Cell Motility and the Cytoskeleton 18:159-163 (1991)
Views and Reviews Genetic Aspects of Microtubule Biology in the Nematode Caenorhabditis elegans Cathy Savage and Martin Chalfie Department of Biolog~calSnences, Columbia University, New York, New York
INTRODUCTION
Microtubules are required for a variety of cellular processes, including mitosis, meiosis, cell motility, morphogenesis, and, in neurons, neurite outgrowth, axonal transport, and sensory transduction. One approach to the study of microtubule biology is to isolate mutations that disrupt microtubules; these mutations should identify genes and gene products that are important for microtubule structure and/or function. The phenotype resulting from the loss of a particular gene product also gives an indication of the role of that product. Genetic approaches have been particularly useful in the study of microtubules in Drosophila [Kemphues et al., 1983; Matthews and Kaufman, 19871, Aspergillus [Burland et al., 1984; Weatherbee et al., 19851, and yeast [Yamamoto, 1980; Hiraoka et al., 1984; Thomas et al., 19851. In this review we summarize genetic and biochemical studies of microtubule function and structure in the nematode Caenorhabditis elegans. MICROTUBULE STRUCTURE
C. elegans has three structurally distinct types of microtubules: two forms of cytoplasmic microtubules and ciliary microtubules [Chalfie and Thomson, 19821. In contrast to most eukaryotic cells, C. elegans cells do not have cytoplasmic microtubules with 13 protofilaments; the majority of the cytoplasmic microtubules of C. elegans have 1 1 protofilaments. These 1 1 -protofilament microtubules are found in all cells except the six cells which mediate a response to gentle touch. The processes of these touch receptor neurons contain 15-protofilament microtubules. The presence of these two unusual microtubule forms may be characteristic of the phylum, as they are found in other nematodes (Chalfie and Thomson, 1982). Both types of cytoplasmic microtubules are short compared to the length of the neuronal 0 1991 Wiley-Liss, Inc.
processes that contain them [Chalfie and Thomson, 19791. Short microtubules might more easily accommodate changes in cell length, such as those that occur during normal sinusoidal movement of the worms. In addition to cytoplasmic microtubules, some sensory neurons have ciliated endings with axonemes consisting of nine outer doublet microtubules and a variable number, up to seven, of inner singlet microtubules [Ward et al., 1975; Ware et al., 1975; Perkins et al., 19861. These sensory cilia are the only axonemal structures in the nematode (the sperm are amoeboid instead of flagellated). The doublet microtubules are similar to those in other organisms, having 13-protofilament A subfibers and 11-protofilament B subfibers. The inner singlet microtubules are unusual in that they have l l protofilaments [Chalfie and Thomson, 19821. No dynein arms or radial spokes are seen. The axonemes have three structurally identifiable regions [Perkins et al., 19861. In the proximal segment, a central cylinder attaches the outer doublet and inner singlet microtubules, and the doublet microtubules attach to the membrane by Y-shaped links. There is no associated basal body in adults. In the middle segment, the doublet microtubules associate with the membrane and the singlet microtubules appear unattached in the center. The outer segment contains only A subfibers and inner singlet microtubules, and no membrane attachments are found. Several microtubule proteins of C . elegans are being characterized. The nematode contains three to five or-tubulin and three to four P-tubulin genes, some of which have been cloned [Gremke, 1986; Savage et al., 1989; Driscoll et a]., 19891. In contrast to bovine brain Accepted November 6 . 1990 Address reprint requests to Cathy Savage and Martin Chalfie, Department of Biological Sciences. 1012 Sherman Fairchild Center, Columbia Univeristy, New York. N Y 10027.
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microtubule proteins which form 13-14 protofilament microtubules in vitro, nematode microtubule proteins polymerize in vitro to form predominantly 9-1 l protofilament structures [Aamodt and Culotti, 19861, suggesting an inherent difference in the microtubule proteins. Tubulins from the 1 I-protofilament microtubules are likely to contribute most to the pool of microtubule proteins because the 15-protofilament touch cell microtubules derive from only six neurons in an animal with 302 neurons and many dividing germ cells. C. eleguns microtubule proteins include several high molecular weight MAPs and a 32 kd MAP. When these proteins are polymerized in vitro, the microtubules are connected by periodic cross-links formed by microtubule-associated proteins [Aamodt and Culotti, 19861. Adligin, the 32 kd MAP, is the major component of the periodic cross-links; adligin, but not the high molecular weight MAPs, forms periodic cross-links when added to purified nematode tubulins [Aamodt et al., 19891. In addition, a major 400 kd MAP has been isolated and shown to be a cytoplasmic dynein-like motor [Lye et al., 19871. MUTATIONS AFFECTING MICROTUBULE STRUCTURE
Several mutations affecting microtubule structure have been identified in screens for mutants defective in behaviors such as chemotaxis, touch sensitivity, and coordinated movement. Defects in microtubule structure in these mutants have usually been characterized by electron microscopy. Other mutants have been identified by their resistance to the anti-microtubule drug benomyl. The structure of the axonemes of sensory cilia is disrupted in some mutants defective in behaviors such as chemotaxis, osmotic avoidahce, and dauer formation (which requires chemosensory detection of a specific pheromone) [Perkins et al., 19861. Mutations in some genes result in the complete loss of all sensory cilia (duf 1 9 ) or of a subset of cilia (che-10).Other mutations result in shortening of the axoneme, again affecting all cilia (che-2, che-3, che-13, duf-10, osm-I, osm-5, and osm-6) or a subset (osm-3). In addition, in cut-6 mutants, supernumerary microtubules normally at the ends of mechanosensory cilia are found throughout the axoneme. The set of genes affecting sensory cilia has probably not been saturated, however, since many of these genes are represented by only one or a few alleles. Also, many proteins found in the axoneme may not be specific to these structures, and mutants would therefore be pleiotropic. unc-33 may be an example of a gene that is required for microtubules in both sensory cilia and neuronal processes [Hedgecock et al., 19851. In unc-33 mutants sensory cilia have elevated numbers of microtu-
bules, many of which have structural abnormalities. Larger-diameter microtubules, microtubules with hooks, and doublet and triplet microtubules are all seen. Since these mutants seem to be overproducing microtubules, the wild-type unc-33 product is thought to regulate the stability or association of microtubule proteins. Neuronal processes are misdirected in these mutants, probably causing the uncoordinated phenotype. Although microtubule organization has not been examined in neuronal processes, similar microtubule defects may be the cause of the outgrowth phenotype. The organization of cleavage divisions in early development also depends on microtubule function [Hyman and White, 19871. The first cell divisions in C. eleguns follow a strict pattern of polarity and asymmetry [Deppe et al., 1978; Laufer et al., 19801 and the large cell size of the early embryo requires a larger spindle [Albertson, 19841. Several mutations disrupt the pattern of the first cleavage [Wood et al., 1980; Cassada et al., 19811; mutations in the gene zyg-9 particularly affect microtubule organization [Kemphues et al., 19861. In zyg-9 mutants, the first cleavage spindle contains short astral rays and supernumerary aster-like microtubule arrays [Albertson, 1984; Kemphues et al., 19861. These results suggest that zyg-9 is necessary for the organization of microtubules in the spindle in early embryos. A specialized gene product may be used in early embryos because of the large cell size and the specific polarity and asymmetry of cleavage. The 15-protofilament touch receptor microtubules can be specifically disrupted by mutations in the genes mec-7 and mec-12. This disruption leads to touch insensitivity but no loss of viability [Chalfie and Thomson, 1982; Chalfie and Au, 19891. The mec-7 gene encodes a 6-tubulin isoform that appears to affect the protofilament number of the touch cell microtubules [Savage et al., 19891. In the absence of mec-7 gene activity, the 15protofilament microtubules are missing and are replaced by a smaller number of 1 1-protofilament microtubules [Chalfie and Thomson, 19821. Thus, although other ptubulins can be expressed in the touch cells and allow for process outgrowth, they are insufficient for the production of 15-protofilament microtubules and fail to support mechanosensory transduction. The exact need for 15protofilament microtubules is not understood; because of their extensive crossbridging, however, they may be used for a structural support needed for mechanosensation. Because microtubule assembly requires the interaction of various components, mutant proteins interfering with microtubule assembly would be expected to produce a dominant phenotype. Approximately 60% of mutations in mec-7 are expressed dominantly [Savage et al., 19891. An even larger percentage of mec-12 alleles
Nematode Microtubule Biology
are expressed dominantly or co-dominantly [Chalfie and Au, 19891, suggesting that mec-12 encodes another component of the 15-protofilament touch cell microtubules, perhaps an a-tubulin. Because mutations in both mec-7 and mec-12 result in the loss of the 15-protofilament microtubules, neither product is sufficient to form these structures. The mec-7 tubulin is very similar to other P-tubulins [Savage et al., 19891; only a few amino acid changes may be needed to permit the assembly of 15-protofilament microtubules. Other than differences in the carboxyl terminus, which is heterogeneous among p-tubulins [Little and Seehaus, 1988; Burland et al., 19881, only seven amino acid residues are unique to mec-7. Which of these residues, if any, are important for protofilament number is not known. One change, however, is suggestive: the substitution of a unique cysteine at residue 293. Residue 293 lies in the “hinge” region between the amino- and carboxyterminal domains. A single amino acid change at position 288 in the hinge region in a Drosophila P-tubulin prevents polymerization of the tubulin into functional microtubules without preventing heterodimer or protofilament formation [Rudolph et al., 19871. Unique amino acids are also found in the hinge region in two P-tubules thought to form 11protofilament microtubules (see below). This region may thus be important for interactions between protofilaments. Resistance to benzimidazoles has been used to select mutations in tubulin genes in several organisms [Borck and Braymer, 1974; Sheir-Neiss et al., 1978; Yamamoto, 1980; Burland et al., 1984; Thomas et al., 19851. Wild-type C. elegans grow slowly and are uncoordinated in the presence of benomyl; the ventral nerve cords of these animals have fewer neuronal processes than those of untreated animals [Chalfie and Thomson, 19821. Studies of benomyl sensitivity in C. elegans have shown that only one gene product, the P-tubulin encoded by ben-1 , confers significant benomyl sensitivity to microtubules [Driscoll et al., 19891. Complete elmination of the ben-1 P-tubulin results in viable animals whose only detectable phenotype is resistance to benomyl and other benzimidazoles. These results suggest that while the ben-1 gene product is normally expressed in a large number of neurons, the ben-1 function can be replaced by other P-tubulins in the worm, as might be expected of a member of a gene family. Because all sequenced ptubulins in C. elegans [Gremke, 1986; Driscoll et al., 1989; Savage et al., 19891 contain the aminoterminal residues MREI needed for tubulin autoregulation [Cleveland, 19891, such autoregulation may compensate, at least in part, for the loss of ben-1 tubulin. Examination of the ben-1 P-tubulin sequence suggests residues that may influence its sensitivity to
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benomyl and its incorporation into 1 1 -protofilament microtubules. ben-l protein differs from tub-l and mec-7 at only six positions in addition to the heterogenous carboxyl terminus [Driscoll et al., 19891, but the significance of these changes for benomyl sensitivity is not known. One striking change found in both ben-1 and tub-I [Driscoll et al., 1989; Gremke, 19861, the two sequenced nematode tubulins thought to be incorporated into 1 1-protofilament microtubules, is the substitution of alanine for an otherwise-invariant proline at position 287. As discussed above, this residue lies in the hinge region that may influence interactions between protofilaments. FUTURE DIRECTIONS
The molecular characterization of genes involved in C. elegans microtubule function has only begun. The structure and function of products of sequenced genes, such as mec-7 and ben-1, can now be studied by DNA transformation of in vitro mutagenized genes. Furthermore, mutant genes can be sequenced to determine which amino acid changes lead to a mutant phenotype. In particular, the 54 mutations in mec-7 should represent many ways to disrupt the function of the mec-7 P-tubulin, and the amino acid changes in these mutants may identify protein domains required for function. Other genes cited above, such as those involved in the structure of the ciliary axoneme, remain to be characterized molecularly. In addition, several uncloned C. elegans genes produce phenotypes that suggest a possible involvement in microtubule biology. These genes may be found to encode microtubule components or motors. For example, him-3 and him-6 are candidates for genes required in meiotic chromosome segregation [Hodgkin et al., 19791. Furthermore, /in-5 mutants are defective in post-embryonic nuclear divisions and cytokinesis [Albertson et al., 19781; this gene may thus be required for mitotic chromosome segregation. Another direction for future research will be the identification of genes of known proteins. In particular, the tubulin gene family remains to be completely characterized. Other proteins to study include nematode homologues of microtubule proteins and proteins identified biochemically, such as the cytoplasmic dynein-like motor [Lye et al., 19871 and the cross-linking protein, adligin [Aamodt et al., 19891. In some cases, the candidate gene may have already been identified by mutation. The mec-7 gene, for example, was identified as a P-tubulin gene by the presence of restriction fragment legnth differences in that P-tubulin gene in mec-7 mutants [Savage et al., 19891. Finally, classical genetic approaches to the study of microtubule biology in C. eleguns will also continue to be productive. In many of the systems described above,
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saturation screens for particular behaviors would identify additional alleles and other genes required for each process. Furthermore, in C. elegans it is relatively easy to screen for suppressor mutations; these should provide a way to study components that interact in both microtubule assembly and function. ACKNOWLEDGMENTS
Work in our laboratory was supported by U.S. Public Health Service grants GM-30997 and AI- 19399 to M.C., and a National Science Foundation graduate fellowship to C.S. NOTE ADDED IN PROOF
The unc-104 gene, mutations in which disrupt functional synapse formation, has been found to encode a kinesin-like protein, suggesting that microtubulebased axonal transport may be necessary for synapse formation (Otsuka et al., 1991). REFERENCES Aamodt, E.J., and Culotti, J . G . (1986): Microtubules and microtubule-associated proteins from the nematode Caenorhabditis eleguns: Periodic cross-links connect microtubules in vitro. J. Cell Biol. 103:23-31. Aamodt, E., Holmgren, R., and Culotti, J. (1989): The isolation and in situ location of adligin: the microtubule cross-linking protein from Caenorhubditis elegans. J. Cell Biol. 108:955-963. Albertson, D.G. (1984): Formation of the first cleavage spindle in nematode embryos. Dev. Biol. 101:61-72. Albertson, D.G., Sulston, J.E., and White, J.G. (1978): Cell cycling and DNA replication in a mutant blocked in cell division in the nematode Caenorhabditis elegans. Dev. Biol. 63: 165-178. Borck, K., and Braymer, H.D. (1974): The genetic analysis of resistance to benomyl in Neurospora crassa. J . Gen. Microbiol. 8 5 5 1-56. Burland, T . G . , Schedl, T., Gull, K., and Dove, W.F. (1984): Genetic analysis of resistance to benzimidazoles in Physarum: differential expression of P-tubulin genes. Genetics 108:123-141. Burland, T.G., Paul, E.C.A., Oetliker, M . , and Dove, W.F. (1988): A gene encoding the major P-tubulin of the mototic spindle in Physarum polvcephalum plasmodia. Mol. Cell Biol. 8: 12751281.
Cassada, R., Isnenghi, E., Culotti, M . , and von Ehrenstein, G . (198 I ) : Genetic analysis of temperature-sensitive embryogenesis mutants in Caenorhabditis elegans. Dev. Biol. 84: 193205.
Chalfie. M., and Au, M. (1989): Genetic control of differentiation of the C. eleguns touch receptor neurons. Science 243: 10271033.
Chalfie, M., and Thomson, J.N. (1979): Organization of neuronal microtubules in the nematode Caenorhubditis elegans. J. Cell Biol. 82:278-289. Chalfie. M., and Thomson, J.N. (1982): Structural and functional diversity in the neuronal microtubules of Cuenorhuhditis d o ,qcm.s. J . Cell Biol. 93:15-23.
Cleveland, D.W. (1989): Autoregulated control of tubulin synthesis in animal cells. Cum. Opinion Cell Biol. 1:10-14. Deppe, U., Schierenberg, E., Cole, T., Krieg, C . , Schmitt, D., Yoder, B., and von Ehrenstein, G . (1978): Cell lineages of the embryo of the nematode Caenorhabdifis elegans. Proc. Natl. Acad. Sci. USA 75:376-380. Driscoll, M., Dean, E., Reilly, E., Bergholz, E., and Chalfie, M. ( 1989): Genetic and molecular analysis of Caenorhabditis eleguns p-tubulin that conveys benzimidazole sensitivity. J. Cell Biol. 109:2993-3003. Gremke, L. (1986): “Cloning and Molecular Characterization of the Tubulin Genes of Caenorhabditis eleguns: Nucleotide Sequence Analysis of a Tubulin Gene.” Ph.D. thesis, Northwestern University, Evanston, Illinois. Hedgecock, E.M., Culotti, J.G., Thomson, J.N., and Perkins, L.A. ( 1985): Axonal guidance mutants of Caenorhubditis elegans identified by filling sensory neurons with fluorescein dyes. Dev. Biol. 11 1:158-170. Hiraoka, T., Toda, T., and Yanagida, M. (1984): The NDA3 gene of fission yeast encodes P-tubulin: A cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39:349-358. Hodgkin, J., Horvitz, H.R., and Brenner, S. (1979): Nondisjunction mutants of the nematode Caenorhabditis elegans. Genetics 91: 67-94.
Hyman, A.A., and White, J.G. (1987): Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans. J. Cell Biol. 105:2123-2135. Kemphues, K.J., Raff, E.C., and Kaufman, T.C. (1983): Genetic analysis of P2t, the structural gene for a testis-specific p-tubulin subunit in Drosophila melanogaster. Genetics 105:345356.
Kemphues, K.J., Wolf, N., Wood, W.B., and Hirsh, D. (1986): Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans. Dev. Biol. 113:449-460. Laufer, J.S., Bazzicalupo, P., and Wood, W.B. (1980): Segregation of developmental potential in early embryos of Caenorhabditis elegans. Cell 19569-577. Little, M., and Seehaus, T. (1988): Comparative analysis of tubulin sequences. Comp. Biochem. Physiol. 90:655-670. Lye, R.J., Porter, M.E., Scholey, J.M., and McIntosh, J.R. (1987): Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell 51:309-318. Matthews, K.A., and Kaufman, T.C. (1987): Developmental consequences of mutations in the 84B a-tubulin of Drosophila melanogaster. Dev. Biol. 119:100-112. Otsuka, A.J., Jeyaprakash, A., Garcia-Anoveros, J . , Tang, L.Z., Fisk, G . , Hartshorne, T., Franco, R., and Born, T. (1991): The C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein. Neuron (in press). Perkins, L.A., Hedgecock, E.M., Thomson, J.N., and Culotti, J.G. (1986): Mutant sensory cilia in the nematode Caenorhabditis eleguns. Dev. Biol. 117:456-487. Rudolph, J.E., Kimble, M . , Hoyle, H . D . , Subler, M.A., and Raff, E.C. ( 1987): Three Drosophila P-tubulin sequence opmentally regulated isoform (P3), the testis-speci (p2). and an assembly-defective mutation of the testis-specific isoform (P2t“) reveal both an ancient divergence in metazoan isotypes and structural constraints for P-tubulin function. Mol. Cell Biol. 7:2231-2242. Savage, C.. Hamelin. M.. Culotti. J.G., Coulson, A , . Albertson, D.G., and Chalfie, M . (1989): mec-7 is a P-tubulin gene required for the production of 15-protofilament microtubules in Caenorhubditis elepu7.s. Genes Dev. 3:870-88 I .
Nematode Microtubule Biology Sheir-Neiss, G., Lai, M.H., and Morris, N.R. (1978): Identification of a gene for @-tubulinin Aspergillus niduluns. Cell 15:639647. Thomas, J.H., Neff, N.H., and Botstein, D. (1985): Isolation and characterization of mutation in the 6-tubulin gene of Sacchurornyces cerevisiue. Genetics 1 12:715 -734. Ward, S . , Thomson, N . , White, J.G., and Brenner, S. (1975): Electron microscopical reconstruction of the anterior sensory anatomy of the nematode Caenorhubdiris elegans. J. Comp. Neurol. 160:313-338. Ware, R.W., Clark, D., Crossland, K., and Russell, R.L. ( 1975): The nerve ring of the nematode Cuenorhubdiris eleguns: Sensory input and motor output. J. Comp. Neurol. 162:71-110.
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Weatherbee, J . A . , May, G.S., Gambino, J., and Morris, N.R. (1985): Involvement of a particular species of P-tubulin (P3) i n conidial development of Aspergillus niduluns. J. Cell Biol. 101: 705-7 1 I . Wood, W.B., Hecht, R., Carr, S., Vanderslice, R . , Wolf, N., and Hirsh, D. (1980): Parental effects and phenotypic characterization of mutations that affect early development i n Cuenorhuhditis elegans. Dev. Biol. 74:446-469. Yamamoto, M. (1980): Genetic analysis of resistant mutants to antimitotic benzimidazole compounds in Schizosucchuromyce.s pornbe. Mol. Gen. Genet. 180:231-234.
Views and Reviews Genetic Aspects of Microtubule Biology in the Nematode Caenorhabditis elegans Cathy Savage and Martin Chalfie Department of Biolog~calSnences, Columbia University, New York, New York
INTRODUCTION
Microtubules are required for a variety of cellular processes, including mitosis, meiosis, cell motility, morphogenesis, and, in neurons, neurite outgrowth, axonal transport, and sensory transduction. One approach to the study of microtubule biology is to isolate mutations that disrupt microtubules; these mutations should identify genes and gene products that are important for microtubule structure and/or function. The phenotype resulting from the loss of a particular gene product also gives an indication of the role of that product. Genetic approaches have been particularly useful in the study of microtubules in Drosophila [Kemphues et al., 1983; Matthews and Kaufman, 19871, Aspergillus [Burland et al., 1984; Weatherbee et al., 19851, and yeast [Yamamoto, 1980; Hiraoka et al., 1984; Thomas et al., 19851. In this review we summarize genetic and biochemical studies of microtubule function and structure in the nematode Caenorhabditis elegans. MICROTUBULE STRUCTURE
C. elegans has three structurally distinct types of microtubules: two forms of cytoplasmic microtubules and ciliary microtubules [Chalfie and Thomson, 19821. In contrast to most eukaryotic cells, C. elegans cells do not have cytoplasmic microtubules with 13 protofilaments; the majority of the cytoplasmic microtubules of C. elegans have 1 1 protofilaments. These 1 1 -protofilament microtubules are found in all cells except the six cells which mediate a response to gentle touch. The processes of these touch receptor neurons contain 15-protofilament microtubules. The presence of these two unusual microtubule forms may be characteristic of the phylum, as they are found in other nematodes (Chalfie and Thomson, 1982). Both types of cytoplasmic microtubules are short compared to the length of the neuronal 0 1991 Wiley-Liss, Inc.
processes that contain them [Chalfie and Thomson, 19791. Short microtubules might more easily accommodate changes in cell length, such as those that occur during normal sinusoidal movement of the worms. In addition to cytoplasmic microtubules, some sensory neurons have ciliated endings with axonemes consisting of nine outer doublet microtubules and a variable number, up to seven, of inner singlet microtubules [Ward et al., 1975; Ware et al., 1975; Perkins et al., 19861. These sensory cilia are the only axonemal structures in the nematode (the sperm are amoeboid instead of flagellated). The doublet microtubules are similar to those in other organisms, having 13-protofilament A subfibers and 11-protofilament B subfibers. The inner singlet microtubules are unusual in that they have l l protofilaments [Chalfie and Thomson, 19821. No dynein arms or radial spokes are seen. The axonemes have three structurally identifiable regions [Perkins et al., 19861. In the proximal segment, a central cylinder attaches the outer doublet and inner singlet microtubules, and the doublet microtubules attach to the membrane by Y-shaped links. There is no associated basal body in adults. In the middle segment, the doublet microtubules associate with the membrane and the singlet microtubules appear unattached in the center. The outer segment contains only A subfibers and inner singlet microtubules, and no membrane attachments are found. Several microtubule proteins of C . elegans are being characterized. The nematode contains three to five or-tubulin and three to four P-tubulin genes, some of which have been cloned [Gremke, 1986; Savage et al., 1989; Driscoll et a]., 19891. In contrast to bovine brain Accepted November 6 . 1990 Address reprint requests to Cathy Savage and Martin Chalfie, Department of Biological Sciences. 1012 Sherman Fairchild Center, Columbia Univeristy, New York. N Y 10027.
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microtubule proteins which form 13-14 protofilament microtubules in vitro, nematode microtubule proteins polymerize in vitro to form predominantly 9-1 l protofilament structures [Aamodt and Culotti, 19861, suggesting an inherent difference in the microtubule proteins. Tubulins from the 1 I-protofilament microtubules are likely to contribute most to the pool of microtubule proteins because the 15-protofilament touch cell microtubules derive from only six neurons in an animal with 302 neurons and many dividing germ cells. C. eleguns microtubule proteins include several high molecular weight MAPs and a 32 kd MAP. When these proteins are polymerized in vitro, the microtubules are connected by periodic cross-links formed by microtubule-associated proteins [Aamodt and Culotti, 19861. Adligin, the 32 kd MAP, is the major component of the periodic cross-links; adligin, but not the high molecular weight MAPs, forms periodic cross-links when added to purified nematode tubulins [Aamodt et al., 19891. In addition, a major 400 kd MAP has been isolated and shown to be a cytoplasmic dynein-like motor [Lye et al., 19871. MUTATIONS AFFECTING MICROTUBULE STRUCTURE
Several mutations affecting microtubule structure have been identified in screens for mutants defective in behaviors such as chemotaxis, touch sensitivity, and coordinated movement. Defects in microtubule structure in these mutants have usually been characterized by electron microscopy. Other mutants have been identified by their resistance to the anti-microtubule drug benomyl. The structure of the axonemes of sensory cilia is disrupted in some mutants defective in behaviors such as chemotaxis, osmotic avoidahce, and dauer formation (which requires chemosensory detection of a specific pheromone) [Perkins et al., 19861. Mutations in some genes result in the complete loss of all sensory cilia (duf 1 9 ) or of a subset of cilia (che-10).Other mutations result in shortening of the axoneme, again affecting all cilia (che-2, che-3, che-13, duf-10, osm-I, osm-5, and osm-6) or a subset (osm-3). In addition, in cut-6 mutants, supernumerary microtubules normally at the ends of mechanosensory cilia are found throughout the axoneme. The set of genes affecting sensory cilia has probably not been saturated, however, since many of these genes are represented by only one or a few alleles. Also, many proteins found in the axoneme may not be specific to these structures, and mutants would therefore be pleiotropic. unc-33 may be an example of a gene that is required for microtubules in both sensory cilia and neuronal processes [Hedgecock et al., 19851. In unc-33 mutants sensory cilia have elevated numbers of microtu-
bules, many of which have structural abnormalities. Larger-diameter microtubules, microtubules with hooks, and doublet and triplet microtubules are all seen. Since these mutants seem to be overproducing microtubules, the wild-type unc-33 product is thought to regulate the stability or association of microtubule proteins. Neuronal processes are misdirected in these mutants, probably causing the uncoordinated phenotype. Although microtubule organization has not been examined in neuronal processes, similar microtubule defects may be the cause of the outgrowth phenotype. The organization of cleavage divisions in early development also depends on microtubule function [Hyman and White, 19871. The first cell divisions in C. eleguns follow a strict pattern of polarity and asymmetry [Deppe et al., 1978; Laufer et al., 19801 and the large cell size of the early embryo requires a larger spindle [Albertson, 19841. Several mutations disrupt the pattern of the first cleavage [Wood et al., 1980; Cassada et al., 19811; mutations in the gene zyg-9 particularly affect microtubule organization [Kemphues et al., 19861. In zyg-9 mutants, the first cleavage spindle contains short astral rays and supernumerary aster-like microtubule arrays [Albertson, 1984; Kemphues et al., 19861. These results suggest that zyg-9 is necessary for the organization of microtubules in the spindle in early embryos. A specialized gene product may be used in early embryos because of the large cell size and the specific polarity and asymmetry of cleavage. The 15-protofilament touch receptor microtubules can be specifically disrupted by mutations in the genes mec-7 and mec-12. This disruption leads to touch insensitivity but no loss of viability [Chalfie and Thomson, 1982; Chalfie and Au, 19891. The mec-7 gene encodes a 6-tubulin isoform that appears to affect the protofilament number of the touch cell microtubules [Savage et al., 19891. In the absence of mec-7 gene activity, the 15protofilament microtubules are missing and are replaced by a smaller number of 1 1-protofilament microtubules [Chalfie and Thomson, 19821. Thus, although other ptubulins can be expressed in the touch cells and allow for process outgrowth, they are insufficient for the production of 15-protofilament microtubules and fail to support mechanosensory transduction. The exact need for 15protofilament microtubules is not understood; because of their extensive crossbridging, however, they may be used for a structural support needed for mechanosensation. Because microtubule assembly requires the interaction of various components, mutant proteins interfering with microtubule assembly would be expected to produce a dominant phenotype. Approximately 60% of mutations in mec-7 are expressed dominantly [Savage et al., 19891. An even larger percentage of mec-12 alleles
Nematode Microtubule Biology
are expressed dominantly or co-dominantly [Chalfie and Au, 19891, suggesting that mec-12 encodes another component of the 15-protofilament touch cell microtubules, perhaps an a-tubulin. Because mutations in both mec-7 and mec-12 result in the loss of the 15-protofilament microtubules, neither product is sufficient to form these structures. The mec-7 tubulin is very similar to other P-tubulins [Savage et al., 19891; only a few amino acid changes may be needed to permit the assembly of 15-protofilament microtubules. Other than differences in the carboxyl terminus, which is heterogeneous among p-tubulins [Little and Seehaus, 1988; Burland et al., 19881, only seven amino acid residues are unique to mec-7. Which of these residues, if any, are important for protofilament number is not known. One change, however, is suggestive: the substitution of a unique cysteine at residue 293. Residue 293 lies in the “hinge” region between the amino- and carboxyterminal domains. A single amino acid change at position 288 in the hinge region in a Drosophila P-tubulin prevents polymerization of the tubulin into functional microtubules without preventing heterodimer or protofilament formation [Rudolph et al., 19871. Unique amino acids are also found in the hinge region in two P-tubules thought to form 11protofilament microtubules (see below). This region may thus be important for interactions between protofilaments. Resistance to benzimidazoles has been used to select mutations in tubulin genes in several organisms [Borck and Braymer, 1974; Sheir-Neiss et al., 1978; Yamamoto, 1980; Burland et al., 1984; Thomas et al., 19851. Wild-type C. elegans grow slowly and are uncoordinated in the presence of benomyl; the ventral nerve cords of these animals have fewer neuronal processes than those of untreated animals [Chalfie and Thomson, 19821. Studies of benomyl sensitivity in C. elegans have shown that only one gene product, the P-tubulin encoded by ben-1 , confers significant benomyl sensitivity to microtubules [Driscoll et al., 19891. Complete elmination of the ben-1 P-tubulin results in viable animals whose only detectable phenotype is resistance to benomyl and other benzimidazoles. These results suggest that while the ben-1 gene product is normally expressed in a large number of neurons, the ben-1 function can be replaced by other P-tubulins in the worm, as might be expected of a member of a gene family. Because all sequenced ptubulins in C. elegans [Gremke, 1986; Driscoll et al., 1989; Savage et al., 19891 contain the aminoterminal residues MREI needed for tubulin autoregulation [Cleveland, 19891, such autoregulation may compensate, at least in part, for the loss of ben-1 tubulin. Examination of the ben-1 P-tubulin sequence suggests residues that may influence its sensitivity to
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benomyl and its incorporation into 1 1 -protofilament microtubules. ben-l protein differs from tub-l and mec-7 at only six positions in addition to the heterogenous carboxyl terminus [Driscoll et al., 19891, but the significance of these changes for benomyl sensitivity is not known. One striking change found in both ben-1 and tub-I [Driscoll et al., 1989; Gremke, 19861, the two sequenced nematode tubulins thought to be incorporated into 1 1-protofilament microtubules, is the substitution of alanine for an otherwise-invariant proline at position 287. As discussed above, this residue lies in the hinge region that may influence interactions between protofilaments. FUTURE DIRECTIONS
The molecular characterization of genes involved in C. elegans microtubule function has only begun. The structure and function of products of sequenced genes, such as mec-7 and ben-1, can now be studied by DNA transformation of in vitro mutagenized genes. Furthermore, mutant genes can be sequenced to determine which amino acid changes lead to a mutant phenotype. In particular, the 54 mutations in mec-7 should represent many ways to disrupt the function of the mec-7 P-tubulin, and the amino acid changes in these mutants may identify protein domains required for function. Other genes cited above, such as those involved in the structure of the ciliary axoneme, remain to be characterized molecularly. In addition, several uncloned C. elegans genes produce phenotypes that suggest a possible involvement in microtubule biology. These genes may be found to encode microtubule components or motors. For example, him-3 and him-6 are candidates for genes required in meiotic chromosome segregation [Hodgkin et al., 19791. Furthermore, /in-5 mutants are defective in post-embryonic nuclear divisions and cytokinesis [Albertson et al., 19781; this gene may thus be required for mitotic chromosome segregation. Another direction for future research will be the identification of genes of known proteins. In particular, the tubulin gene family remains to be completely characterized. Other proteins to study include nematode homologues of microtubule proteins and proteins identified biochemically, such as the cytoplasmic dynein-like motor [Lye et al., 19871 and the cross-linking protein, adligin [Aamodt et al., 19891. In some cases, the candidate gene may have already been identified by mutation. The mec-7 gene, for example, was identified as a P-tubulin gene by the presence of restriction fragment legnth differences in that P-tubulin gene in mec-7 mutants [Savage et al., 19891. Finally, classical genetic approaches to the study of microtubule biology in C. eleguns will also continue to be productive. In many of the systems described above,
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saturation screens for particular behaviors would identify additional alleles and other genes required for each process. Furthermore, in C. elegans it is relatively easy to screen for suppressor mutations; these should provide a way to study components that interact in both microtubule assembly and function. ACKNOWLEDGMENTS
Work in our laboratory was supported by U.S. Public Health Service grants GM-30997 and AI- 19399 to M.C., and a National Science Foundation graduate fellowship to C.S. NOTE ADDED IN PROOF
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