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~cal Snences, Columbia University, New York, New York

INTRODUCTION

Microtubules are required for a variety of cellular processes, including mitosis, meiosis, cell motility, mor- phogenesis, 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 microtu- bule 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 micro- tubule function and structure in the nematode Cae- norhabditis 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 protofila- ments; the majority of the cytoplasmic microtubules of C. elegans have 1 1 protofilaments. These 1 1 -protofila- ment microtubules are found in all cells except the six cells which mediate a response to gentle touch. The pro- cesses of these touch receptor neurons contain 15-proto- filament microtubules. The presence of these two un- usual microtubule forms may be characteristic of the phylum, as they are found in other nematodes (Chalfie and Thomson, 1982). Both types of cytoplasmic micro- tubules are short compared to the length of the neuronal

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processes that contain them [Chalfie and Thomson, 19791. Short microtubules might more easily accommo- date changes in cell length, such as those that occur during normal sinusoidal movement of the worms.

In addition to cytoplasmic microtubules, some sen- sory neurons have ciliated endings with axonemes con- sisting 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 flag- ellated). The doublet microtubules are similar to those in other organisms, having 13-protofilament A subfibers and 1 1-protofilament B subfibers. The inner singlet mi- crotubules are unusual in that they have l l protofila- ments [Chalfie and Thomson, 19821. No dynein arms or radial spokes are seen. The axonemes have three struc- turally 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 unat- tached in the center. The outer segment contains only A subfibers and inner singlet microtubules, and no mem- brane attachments are found.

Several microtubule proteins of C . elegans are be- ing 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, Depart- ment of Biological Sciences. 101 2 Sherman Fairchild Center, Colum- bia Univeristy, New York. N Y 10027.

160 Savage and Chalfie

microtubule proteins which form 13-14 protofilament microtubules in vitro, nematode microtubule proteins po- lymerize in vitro to form predominantly 9-1 l protofila- ment structures [Aamodt and Culotti, 19861, suggesting an inherent difference in the microtubule proteins. Tu- bulins from the 1 I-protofilament microtubules are likely to contribute most to the pool of microtubule proteins because the 15-protofilament touch cell microtubules de- rive 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 micro- tubule-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 co- ordinated movement. Defects in microtubule structure in these mutants have usually been characterized by elec- tron 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, su- pernumerary microtubules normally at the ends of mech- anosensory 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 rep- resented by only one or a few alleles. Also, many pro- teins 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 re- quired for microtubules in both sensory cilia and neu- ronal 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 micro- tubule 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 de- velopment also depends on microtubule function [Hy- man 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 ar- rays [Albertson, 1984; Kemphues et al., 19861. These results suggest that zyg-9 is necessary for the organiza- tion 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 insen- sitivity 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 15- protofilament microtubules are missing and are replaced by a smaller number of 1 1 -protofilament microtubules [Chalfie and Thomson, 19821. Thus, although other p- tubulins can be expressed in the touch cells and allow for process outgrowth, they are insufficient for the produc- tion of 15-protofilament microtubules and fail to support mechanosensory transduction. The exact need for 15- protofilament microtubules is not understood; because of their extensive crossbridging, however, they may be used for a structural support needed for mechanosensa- tion.

Because microtubule assembly requires the inter- action of various components, mutant proteins interfer- ing 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 161

benomyl and its incorporation into 1 1 -protofilament mi- crotubules. ben-l protein differs from tub-l and mec-7 at only six positions in addition to the heterogenous car- boxyl terminus [Driscoll et al., 19891, but the signifi- cance 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 protofil- aments.

are expressed dominantly or co-dominantly [Chalfie and Au, 19891, suggesting that mec-12 encodes another com- ponent 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-tubu- lins [Savage et al., 19891; only a few amino acid changes may be needed to permit the assembly of 15-protofila- ment microtubules. Other than differences in the car- boxyl terminus, which is heterogeneous among p-tubu- lins [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 proto- filament 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 polymeriza- tion of the tubulin into functional microtubules without preventing heterodimer or protofilament formation [Ru- dolph et al., 19871. Unique amino acids are also found in the hinge region in two P-tubules thought to form 11- protofilament microtubules (see below). This region may thus be important for interactions between protofila- ments.

Resistance to benzimidazoles has been used to se- lect 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 unco- ordinated 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 mi- crotubules [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 p- tubulins in C. elegans [Gremke, 1986; Driscoll et al., 1989; Savage et al., 19891 contain the aminoterminal residues MREI needed for tubulin autoregulation [Cleve- land, 19891, such autoregulation may compensate, at least in part, for the loss of ben-1 tubulin.

Examination of the ben-1 P-tubulin sequence sug- gests residues that may influence its sensitivity to

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. Further- more, 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-tubu- lin, 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 char- acterized molecularly. In addition, several uncloned C. elegans genes produce phenotypes that suggest a possi- ble involvement in microtubule biology. These genes may be found to encode microtubule components or mo- tors. 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 cyto- kinesis [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 mi- crotubule 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 pres- ence 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,

162 Savage and Chalfie

saturation screens for particular behaviors would identify additional alleles and other genes required for each pro- cess. 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 microtu- bule assembly and function.

ACKNOWLEDGMENTS

Work in our laboratory was supported by U.S. Pub- lic Health Service grants GM-30997 and AI- 19399 to M.C., and a National Science Foundation graduate fel- lowship 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 microtubule- based axonal transport may be necessary for synapse formation (Otsuka et al., 1991).

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