can be surmised that the distribution of certain proteins in the plasma membrane is of prime importance in this respect. For instance, the suspected spatial distribution


73

of proton pumps in the hyphal plasma membrane creates proton currents which in turn may cause the maintenance of the spatial distribution of proton pumps and possibly other proteins in the plasma membrane. The apical gradients in protons and other ions such as Ca2+ could also be instrumental in organizing the cytoskeleton at the apex and allow for the proper interactions of the vesicles with the cytoskeleton to effect their transport to the extreme apex and subsequent fusion with the plasma membrane. Both the polarized distribution of proteins in the plasma membrane and the polarized delivery of vesicles are likely to play a role in the gradient of wall-synthesizing activity generated at the apex. Because of the high turgor pressure the tubular form of the hypha can be maintained only by a rigid wall generated at the apex. It is not certain, however, that the full turgor pressure also acts on the wall at the apex because of the cytoskeletal organization in this region. With regard to the type of wall synthesized at the apex fungi exhibit wide variations. It is clear, however, that a fruitful approach to the study of wall biosynthesis can be expected only after sufficient knowledge is obtained about the details of the structure of a particular wall. In fact, such a structural analysis can reveal clues as to possible mechanisms involved in the synthesis of the wall which then hopefully can be generalized. Growth of the wall at the hyphal apex requires that the wall in this region has plastic properties which contrasts with the requirement of rigidity elsewhere in the hypha. A widely held view involves the participation of wall-lytic enzymes in plasticizing the wall at the apex and in allowing new wall material to be inserted. A critical evaluation of the evidence presented to support this view makes this hypothesis less attractive. As an alternative a steady-state model is discussed based on recent observations in the author’s laboratory. In essence this model holds that the assemblage of polymers synthesized at the apex is inherently plastic. However, this assemblage develops rigidity by interactions, in the wall, between and among the various individual polymers present while the wall segment moves in subapical directions during elongation. This model seems to fit many of the original observations made on living hyphae.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 104

Connectin, an Elastic Filamentous Protein of Striated Muscle KOSCAK MARUYAMA Department of Biology, Faculty of Science, Chiba University, Chibn 260, Japan

I. Introduction Connectin, also called titin, is a very long, flexible filamentous protein of striated muscle that links thick myosin filaments to Z disks in a sarcomere. Despite its presence in rather high amount (about 10%) in myofibrils next to myosin (43%) and actin (22%), as listed in Table I, connectin has only recently been characterized. This was because physicochemical investigations have been very difficult due to its huge molecular weight of a few million. At present, a proteolytic product of the mother molecule (a-connectin, T,), p-connectin or T, has been purified as native form, but a-connectin has not yet been isolated. Connectin superthin filaments are shown to serve as an elastic component of striated muscle. Therefore, it is regarded as the fourth type of cytoskeletal structure following microtubules, intermediate filaments, and actin filaments. The problem is whether connectin-like filaments are present in nonmuscle cells or not. The present review deals with this problem after a full description of muscle connectin (cf. Wang, 1984, 1985; Locker, 1984a; Maruyama and Kimura, 1985; Ohtsuki et af., 1986).

11. The Third Filament? Over a century ago, a great German physiologist Johannes Miiller indicated in his famous textbook on human physiology that skeletal muscle could be considered to be elastic bodies (Miiller, 1840). Since then, it has long been assumed that muscle consists of two components, contractile and elastic. The elastic property of muscle was ascribed to the function of extracellular collagen fibers attached to muscle cell membranes (sarcolemma). In 1954, Natori first demonstrated that an elastic component exists in muscle cells using his famous demembraned fibers of the Natori type (Natori, 1954). He postulated the presence of an “internal elastic structure.” In the same year, H. E. Huxley and Hanson, together with A. F. Huxley and Niedergerke (1954), proposed the sliding theory based on the movement of the thin (actin) filaments relative to the thick (myosin) 81 Copyrighl 0 1986 by Academic Press. Inc. All rights or reprduclion in any form reserved.

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KOSCAK MARUYAMA TABLE I MYOFIBRILLAR PROTEIN OF VERTEBRATE SKELETAL MUSCLE“

Protein Contractile Myosin Actin Regulatory Troponin Tropomyosin M-Protein C-F’rotein a-Actinin Cytoskeletal Connectin Nebulin

Molecular weight (x 103)

Content wt%

Localization (band)

520 42

43 22

A I I I M line A Z line

70

33 x 2 165 135 95 x 2 2800 750

10 5

A-I I

OMinor structural proteins less than 1% of the total proteins are omitted. For a complete list, see Ohtsuki er al. (1986).

filaments in a sarcomere (H. E. Huxley and Hanson, 1954). In order to explain the continuity of myosin-removed myofibrils, they assumed the presence of an elastic filament called an S-filament linking the free ends of actin filaments in a sarcomere. However, this elastic filament model was not subsequently mentioned in the development of the sliding theory. In 1962, Sjostrand observed very thin filaments at the gap region between myosin and actin filaments when muscle was extremely stretched beyond the overlap of the two sets of filaments. Sjostrand (1962) called these filaments that were thinner than actin filaments “gap filaments,” and assumed that they were continuous with the tapered ends of myosin filaments. A year earlier, A. F. Huxley and Peachy (1961) mentioned the possible presence of “fine filaments” connecting the ends of both myosin and actin filaments from their observations of highly stretched muscle fibers. Graham Hoyle and his associates examined fine structures of a variety of striated muscles both in invertebrates and vertebrates and reached the conclusion that there were “superthin” or T-filaments (3 nm in width) connecting adjacent Z lines in a sarcomere (McNeill and Hoyle, 1967; Hoyle et af., 1968). Guba et al. (1968) reported that there were residual filaments after the extraction of myosin and actin. They claimed that those superthin filaments consisted of a protein designated fibrillen. dos Remedios (1969) observed that there was a third filament in addition to the myosin and actin filaments in a sarcomere that was resistant to salt extraction (dos Remedios and Gilmour, 1978). Revival of Sjostrand’s gap filament was brought about by Locker in 1975.

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

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TABLE II CHRONOLOGICAL LIST OF THE CONCEFT OF SUPERTHIN FILAMENT IN STRIATED MUSCLE Reference Inner elastic structure S filament connecting actin filaments Fine filament connecting myosin and actin filaments Gap filaments connecting myosin and actin filaments Connecting filaments connecting myosin and Z disk in insect muscle Superthin T filament running from Z to Z disks Fibrillen Third thin filament running from Z to Z disks G-filament connecting myosin and Z disks Connectin 10-nm filament connecting Z to Z disks Titin Projectin in insect muscle Cross-linked matrix

Natori (1954) H. E. Huxley and Hanson (1954) A. F. Huxley and Peachy (1961) Sjostrand (1962) Auber and Couteaux (1963) McNeill and Hoyle (1967) Guba er al. (1968) dos Remedios (1969); dos Remedios and Gilmour (1978) Locker and Leet (1975) Maruyama et al. (1976, 1977a) Price and Sanger (1977) Wang et al. (1979) Saide (1981) Lowey et al. (1983)

Locker and Leet (1975) first observed that bovine neck muscle (sternomandibularis) can be stretched to five times its rest length, up to 12 pm between the neighboring Z disks, spanned by very thin gap filaments, continuous with the thick filaments (Locker and h a t , 1976a,b). Locker called them G-filaments and assumed that they formed cores of myosin filaments, emerging at one end only, and arriving at the Z disk (cf. Locker, 1984a). Thus, as listed in Table 11, morphological and physiological observations strongly suggested the presence of the third superthin filament in addition to the thick and thin filaments in a sarcomere of vertebrate striated muscles. However, this was not widely accepted. One reason was, and still is, the lack of a clear-cut image of the compatibility of the third filament model toward the sliding theory of muscle contraction. Finally, it is to be noted that there are third filaments connecting the edges of myosin filaments to Z disks in fibrillar flight muscles of some insects, e.g., bee, fly, waterbug, etc. that rhythmically and quickly repeat contraction and relaxation (Auber and Couteaux, 1963; reviewed by Pringle, 1978). Projectin, the connecting filament protein, was isolated from honeybee thoracic muscle (Saide, 1981). 111. Connectin versus Titin

Stimulated by Natori’s pioneering work (Natori, 1954), the present writer began to identify the chemical entity of the elastic filament connecting Z disks in myofibrils of vertebrate skeletal muscle after removal of myosin and actin (rab-

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bit, chicken, and frog) in 1975. The residue was completely insoluble in salt solution, e.g., 1 M K1, LiBr, KSCN, etc. Dilute acid (1 N acetic acid, 0.1 N HCl, etc.) or alkali (0.01 and 0.1 N NaOH) also failed to solubilize. Most of the residue was resistant to even 0.1% SDS or 8 M urea. Alkali-treated ghost skinned fibers of frog muscle behaved just like rubber (Maruyama et al., 1976). When the insoluble residue, after removal of myosin, actin, and regulatory proteins and also connective tissue, was washed with water and then solubilized in I % SDS solution, the SDS-gel electrophoresis pattern showed that the main components were a high-molecular-weight (HMW) component which hardly moved and a 42kDa band (Maruyama et al., 1977a). The HMW component was cut out of the gel and used as an antigen. The FITC-labeled antiserum stained the filamentous material in the muscle residues (Maruyama et al. 1977a). Immunofluorescence observation showed that the A-I junction area of a sarcomere was most intensely stained (Maruyama et al., 1980). The 42-kDa component was nothing but denatured actin (Maruyama et al., 1983). King and Kurth (1980) isolated HMW connectin by chromatography on DEAE-Sepharose CL-6B in the presence of guanidine-HC1 and urea. Locker and Daines (1980) separated maleylated protein by DEAE-cellulose chromatography. The relationship between ‘‘gap filaments” and salt-insoluble connectin was discussed by Locker and Daines (1980). It is worth mentioning the peculiar solubility behavior of muscle structural proteins. Myosin can be easily extracted with a large volume of HasselbachSchneider solution: the remaining myosin would be very small in amount, if extraction is repeated several times. Actin extraction with 0.6 M KI is always incomplete. Some actin is left behind. If myofibrils are treated with 0.6 M KI from the beginning, a large amount of actin remains unextracted together with myosin. The proteins aggregate around deteriorated Z disks as first pointed out by Granger and Lazarides (1978). The most likely situation is that actin, myosin, and other proteins are bound to free connectin filaments extending from the Z disk and the whole mass aggregates near the Z disk. These become completely insoluble in salt solutions and some of them are not soluble even in a SDS solution. This explains the formation of the “elastic matrix” mentioned by Lowey et al. (1983). Quite independent of the connectin work aimed at the elastic filament of skeletal muscle, Kuan Wang accidentally discovered a giant protein by SDS-gel electrophoresis of total SDS e.xtract of whole muscle (Wang et al., 1979). Originally, he intended to find smooth muscle actin-binding protein (filamin according to Wang’) in skeletal muscle without success. Instead, he found at least three

’

Actin-binding protein (ABP) was first purified from rabbit lung macrophages (Hartwig and Stossel, 1975; Stossel and Hartwig, 1975). Chicken gizzard ABP was isolated by Wang (1977; cf. Wang er al., 1975) and named filamin.


85

1

2

3

4

a

b

c

d

FIG. 1. SDS-gel electrophoresis patterns of a direct SDS extract of chicken breast muscle myofibrils. (a) 2.8% polydcrylamide gels, (b) 2.5%. (c) 2.3%,(d) 2.0%.( I ) a-Connectin (titin I ) , (2) p-connectin (titin 2), (3) nebulin, (4) myosin heavy chain. D. H. Hu (unpublished).

high-molecular-weight protein bands (bands 1, 2, and 3). Figure 1 shows SDSgel electrophoresis pakerns of a SDS extract of chicken breast muscle. By gel filtration in the presence of 0.1% SDS, Wang was able to isolate bands 1 and 2 in a denatured state and to raise antibodies against them. Bands 1 and 2 were named titin because of their huge molecular weight of one million. In myofibrils, they were located in the A - I junction area and also in Z disks as revealed by an immunofluorescence study. The content of titin was as large as 10% of the total myofibrillar proteins, and therefore Wang called it the third major structural protein of muscle. We immediately confirmed that Wang’s titin was identical to connectin (Maruyama et al., 1981a). Although King and Kurth (1980) separated connectin by gel filtration from guanidine- and HCI-solubilized muscle residues, Wang’s procedure was simpler and more reproducible. Then, our efforts were concentrated on the isolation of connectin in a native form. We noticed that some amount of connectin is soluble in a salt solution, coextractable with myosin in Guba-Straub solution (Maruyama et al., 1981a). It turned out that this is p-connectin (T,), derived from a-connectin (T2) by endog-

86

KOSCAK MARUYAMA

enous protease. a-Connectin is not soluble. We have selected the conditions in which myosin is not solubilized and isolated native p-connectin (Kimura et al., 1982; Kimura and Maruyama, 1983a; Kimura et al., 1984b). Meanwhile, Wang’s group in the United States and Trinick’s group in the United Kingdom were successful in isolating connectin by different means (Wang et al., 1984; Trinick et al., 1984).

IV. Native Connectin A. PREPARATION With our observations that some connectin is solubilized from chicken breast muscle together with myosin in Guba-Straub or Hasselbach-Schneider solution (Maruyama et al. 1981a), we sought the conditions in which connectin alone is soluble without myosin. First, it was ascertained that connectin is soluble in the presence of 0.2 M NaCl (pH 7.0) or 0.075 M phosphate buffer (pH 6.5). With 0.1 M sodium phosphate buffer, connectin was solubilized above pH 6.5. At pH 7.0, both connectin and myosin were soluble, but the latter was not soluble at pH 6.5. However, a-actinin, actin, and other proteins were also extracted by 0.1 M phosphate buffer (pH 6.5). Therefore, myofibrils were first washed well with 5 mM NaHCO, followed by extraction with 0.1 M phosphate buffer at pH 5.6. These procedures removed a-actinin and other proteins. The precipitate was briefly washed with water and then extracted with 0.1 M phosphate buffer, pH 6.6. The filtrate consisted largely of connectin (Kimura and Maruyama, 1983a). For further purification, hydroxyapatite chromatography is highly recommended. However, it was noticed that fresh myofibrillar preparations resulted in a low yield of connectin. Therefore, myofibrils were prepared from muscle strips stored overnight at 0°C (Kimura et al., 1984b). This was due to proteolysis of aconnectin to p-connectin (see Section IV). The native connectin we obtained was p-connectin. By this procedure the yield was as high as 400 mg starting from 100 g of muscle. This is approximately 40% of the original content of connectin. Trinick and his associates (1984) have separated connectin from myosin in a salt extract of rabbit psoas myofibrils by sedimenting myosin in 0.2 M KCI. Contaminated C-protein and other proteins can be removed by DEAE-cellulose column chromatography. The eluted connectin is precipitated by (NH,),SO, at 35% saturation and it was dissolved in 0.5 M KCI containing 50 mM Tris-HC1 buffer, pH 7.9. This procedure is very convenient for concentrating connectin. Pure connectin can be obtained by gel filtration. The yield is approximately 200 mg from 100 g of muscle. Wang’s procedure (Wang er al., 1984) was similar to the procedures of C-protein purification (Reinach et al., 1982) and the yield was low: 100 mg from 100 g of muscle. Crude myosin preparations were subjected to


87

DEAE-Sephadex chromatography to remove myosin. The unabsorbed material containing C-protein and connectin was then subjected to hydroxyapatite chromatography. Connectin tends to degrade during this preparation procedure, unless the procedure is performed quickly in the cold. B. SIZEAND SHAPE The mobility of connectin in SDS-gel electrophoresis is very slow: in a 510% polyacrylamide gel, connectin hardly moves and remains at the top of the gel. In 2-3% polyacrylamide gels it moves slowly (cf. Fig. 1). Since the apparent molecular weight (MW) of connectin is very large, appropriate markers for MW determination are not available. Therefore, artificial markers were prepared by cross-linking myosin heavy chains (MHC) with a maleimide derivative (Knight and Offer, 1978). Myosin heavy chains were rapidly dimerized by the cross-linking reaction of their SH groups with the maleimide reagent and within a few minutes of incubation monomers completely disappeared. Dimers were further cross-linked with each other forming 2 X dimers, 4 X dimers, etc. A linear relationship between logarithms of MWs and electrophoretic mobilities was observed up to 3200 kDa (8 X dimers) using 2.0% polyacrylamide gels, as seen in Fig. 2. From the mobility of isolated native connectin, its MW was roughly estimated to be 2100 kDa between 5 X dimers (2000 kDa) and 6 X dimers (2400 kDa) of MHC. This native connectin corresponds to the lower band (p-connectin) of direct SDS extract of intact myofibrils. The upper band (a-connectin) had an apparent MW of 2800 kDa. Wang (1982) claimed the MWs of 1800 and 1200 kDa for T , and T, (a-and p-connectins) by a similiar procedure, but he assumed that dimers, trimers, etc. had been formed by cross-linking reaction of MHC. Our sedimentation equilibrium measurements of MW of p-connectin in 0.5 M KCl and 0.1 M phosphate buffer (pH 7.0) also showed a value of 2700 kDa (Maruyama et al., 1984a). The sedimentation pattern was a single hypersharp peak having a sedimentation coefficient of 17 S in 0.1 M phosphate buffer, pH 7.0, in agreement with the value of 13.4 S in 0.5 A4 KCI and 0.05 M phosphate buffer, pH 7.5 (Trinick et al.. 1984). The shape of the sedimentation pattern showed that the connectin molecule is highly asymmetric. At the same time, the presence of a smaller peak of approximately 30-40 S suggested formation of side-by-side aggregates. An interesting observation was made in the presence of 1% SDS: the sedimentation coefficient of 15 S in the native state changed to 11 S in the denatured state. Since the peak became more hypersharp, it is likely that the change in the sedimentation coefficient was not due to subunit dissociation, but to a shape change in the presence of SDS. King (1984) estimated the MW of connectin in SDS to be lo6 by lightscattering technique. Electron microscopic images by a low angle rotary shadowing procedure showed that there were more straight rods in the presence of SDS

88

KOSCAK MARUYAMA

a

C

e

rno bil it y FIG. 2. Molecular weight determinations of a- and p-connectins by SDS-gel electrophoresis: 2% polyacrylamide gels. The two arrows indicate a- and p-connectins. (a) Cross-linked myosin heavy chains, (b) isolated native connectin, (c) a + b, (d) direct SDS extract of chicken breast muscle. Myosin oligomers, (0)albumin oligomers. Modified (e) cross-linked bovine serum albumin. (0) from Maruyama ei al. (1984a).

than in its absence (H. Sawada and S. Kimura, unpublished). From the asymmetric nature of connectin, it was expected that the viscosity value was large. In a conventional Ostwald type viscometer, the value of intrinsic viscosity was less than 2 (g/dl) suggesting an axial ratio of about 50. However, it turned out that the viscosity of connectin greatly depended on the velocity gradient in the measurements. Thus at a very low velocity gradient of 0.0007 second-', a connectin solution of 0.3 mg/ml had a viscosity value of as high as 17,000 CPand the value dropped to 230 CP at 0.08 second-'. This is a thyxotropic nature owing to an entanglement of very thin filaments that can be easily disentangled by weak force. Electron microscopic observations have revealed that connectin is a very long,


89

flexible filament (Maruyama et al., 1984a; Wang et al., 1984; Trinick ef al., 1984). A low angle rotary shadowing method gave various kinds of images: from straight filament to entangled knitting wool (Fig. 3). The length distribution was heterogeneous ranging from 0.2 to 1 pm. Flow birefringence measurements

FIG. 3. Low angle shadowing images of connectin filaments. Note that myosin molecule (160 nm long) is included. Bar, 0.2 Fm. From H . Sawada and S. Kimura (unpublished; cf. Maruyarna er a / . , 1984a).

90

KOSCAK MARUYAMA

suggested an approximate length of 0.4 p n in solution. Electron microscopic pictures showed that there were some beaded structures in the filaments. Trinick et al. (1984) observed that the width of the connectin filament was 4-5 nm in negatively stained samples. C. OTHERPROPERTIES Connectin is soluble as filaments in KCl concentrations higher than 0.2 M at pH 7-8 and aggregates in lower concentrations of KCl. At 0.05 M KCl it precipitates. Even in 0.2 M KCl, shaking or agitation results in fiber-like aggregate formation. On concentration by a rotary evaporator, lateral association occurs leading to formation of an elastic rubber-like bundle. UV absorption spectra are of a protein nature with a maximum at 280 nm. The value of A,, at 1 mg/ml was approximately 1.2 (light path, 1 cm). A slight shoulder around 290 nm is always seen, but its origin is unknown. Amino acid composition shows that connectin is an acidic protein (Table 111). On the whole, connectin is similar to actin in amino acid composition: proline, TABLE 111 AMINOACIDCOMPOSITION OF CONNECTIN FROM CHICKEN BREASTMUSCLP

Native connectin Asx Thr Ser Glx Pro GlY Ala Cysl2

Val Met Ile Leu TYr Phe LYS

His Arg

96 76 60 111 74 74 65 2 87 12 60

66 31 26 86 15 59

Denatured connectinb 1

2

95 75 69 116 74 71 62 II 85 10 59 67 30 27 82 15 55

93 66 67 I I8 67 76 75 6 78 16 56 76 30 29 79

uNumber of residues per lo00 residues. bPrepared according to Wang et al. (1979).

18

50

91

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN TABLE IV A M I N OACID COMFQSITIONS OF CONNECTIN, C-PROTEIN, AND PROJECT IN",^

Asx Thr Ser Glx Pro GlY Ala Cysl2 Val Met Ile Leu TYr Phe LYS His Arg

T

co

CI

c2

c3

P

95 75 69 I16 74 71 62 I1 85 10 59 67 30 27 82 15 55

93 66 67 118 67 76 75 6 78 16 56 76 30 29 79 18 50

96 59 57 I I7 71 71 66 14 I 04 17 51 69 29 37 86 14 44

99 58 62 I24 69 85 74 ND 85 13 43 68 27 35 89 17 52

I07 69 58 I I9 70 70 80 ND 68 18 62

I09 66 74 I25 76 93 68 8 53 12 36 69 27 32 87 23 51

64 35 32 93 15 48

UNumber of residues per 1000 residues. "T, Denatured connectin (chicken breast) (Maruyama c/ a/.. 1981); CO, native connectin (chicken breast) (Maruyama CI a / ., 1981 a); C I, C-protein (rabbit skeletal) (Offer cr a/.. 1973); C2, C-protein (rabbit white) (Callaway and Bechtel, 1981); C3, C-protein (rabbit red) (Callaway and Bechtel, 1981); P, projectin (Honeybee flight) (Saide, 1981); ND, not determined.

valine, and lysine are abundant and alanine, methionine, isoleucine, and tyrosine occur less in connectin than in actin. Methylhistidine is not present in connectin. A rather high content of proline (-9%) is in parallel with the lack in the a-helix portion (see below). The fact that connectin is rich in nonpolar amino acids may be related to its tendency to form salt-insoluble aggregates. A striking fact in the amino acid composition is that connectin is almost identical with C-protein (Table IV; Fig. 4). Although immunological crossreactivity was not detected using both antisera against connectin and C-protein, this fact is of some interest in view of their localizations in myofibrils (see Section VI). The amino acid composition of connectin is also very similar to projectin, an elastic protein of insect flight muscle (Table 1V; Fig. 4), whose MW is 360 kDa (Saide, 1981). This is of special interest in that both proteins serve as the elastic component of muscle. It is generally thought that a polypeptide of such a huge MW as connectin does not consist of a single peptide. It is reasonable to assume that several peptides are cross-linked as in collagen or elastin. The presence of hydroxylysinonorleucine,

92

KOSCAK MARUYAMA

FIG. 4. Star diagrams of the amino acid compositions of connectin, C-protein, and projectin. Relative contents of the amino acids listed in Table IV are presented in star diagrams. ( I ) Denatured connectin (a + p). (2) native p-connectin, (3) projectin, (4) C-protein (rabbit skeletal), (5) C-protein (rabbit white), and (6) C-protein (rabbit red).

a cross-linker in collagen and elastin, was suggested by tritium incorporation experiments (Fujii and Maruyama, 1982). However, the amino acid analyses showed that the presence of the diamino acid was negligible (Maruyama et al., 1983). Gruen et al. (1982) also denied its presence. They could not detect glutamyllysine either. The carbohydrate content is very small and less than 1% by weight (Table V). Recently, Gassner et al. (1985) have emphasized the possibility of connectin as a glycoprotein. It is not clear whether the small amount of carbohydrates is covalently bonded to connectin or not (cf. Gassner, 1986). Circular dichroism measurements led Trinick and his associate (1984) to the conclusion that connectin completely consists of a random coil. This was an important finding. However, when we tried to measure circular dichroism in

CONNECTIN. AN ELASTIC FILAMENTOUS PROTEIN

93

TABLE V CARBOHYDRATE CONTENTSI N CONNECTIN FROM CHICKEN BREASTMUSCLE"

Fucose Mannose Galactose Glucose GluNAc NeuNAc

Native connectin

Denatured connectinb

0.38 0.48 0.81 5.77 0.34 0

0.68 0.95 0.91 9.41

0.12 0

40 mV) to the cytoplasm but still negative to the external solution (Bates el al., 1982). It is now well established that ATPases, which perhaps pump protons, exist at the tonoplast and other vacuolar membranes (Matile, 1978; Doll, 1979; Guy er al., 1979; Marin, 1980). The proton pumps of the plasmalemma and the tonoplast of higher plants have recently been reviewed by Marrk and Ballarin-Denti (1985). Most lysosomal enzymes, when isolated, exhibit a pronounced pH optimum. Coffey and de Duve (1968), therefore, inferred that the intralysosomal pH would probably be low, in general lower than the average cytoplasmic pH. Okhuma and Poole (1978) and Poole and Okhuma (1981), using a pH-dependent fluorescent probe, reported that the intralysosomal pH in living cells is maintained in the range 4.7-4.8. According to Szego and Pietras (1984) two mechanisms for regulating the pH difference across lysosomal membranes have been postulated. The first one is a Donnan-type equilibrium system due to the presence of nondiffusible negatively charged groups within the lysosomes (i.e., acidic glyco+

THE ELECTRICAL DIMENSION OF CELLS

277

lipids, anionic lipoproteins, and glycoproteins with relatively low isoelectric points). According to Hollemans et al. (1980) this mechanism is used in rat liver lysosomes. The second one, more and more supported by strong experimental evidence, states that a lysosomal proton pump, driven by Mg2+ and ATP, is involved (Schneider et al., 1978; Schneider, 1981; Okhuma et al., 1982). Whether the lysosomal H -ATPase may be related to the well-characterized H -ATPase found in secretion granules of adrenal chromaffin cells (Johnson and Scarpa, 1976; Pollard et al., 1979; Geisow, 1982) remains to be confirmed. Recently, Reggio et al. (1984) reported that antibodies against lysosomal membranes of rat liver revealed a 100,000 Da protein that cross-reacts with purified H+,K+-ATPase from gastric mucosa. The antigen was also found in those compartments that have recently been demonstrated to be acidified by an ATP driven pump (some compartments of an endocytic pathway in macrophages, small amounts in the plasma membrane of liver, but large amounts in coated vesicles, some parts of the Golgi complex, etc.). For the role played by the low pH in lysosomes, we refer to the review of Szego and Pietras (1984). Golgi vesicles of liver (Zhang and Schneider, 1983) and those from lactating rat mammary glands (Virk et al., 1985) possess a ApH of about 1 pH unit. Golgi system membranes of adipocytes seem to have a glucose transport activity similar to the plasma membrane (Smith et al., 1984). Lutoids, which are abundant in some plants, e.g., Hevea brasiliensis, are single-membrane microvacuoles with lysosomal characteristics. They have a lower pH (about 5.5) than that of their cytoplasmic environment (about 7) and accumulate numerous mineral and organic ions. Critin (1982) presented data strongly supporting the presence of an inward electrogenic proton-translocating ATPase on the membrane of intact lutoids. Newly formed and especially aged neurohypophyseal neurosecretory granules are more internally acidic than the cytoplasm, but this seems to originate from a Donnan equilibrium (Scherman and Nordmann, 1982). Clathrin-coated vesicles contain an ATP-dependent proton pump that may play a role in the acidification events that are essential in receptor-mediated endocytosis (Forgac et al., 1983). +

+

3. Mitochondria The question about the “right” membrane potential of mitochondria is also as yet not settled (Tedeschi, 1980). The chemiosmotic hypothesis of the coupling mechanism states that oxidative phosphorylation requires that there should be a total promotive force of 210-270 mV across the osmotic barrier of the cristae membrane of respiring mitochondria (electrically negative inside the mitochondrion, Mitchell, 1961, 1966, 1967; Mitchell and Moyle, 1969). Gulian and Diacumakos (1977) measured in HeLa cells in situ only -2 I mV for the impaled mitochondria. Experiments with isolated giant mitochondria of Drosophila yielded values of 10-20 mV, positive inside (Tupper and Tedeschi, 1969).

278

ARNOLDDELOOF

Similar positive values were found in the metabolically viable giant mitochondria isolated from the liver of cuprizone-fed mice (Maloff er al., 1978a). The reason why the values are positive might be that the ionic composition of the incubation medium is quite different from that of the in vivo cytoplasm. If impalement had caused a substantial leak, which is of course possible with such small organelles, then the metabolic viability, according to Mitchell’s hypothesis, would have been lost. It, however, remained intact as revealed by biochemical analyses (Maloff er al., 1978b). During the recent decade, indirect methods have been used for estimating the membrane potential of mitochondria and, depending upon the conditions, values closer to the ones predicted by Mitchell have been reported (see, e.g., Rottenberg, 1984). According to Ling (1981) the discrepancy between calculated and actually measured values is due to the fact that several premises of the chemiosmotic theory are incorrect: mitochondria1 functioning could be explained by his association-induction hypothesis. Chloroplasts also seem to be able to create their own ionic environment (Robinson and Downton, 1984). 4. Sarcoplasmic Reticulum The sarcoplasmic reticulum of skeletal muscle cells is very rich in Ca2+ATPase and is a storage site for Ca2 , the essential ion controlling muscle contraction. Merocyanin 540 is a potential sensitive dye. Shifts in fluorescence have been observed during Ca transport in reconstituted vesicles of sarcoplasmic reticulum (Haeyaert et al., 1980). This suggests that there is a charge deficit, despite the simultaneous transport of Mg2 , K ,and, perhaps, H and organic ions (Somlyo et al., 1981). +

+

+

+

5 . Nucleus The question of whether there is also a potential difference across the nuclear envelope has as yet received little attention as it is intuitively assumed that because of the presence of the numerous “pores,” such a potential difference cannot be built up. The scarce experimental data suggest that the nuclei of some somatic cells, at least, may be electrically and ionically compartmentalizedfrom the cytoplasm. Loewenstein and Kanno (1963) examined the electrical potential and resistance in situ and in isolated giant nuclei of salivary glands of Drosophila flavorepleta, with microelectrodes of 10-35 M a , and tip potentials lower than 2.5 mV. The measured resistance of the nuclear envelope was of the order of 1 fl cm2. This is smaller than that of the cell membrane but still large enough to represent a formidable barrier to ion diffusion. The nucleoplasm was, on the average, 15 mV negative with respect to the cytoplasm. The potential declined to zero and the resistance to a fraction of its original value when the nuclear membrane was experimentally perforated by repeatedly driving an empty micro-

279

THE ELECTRICAL DIMENSION OF CELLS

pipette through it. From these experiments Loewenstein and Kanno concluded that it was unlikely that the sites where the double nuclear envelope fuses together (later called the pore complexes) are bridged by nucleoplasm or cytoplasm. They speculated that these “pores” might be additional barriers which confer its high electrical resistance upon the nuclear envelope. In salivary gland cells of another insect, Chironomus thummi, the nucleoplasm was found to be 2-5 mV negative to the cytoplasm (Ito and Loewenstein, 1965). We will refer to nuclei which are able to maintain a potential difference over the nuclear envelope as “closed” nuclei. When Kanno and Loewenstein (1963) performed similar experiments on the germinal vesicle of an amphibian oocyte, they found that this type of nucleus was a rather permeable structure, its resistance was indistinguishable from that of the cytoplasm and nucleoplasm, and no nucleuspotential could be measured. Such a type of nucleus will be referred to as “open.” If there is indeed a potential difference between nucleoplasm and cytoplasm, one would expect to find differences in ionic concentrations-or better activities-in both compartments. Such differences in ionic concentrations between nucleus and cytoplasm of frog oocytes were already reported in 1962 by Naora et al. who dissected out clean and whole nuclei from frozen oocytes. The Na+ and K + content (microequivalents per gram water) of the nucleus were respectively 3.2 and 2.4 times higher than that of the cytoplasm (excluding yolk platelets). The Na+ :K ratio of the nucleus was 1.1 and that of the cytoplasm (excluding yolk platelets) was 0.72. Autoradiographic experiments after incorporation using [22Na, 42K, 14C]leucine and [t4C]alanine showed, in each case, a marked accumulation within the nucleus. Only a very small fraction of the amino acids which accumulated in the nucleus could be precipitated by trichloroacetic acid, which indicates that most amino acids were not incorporated into proteins. These data suggested that, at the level of the nuclear envelope, active mechanisms might be present for uphill movement of ions and amino acids and that the germinal vesicle is of the “closed” type in the developmental stage used by Naora et a f . (1962). However, these data are not supported by more recent work on concentrations and activities of K and Na in oocytes of R a m pipiens. In small oocytes (

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