Mutations Affecting Microtubule Structure in Caenorhabditis Elegans

M A R T I N C H A L F I E 0 E L L E N D E A N 0 E V E L Y N R E I L L Y 0 K A Y B U C K 0 t 0 n 0 J . N IC H O L T H O M S O N 0 0 1D epartm ent o f Biological Sciences, Columbia University, N ew York, N Y 10027, USA 2M RC Laboratory o f M olecular Biology , Hills Road, Cambridge CB2 2QH, U K S U M M A R Y Three types of microtubules are seen in the neuronal processes of the nematode Caenorhabditis elegans. Single cytoplasmic microtubules of most neurones have 11 protofilaments whereas those of six touch receptor cells have IS protofilaments. The axonemes of sensory cilia have nine outer doublets with a variable number (up to seven) of singlet microtubules. Mutations in 11 genes affect the appearance of these microtubules. I N T R O D U C T I O N M. Chalfie a nd others et al. 1975; Ware et al. 1975; Perkins et al. 1986). T h e doublet microtubules have A subfibres with 13 protofilaments and B subfibres with 11 protofilaments; the singlet m icrotubules have 11 protofilaments (Chalfie & Thom son, 1982). Dynein arms, nexin links and radial spokes are not seen. Perkins et al. (1986) have described the sensory cilia in great detail. T h e axonemes in adults have three structurally identifiable regions (Fig. 3). In the most proximal region, the transition zone, the doublets are attached to the plasma membrane by Y-shaped links; the singlet microtubules are attached to the inside of a central cylinder that also links the doublets. T h ere is no associated basal body in adults. In the next axonemal region, the middle segment, the diameter of the ring of doublets is larger, and the doublets are directly associated with the plasma membrane. T h e singlets appear unattached in the centre of the axoneme. In the most distal, or term inal, segment there are no doublets; only the A subfibres and the singlets remain. Perkins et al. (1986) noted differences among the axonemes of different neurones. In some axonemes only four or five of the doublets extend through the length of the cilium ; in others the doublets are filled with electron-dense material. Still other axonemes are associated with supernumerary microtubules or striated rootlets. T h e significance of these structural differences and of the variable number of inner singlet microtubules is not known. Fig. 1. Electron micrographs of cytoplasmic microtubules in C. elegans. Preparations were stained with tannic acid to reveal the protofilament structure of microtubules in a ventral cord neurone (a) and a touch cell (b). X 1 600 00. Inset, X 6 0 000 0. (Reproduced from J . Cell Biol. 93, 1 5 -2 3 , by copyright permission of The Rockefeller University Press.) not associate with one another and do not maintain a set spatial relationship with each other. Because of their orderly arrangement, the 15-protofilament microtubules can be followed easily in electron micrographs of serial sections. These microtubules do not span the entire length of the neuroneal process: the touch cell process is 400500 /im long whereas the microtubules are 10-20 pim. long (Chalfie & Thomson, 1982). (The 11-protofilament microtubules are also short compared to the length of the neuroneal processes that contain them.) We have postulated that such short microtubules could slide relative to each other and thus permit stable microtubule organization in the face of changes in cell length (such as those that probably occur during the sinusoidal bending of the animal). Interestingly, the end of the 15-protofilament microtubule that is distal to the cell body is always found on the outside of the bundle of microtubules; the proximal end is preferentially found within the bundle. T he proximal and distal ends also differ structurally (Fig. 4). The significance of the closeness of the distal microtubule end to the plasma membrane is not known. D R U G S A F F E C T I N G N E U R O N A L M I C R O T U B U L E S I N C. E L E G A N S A number of anti-mitotic drugs have been tested on C. elegans. A striking effect is seen when animals are grown in the presence of benomyl or other benzamidazole carbamates. Such animals are paralysed, contorted, and grow slowly (Fig. 5; Chalfie & Thomson, 1982). T h e ventral nerve cords of these animals have fewer neuroneal processes than those of untreated animals. Presumably the drugs interfere with microtubule stability and, thereby, prevent process extension. Experiments with benomyl-resistant mutants (see below) support this view. In contrast, the 15-proto filament microtubules of the touch cells appear to be unaffected by benomyl at concentrations that cause great reduction in process outgrowth in the ventral cord neurones. Fig. 3. Diagram of a C. elegans cilium. Cross-sections at three intervals are shown. See the text for details. Dotted lines indicate the extent of axonemes in various mutants. Fig. 2. Tannic acid fixation of the ciliated endings of amphidial neurones. These neurones are putative chemosensory cells in the head of the nematode. Doublet (d ) and singlet (s) microtubules as well as microtubules with hooks (h ) are seen. These latter microtubules are doublets that are in the process of losing their B subfibres. The resulting microtubule after the loss of the B subfibre has 13 protofilaments (a). X 2 700 00. (Reproduced from J . Cell Biol. 93, 1 5 -2 3 , by copyright permission of The Rockefeller University Press.) T h e third microtubule form, a cytoplasmic microtubule with 15 protofilaments, is found only in the six neurones that serve as receptors for gentle touch (Fig. 1; Chalfie & Thom son, 1982). Only rarely (about one in 450) is an 11-protofilament microtubule seen in the processes of these touch cells. T h e 15-proto filament m icro tubules differ from the 11-protofilament microtubules in several respects: (1) they are less cold-labile; (2) they are stable during osmium fixation; (3) they are differ entially sensitive to anti-microtubule drugs; and (4) they are differentially disrupted by mutation (see below). Moreover, the arrangement of the 15-protofilament m icro tubules within the cell is more orderly than that of the 11-protofilament m icro tubules. T h e larger microtubules form bundles in which each microtubule maintains its relative position. T h e smaller micro tubules found in other C. elegans neurones do Fig. 4. Arrangement of microtubules in the touch cell process. Two sets (a -e and f-k ) of serial sections are shown, anterior (distal) to the right. Distal ends of the microtubules (2 -5 ) are always found on the outside of the microtubule bundle and are often associated with diffusely staining material that appears to contact the plasma membrane. Proximal ends of the microtubules (1 and 6) are found preferentially within the microtubule bundle and often haveafilled appearance. X 116000. (Reproduced from J . Cell Biol. 93, 15 -23 , by copyright permission of T he Rockefeller University Press.) Colchicine has a strikingly different effect on C. elegans. Animals grown in 0-52-0m M -co (...truncated)