Molecular genetics: a key to the cytoskeleton's closet

Molecular genetics: a key to the cytoskeleton's closet M.A. Titus, H.M. Warrick and J.A. Spudich Departments of Cell and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA Current Opinion in Cell Biology 1990, 2:116-120

W h a t is the cytoskeleton? The cytoskeleton is a dynamic network of both structural and motor proteins which are thought to play important roles in maintaining the integrity of the cell as well as in generating various cellular movements. These proteins have been the subject of intense scrutiny due to their ubiquitous nature and importance in cell behavior. Furthermore, they can be rapidly assembled and disassembled during various cellular activities and therefore are ideal proteins for the study of principles of self-assembly and of spatial and temporal controls operating on proteins within the cell. The best known of the cytoskeletal proteins are actin, actin-associated proteins, the actin-based motor myosin, micrombules, the microtubule-based motors dynein and kinesin, and intermediate filaments. In the last 40 years or so, the structural and functional characteristics of many cytoskeletal proteins were elucidated by fruitful investigations into systems which contained these proteins in high abundance and in an ordered array, such as cilia, flagella and skeletal muscle. Actin, myosin and a host of other Cytoskeletal proteins were discovered in a variety of cell types beginning in the late 1960s, stimulating investigators to take on the challenge of understanding how a seemingly disorganized cytoplasm can assume polarity, move vesicles and organelles in a directed fashion and organize the necessary machinery to propel the cell across a substrate. The recent marriage initiated between classic biochemical and structural techniques and the rapidly evolving use of molecular genetics has resulted in an explosion in our knowledge of the structure, assembly and role of the cytoskeleton in the cell.

Is molecular genetics a tool of choice? There are several questions that are raised by the entrance o f this m o d e m technology into the world of the biochemist/cell biologist. Most importantly, is molecular genetics needed to clearly identify the function of a eukaryotic protein? There are clear examples in the biology of the cytoskeleton where the answer to this question

is a resounding yes--such as the genetic dissection of tubulin isotype function in yeast and Aspergillus, the role of actin in yeast, and myosin function in DicOostelium (discussed below). There are other equally compelling examples which lead to the opposite conclusion, such as the apparent lack of effect in DicOx~steliumwhen the major actin-binding proteins are removed. One must bear in mind, however, that this latter result suggests the existence of a complex network of actin-binding proteins in DicO~stelium and thereby provides investigators with clues to further unravel the mysteries of the cytoskeleton. Every system offers its own challenge to the investigator. The absence of a clear phenotypic change following a mutation may require the development of more sophisticated and innovative assays for cellular behavior. Many of the topics highlighted in this review have been presented in a recent issue of CellMotility and the Cytoskeleton [1], and the reader is referred to that volume for a more detailed discussion. Here we focus on two types of systems for using molecular genetics to study any protein of interest: the systems that have been used successfully to study protein structure and function in vitro and those that allow one to examine the role of such proteins in vivo. While a perfect system probably does not exist, the recent rapid progress in a number of systems makes it possible to pick and choose that which best suits the investigator's needs, or to combine the best aspects of more than one. The molecular genetic study of any cytoskeletal protein (or any other protein for that matter) ideally progresses through four basic stages. The first step is the cloning and sequencing of the gene of interest. The cloned gene can then be expressed in a host system in sufficient quantities for biochemical study of the gene product. Following the expression of the wild type gene, mutations can be introduced into the gene and the effect of these alterations on the properties of the gene product can be investigated in vitro. The removal of the protein from a cell can also be carried out and the phenotype of the resultant cell examined. This approach may be coupled with the introduction of mutated proteins in place of wild type proteins to determine the effect of such alteration on the cell's behavior.

Abbreviation

HMM--heavy meromyosin. 116

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Are all expression systems created equal? There have been remarkable advances in understanding structure-function relationships in b o t h structural and motor proteins using sequence analyses and subsequent expression of the cloned genes in an appropriate h6st cell. An instructive example is the family of actin crosslinking proteins, the most familiar of which is 0uactinin. The successful cloning of rt-actinin genes and also other gelation factors and the subsequent sequence comparisons [2] revealed the presence of a common domain believed to be involved in actin binding (Fig. la). Surprisingly, a search of other protein sequences uncovered a similar domain in dystrophin, a protein linked to muscular dystrophy (Fig. lb). This is a case where the function of an uncharacterized protein can be inferred from the conservation of sequences thought to play a role in an event such as actin binding. The genes encoding three well-known actin severing proteins, severin, gelsolin and villin have also been cloned and a comparison of their sequences (Andre et aL, J Biol Cbem 1988, 263:722-727)

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reveals a c o m m o n actin-severing domain (Fig. lc). Comparisons of the N-terminal portions of a number of actinbased motors, such as the conventional myosin (whether it be from a skeletal muscle or a non-muscle cell) and the smaller actin-based motors such as myosin I, reveal a striking conser~ution through the region often referred to as the head domain and a sharp transition in which tail sequences are totally divergent (Fig. ld). This clearly implicates the N-terminal portion of these molecules as that region which is critical for the generation of movement by these proteins, as has been directly demonstrated using an hz vitro motility assay (Toyoshima et al., N a t u r e 1987, 328:536-539). Indeed, such sequence similarities can be exploited by using heterologous probes or the polymerase chain reactions to clone related genes.

The development of the technology to express altered forms of a protein in an appropriate host facilitates the hi vitro characterization of any protein. The systems used to date each have advantages and disadvantages that one must be aware of when embarking on these studies.

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Fig. 1. Dot matrix comparison of several cytoskeletal proteins. (a) Dictyostelium gelation factor [2] versus Dictyostelium 0~-actinin (Noegel et al., F£BSI_ett 1987, 221:391-396); (b) human dystrophin (Koenig et al., Cell 1988, 53:219-228) versus Dictyostelium ct-actinin; (c) vertebrate gelsolin (Kwiatkowski et al., Nature 1986, 323:455-458) versus Dictyostelium severin (Andre el al., l Biol Chem 1988, 263:722-727), and (d) Dictyostelium conventional myosin, mhcA ONarrick et al., Proc Natl Acad Sci USA 1986, 83:9433-9437) versus Dictyostelium abmA [16]. The matrices shown were generated by placing a dot every time 10 residues in a window of 16 matched exactly. The numbers on the axes indicate the position in the deduced amino acid sequence.

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Cytoplasmand cell motility The most commonly used expression system for cloned genes has been Escbericbia col~ Several cytoskeletal proteins have been successfully expressed in E. coli in a functional form. The difficulties in using £ coli are the tendency for expressed protein to be found in an insoluble state and the lack of post-translational modifications. While some proteins can survive the harsh treatment required for solubilization, many o f the proteins o f interest can not. It is not unusual to have to express a protein as a fusion product with a bacterial protein in order to obtain a soluble product. As it is not yet possible to predict how successful the expression of a particular protein in E. coliwill be, one must proceed on a trial basis for each protein. One of the more remarkable success stories of expression in E coli has been the ease with which functional Drosophila kinesin has been obtained [3]. Progress in dissecting the functional domains of kinesin has proceeded rapidly following the application of molecular genetics to the study of this molecular motor. The recombinant Drosophila kinesin has been shown to consist of three distinct domains based on deletion analysis. The N-terminal region of the molecule contains a nucleotide binding site and has been shown to have nucleotidedependent microtubule binding; it is likely that this region is the motor domain. The middle segment of the molecule has an ¢*-helical structure, most likely forming the stalk of kinesin, and the small C-terminal tail portion is where the kinesin light chains bind. The dissection of the important functional segments using in vitro expression allows one to proceed into a detailed analysis o f the particular amino acid residues required for each function. Another successful examination of functional domains using in vitro expression of a cloned protein has been in the case of gelsolin. Two distinct expression systems were employed to produce sufficient quantities of this protein to map out the calcium-binding and the actinbinding, severing and nucleating regions. Gelsolln was expressed in E. colias a soluble fusion protein which has all of the expected properties of the native protein [4]. Transfection of Cos cells with cloned gelsolin resulted in the secretion of gelsolin into the extracellular media [5]. Collection of this secreted gelsolin greatly reduced the number of subsequent purification steps, emphasizing one major advantage of the Cos cell expression system. Also, all of the necessary post-translational modifications are provided by the cell line. The drawbacks to the Cos cell system are that the expression of the proteins is transient and maintaining the ceil lines is more costly and laborious than for E: coll. Dico~stelium has only recently been used for the expression of heterologous genes. Several drug resistance phophostransferases and pyrimidine pathway enzymes have been successfully used as transformation markers in DicO~ostelium. One advantage of using DicO~gstelium as a host expression system is that post-translational modifications are available. Another ad~mtage is that this cell line is easily maintained and inexpensive to grow in large quantities. The use of DicO~stelium as a host for expression of eukat3'otic genes looks promising and is awaiting

further exploitation. Two DicO~stelium myosin subfragments equivalent to S1 and hea W meromyosin (HblM) have been obtained by expression #l vivo [6,7]. The resultant proteins can be isolated in milligram quantities and these proteins have the expected biochemical attributes of the native protein. Additionally, these proteins are capable of producing movement in an hz vitro motility assay. It is now possible to generate mutants in regions of interest, such as the actin-binding site and ATPase site, to help clarify the mechanism by which mechanochemical coupling takes place.

What can we learn from manipulation of cytoskeletal genes in situ? While the functional dissection of proteins ht vitro is a powerful tool for the investigation of the mechanism by which a protein works, a question of major interest to many cell biologists is how a particular protein interacts with others in a cell to generate a given behavior. Ideally, one would like to combine the knowledge gained from in vitro mutagenesis studies with in v&o manipulation of those proteins to fully understand the mechanism by which a cell marshalls its internal machinery to generate a wide variety of movements. While the in vitro behavior of a protein may suggest its role in vivo, genetic manipulation of a protein in situ is a powerful technique for elucidating the actual function of a protein. The lower eukaryotes yeast and Aspergilhts have been shown to be among the most powerful sTstems available for using classic genetics to study cytoskeletal proteins. These organisms have been successfully used to study the ht vivo role of several cytoskeletal proteins (Huffaker et al., A n n u Rev Genet 1987, 21:259-284) [8]. The simplicity of their genomes allows one to study, in a specific manner, a limited number of genes encoding a particular protein. The ability to target genes of interest in both yeast and Aspergillus and to cart3, any lethal mutation which is recessive has enabled researchers to dissect out the role of such fundamental proteins as actin and tubulin. Yeast cells which contain mutant actin are defective in a wide ~:ariety of cell behaviors such as secretion and chitin localization and exist largely in an unbudded state. The tubulin mutants have clearly defined phenotypes: nuclear migration, chromosome separation and nuclear fusion are all affected. Suppressor mutants can also be isolated and therefore make it possible to study the battery of actin- or tubulin-associating proteins which may play an important role in modulating the function of these important cytoskeletal proteins [9]. One can also rescue cells defective in processes which are believed to be performed by cytoskeletal proteins by transformation with a genomic library constructed in an autonomously replicaring plasmid. The gene responsible for the rescue can then be subsequently cloned out and identified. An example of this is the yeast kawogamy (KAR) mutants where the KAR3 gene product has been shown to be a ~riant

Genetic approaches to the c'yloskelelon Titus, Warrick and Spudich of the microtubule motor kinesin (Rose, personal communication). The nature of the motile behaviors of both Aspergillus and yeast is, howex'er, limited. They undergo cytokinesis but do not exhibit gross movements typical of higher eukaryotic cells such as chemotaxis and major alterations of cell shape as development proceeds. Th~ simplicity in cellular behavior has been advantageous for examining the role of cytoskeletal proteins in processes such as intracellular transport which may not be amenable to detailed scruti W in other organisms. The two higher eukaryotes, Drosophila and Caenorhab ditis elegans, are genetic systems which allow one to address the assembly and role of the cytoskeleton in sophisticated developmental processes and in highly organized cellular structures (e.g. muscle). The complexity of these organisms allows the investigator to examine the role of a protein of interest in a structure which may not be essential for survival, e.g. the flight muscle of Drosophila and the body wall muscle of C elegans Although the ability to specifically target and remove or alter genes of interest is lacking, both of these organisms can be stably transformed and mutant cell lines rescued by the introduction of cloned genes. This allows one to use mutagenesis to study important domains of a cytoskeletal protein using an in vivo assay, Two recent examples demonstrating the potential of this molecular genetic method are the rescue of Drosophila kinesin mutants by the introduction of the wild type gene [10] and the complementation of C elegans myosin null mutants with the cloned unc-54 and m3~3 genes i11]. Another interesting example of using an in vivo assay to potentially ex@uate functional domains of a cytoskeletal protein is the heterologous expression of chicken villin in fibroblasts devoid of this protein. The villin expression which induces the formation of microvillus-like structures on the fibroblast surface [12]. Studies of the cytoskeleton of the simple eukaryote D/cO~3stelium have provided several surprises. Homologous recombination has been demonstrated in this organism, the first studies being carried out with the major actin-based motor protein myosin (De Lozanne and Spudich, Science 1987, 236:1086-1091). It had long been postulated that myosin plays a major role in generating cellular movement and microinjection experiments with anti-myosin had shown that this motor protein is essential for cytokinesis in echinoderm eggs (Mabuchi and Okuno, J Cell Biol 1977, 74:251-263; Kiehart et al., J Cell Biol 1982, 94:165-178). The striking colocalization of myosin and actin in actively moving ceils had led to the assumption that these two proteins interact to propel a cell forward across a substrate. Replacement of myosin with a soluble subfragment of myosin (HMbi) by gene targeting or the removal of myosin altogether by anti-sense transformation or by gene replacement led to the startling discovery that cells are still capable of undergoing locomotion--albeit at a reduced speed--thus shattering preconceived notions about the role of this major motor protein in cell migration (De Lozarme and Spudich, 1987; Knecht and Ix3omis, Science 1987, 236:1081-1086) [13]. Other crucial cellular func-

tions do, however, require myosin and it is important to note that replacing the endogenous DicO~stelium myosin with a soluble subfragment (equivalent to HbD,i) resuits in cells whose altered phenotype is the same as that found for cells that are missing the entire protein (De Lozanne and Spudich, 1987; Knecht and Itx~mis, 1987) [13]. These results clearly implicate the filament-forming portion of myosin in the cellular functions of this major motor protein. This is an example of how the in vivo alteration of a cytoskeletal protein can aid in elucidation of its function. The next surprise that Dicgvstelium had in store for researchers was that the complete removal of any of three major actin-associating proteins, ¢x-actinin,gelation factor and severin has no significant effect on the behavior of the cell (Schleicher et al., Protoplasma 1988, 2:22-26) [14]. Additionally, the removal of myosin I, the smaller actin-based motor, does not appear to have significant effects on cellular movements [15]. While these particular experiments provide clues about the existence of a complex network of cytoskeletal proteins involved in cellular movement, they indicate that it may not be possible to easily discern the interactions of a single protein in this nem'ork. Alternate approaches to their in vivo functions are required. There may be a significant change in the cell's behavior that is going undetected at present, indicating distinct new assays may have to be developed. The advances being made using molecular genetics in the systems described have made real the promise of a more thorough understanding of the role of cytoskeletal proteins in cellular events. Molecular genetics is not the only key needed to unlock the cytoskeleton's closet but without this powerful tool the mysteries of the cytoskeleton will linger. The information obtained from the molecular genetic experiments has expanded the results from biochemical, structural and other types of studies, confirming the importance of a multi-faceted approach to study a protein or the interactions between a group of proteins. Just as molecular genetics is not the only means by which to address a problem, the in-depth examination of the cytoskeletal proteins in a single biological system will also not uncover the in vivo function of all of the cytoskeletal proteins. It is important to note that research into the cytoskeleton has benefited from the fact that investigators have focused on a limited number of model organisms. Yet, a balance should be maintained between the depth of investigation into a single organism and the breadth of organisms chosen for such studies. The lessons gleaned from the studies to date indicate that where one system may be ad~Jatageous for the studies of a particular protein (such as in the case of actin and ttrbulin in yeast and Aspergillus) studies in another may not be so informative (such as in DicOostelium where these are at least 16 actin genes). The study of the interactions of a given protein with its cellular environment will require a broad approach, including molecular genetics, in which several well-characterized organisms are exploited.

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Cyloplasmand cell motility Annotated references and recommended reading • ••

Of interest Of outstanding interest

8PUDICtlJA (ED): Molecular genetic approaches to protein structure and function. Applications to cell and developmental biology. Cell Motil Cytoskeleton 1989, 14:1-163. A volume ~ l i c h contains a current collection of detailed reviews covering the application of molecular genetic approaches to the stud)' of the c~toskeleton in a number of diverse systems. 1. • •

2.

NOEGELAA, RAPP $, LO'ITSPEICH F, SCIILEICttER M, STEWARTM:

•

The Dictyostelium gelation factor share a putative actin

binding site with ~x.actinins and dystrophin and also has a rod domain containing six lO0.residue motifs that appear to have a cross-beta conformation. J Cell Biol 1989, 109:607-618. Reports the primary sequence of an actin crosslinking protein and its anal)sis. Highlights cortser~ution of a common puta~-e actin-binding domain as well as features telex:ant to the possible function of this unique protein. 3. ••

YANG Jr, LAYMONRA, GOLDSTE~ LSB: A three-domain struc. ture of kinesin hea~T chain revealed by DNA sequence and microtuhule binding analyses. Cell 1989, 56:879--889. The first report of the primary sequence of a microtubule-based motor. In addition to mapping function through the anab'sis of the sequence, deletions of the protein were expressed in/~ coliand actMties ass~-ed. 4. ••

WAY M, GO(~H J, POPE B, WEEDSAG: Expression of human plasma gelsolin in Escherichia colt and dissection of actin binding sites by segmental deletion mutagenesis. J Cell Biol 1989, 109:593-605. An elegant examination of the domains of an actin severing and capping protein using expression of rearranged forms in E coil 5•

KXXaATKOXXSKI DJ, JANMEYPA, YL'~ IlL: Identification of critical functional and regulatory domains in gelsolin. J Cell Biol 1989, 108:1717-1726. The anab'sis o f an actin severing and capping protein using a series of carboxy-terminal deletions which were obtained secreted from transected Cos ceils. 6. •

M.Z'~STE~DJ, RtJPPEL KM, SPUDICtl JA: Expression and characterization o f a functional myosin head fragment in Dictyostelium discotdeum.. Science 1989, 246:656-658. The over-expression and isolation of an active motor domain of myosin, the preface to mutational dissection o f function.

7. •

RUPPELK.M, EGELHOFFTT, SPUDtCHJA: Purification of a functional recombinant myosin hagment from. Dictyostelium discoideurrt Ann NY Acad Sci 1990, in press. The first report of the isolation and purification o f a recombinant fragment (HS~I) o f the myosin molecule. The expressed tL~,LMpossesses an actin-ac~-ated ATPase.

MORRISIN'R, OS.~L~NISA, ENGLE DB, Doo.'odq Jtl: The genetic anal)sis o f mitosis in Aspergillus ntdulan~ Bioessa3* 1989, 10:196-201. A review covering the use o f molecular genetic techniques to study processes heavily dependent on the c)toskeleton in the filamentous fungus 8. •

A.$pergilh~ ADAMSAEM, BOTSTEIN D, DRUBIN DG: A yeast actin-binding protein is e n c o d e d by SAC6, a gene found by suppression of an actin mutation. Science 1989, 243:231-233. An example o f the power of molecular genetics techniques (in particular suppressor anab~is) ~ h e n used to probe the ~-toskeleton in ~x'ast. 9. •

10. •

SAx-roNWM, RAI:F EC: Weltschmertz displayed by kinesin mutant: the kinesin hea~T chain is essential in Drosophil~ J Cell Biol 1989, 109:281a. The anab~is of the phenot)pes produced in flies which are not expressing the micrombule-based motor kinesin. 11. FIRE A, X,Y]ATERSTONRI'|: Proper expression o f myosin genes • in transgenic nematodes. EtlBOJ 1989, 8:3419-3428. The rescue of C elegans m)~osin mutants through the reintroduction o f normal genes into the germline. This sets the stage for the anab'sis of function through the reintroduction o f modified forms. Also presents an example of an oxxrexpression phenotype. 12. •

FRIEDERICtlE, ttUET C, ARPL'~ M, I_OLA'ARDD: Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell 1989, 59:461-475. The expression o f the gene for a cytoskeletal protein that is usually found only in a few specialized cell structures, in a non-specialized cell resulted in a rearrangement of the normal actin distribution in that ceil. This assay ~ t s used to anabxe earboxT-terminal deletions to map the morphogenic domain. 13. •e

MANSTEL',IDJ, Tn-us MA, DE LOZA.'~X A, SPUDICH JA: Gene replacement in DicO'osteliurr~ generation of myosin null mutants. E~IBO J 1989, 8:923-932. The application of homologous recombination to create a mutant de-" void of all muscle-t3pe myosin sequences in DicO~stelium. A p~'otal study that further elucides the role of myosin in non-mttscle cells ushag molecular generics, and a demonstration of the xxrsatility of Dic-

O~teliun~ 14. •

ANDRE E, BRL\'K M, GERISCH G, ISE.~;BERG G, NOEGEL A, SCItlEICItERM, 8EG.MI.JE, WMJJLkFF E: A DicOx)stelium mutam deficient in severin, and F-actin fragmenting protein, shows normal motility and chemotaxis. J Cell Biol 1989, 108:985--995. The apparent absence of a phenot)pe;in a mutant of DicO~gstelium which is lacking a major actin-severing protein. 15. JUNGG, It.~.~t~iERJA Ili: Creation o f DicO'ostelium myosin I • mutants. J Cell Biol 1989, I09:281a. A mutant of Dico~stelium created by homologous recombination w~th the sequence of one of the small myosins has a phenotype of partially impaired phagoc)xosis. 16. TITUSMA, ~rARRICKItM, 8PUDICHJA: Multiple actin-based mo• tors in DicO'ostelium. Cell Reg 1989, 1:55--63. Evidence for a network of muldple small myosin genes in DicO~stelium.