Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans

Cell, Vol. 65, 837447,

May 31,

1991, Copyright

0 1991 by Cell Press

Kinesin-Related Gene uric-104 Is Required for Axonal Transport of Synaptic Vesicles in C. elegans David H. Hall’ and Edward M. Hedgecocktt *Department of Neuroscience Albert Einstein College of Medicine Bronx, New York 16461 tDepartment of Cell and Developmental Biology Roche Institute of Molecular Biology Nutley, New Jersey 07110 *Department of Biology Johns Hopkins University Baltimore, Maryland 21216

Summary uric-104 encodes a novel kinesin paralog that may act as a microtubule-based motor In the nervous system. Neuronal cell lineages and axonogenesis are normal in uric-704 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC104 Is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal mlcrotubules. Other membrane-bounded organelles are transported normally.

have been isolated from all animal phylaexamined. Arthropod and mollusk kinesin heavy chains have been sequenced from cDNAs and prove to be highly similar along their entire lengths (Kosik et al., 1990). Tentatively, the kinesins isolated from various animal phyla are orthologs, i.e., homologs with conserved function that arose by speciation. Molecular cloning of genes from fungi and animals has revealed a superfamily of kinesin-related proteins sharing only the motor domain of the kinesin heavy chain (Rose, 1991). These proteins are kinesin paralogs, i.e., homologs with divergent functions that arose by gene duplication (perhaps with exon shuffling), rather than orthologs. Several kinesin paralogs are implicated in microtubule-based translocation of chromosomes, microtubules, or spindle pole bodies. The uric-104 gene of the nematode Caenorhabditis elegans encodes a novel kinesin paralog with a kinesin-like motor domain at its N-terminus but with no further homology to known members of the kinesin superfamily (Otsuka et al., 1991). By inference, UNC-104 works as a microtubule-based molecular motor in the nervoussystem. Here we describe the behavioral and cellular phenotypes of uric-104 mutants. Based on these phenotypes, we propose that UNC-104 is a neuron-specific motor protein used for anterograde axonal transport of synaptic vesicles but not other classes of MBOs.

Introduction Neurons communicate by extending long cytoplasmic processes (i.e., axons and dendrites) that form synapses with distant cells. Genesis and maintenance of these neurites pose several problems of intracellular transport. Many materials are made only within the cell body and must be transported anterograde along the neurite before final assembly. For example, membrane and secreted proteins are sorted into several types of vesicles (i.e., lysosomes, constitutive vesi$les, synaptic vesicles, secretory granules)via the trans Golgi network within the cell body. Similarly, mitochondria may replicate within the cell body. These membrane-bounded organelles (MBOs) are then translocated along axonal microtubules to their sites of utilization. Although diverse MBOs move at comparable speeds, it is unknown whether all MBOs share a common microtubule-dependent motor protein for fast axonal transport (see Vallee and Shpetner, 1996). Kinesin, a multimeric enzyme isolated from many cell types including axoplasm, can translocate artificial organelles along microtubules in vitro (see Vallee and Shpetner, 1996). The speed and direction of kinesin-mediated movement are consistent with a role in anterograde axonal transport in vivo. Motor activity requires only the microtubule-dependent ATPase domain at the N-terminus of the kinesin heavy chain (Yang et al., 1996). Other regions of the heavy chain are responsible for heavy chain dimerization, assembly with light chains, and perhaps docking with MBOs. Motor proteins with similar biochemical properties

Results The origins and behavioral phenotypes of 17 recessive uric-704 alleles are summarized in Table 1. These mutations form a single allelic series for all parameters examined. Moderate alleles (e.g., 87265) have paralyzed locomotion but robust feeding, growth, and reproduction in laboratory culture. More severe alleles (e.g., rh43) have slowed feeding, and poor growth and reproduction as a consequence. Null alleles (e.g., rh742) have only sporadic, ineffectual feeding and defecation. Because most homozygotes arrest before maturity, these strains are maintained only as heterozygotes. The development, behavior, and neuronal ultrastructure of these representative alleles are compared in detail below. Embryogenesis, Larval Growth, and Adult Fertility uric-704 is dispensable for embryogenesis, but mutant larvae are paralyzed and grow poorly after hatching. Maternal gene expression, if it occurs, is not required for wildtype behavior in larvae carrying an uric-704(+) allele, nor does it restore any wild-type behavior in homozygous mutant larvae. Whereas moderate alleles (e.g., e7265) are more severe when placed opposite a deletion of the unc704 region, the subviable allele rh742 is not further enhanced (Sigurdson et al., 1964). Therefore, rh742, and perhaps the other subviable alleles, are likely to be null alleles (Table 1).

Cell 030

Table

1. Comparison

of uric-104

Alleles

Allele”

Origir?

LocomotionC

Wild type e1265 rhl23rv 91265 rhl24rv rh43 rhl26rv, rh43 rhl271-v el265 rhl25tv rh43 rhl28rv, rh1077 rhlO43 e1265 rhlOl6 rhl41 e2164 rh43 rh142 rhl40 rh143 rh144

W4

3 3 3 3 3 2 2 2 1 1 1 1 1 1 0 0 0 0

hs

cs

EMS EMS EMS EMS EMS SPONT NJ62 Rw7097 NG NJ82 EMS TR679 EMS EMS EMS EMS EMS

Feeding Rated (pumpslmin) 123 f

I5

128 115 113 72 64 63 34 10

23 23 14 22 24 27 22 10

f f f f f f f *

Defecation (seconds)

Period’ Brood Size’

47 + 2

246 f

40 (22)

55 f

2

160 f

81 (8)

76 f 136 f -

8 54

75 f 56(11) Subviable Subviable Subviable Subviable

-

-

-

’ Alleles e1265 and 82184 were obtained from D. Riddle (University of Missouri-Columbia) and M. Shen (MRC Laboratory of Molecular Biology), respectively. Isolation of intragenic revertants and subviable alleles is described in the Experimental Procedures. Abbreviations: rv, revertant; hs, heat sensitive; cs, cold sensitive. b Forward mutations were induced by nitrosoguanidine (NG), EMS, or arose spontaneously in transposon-active strains (NJ82, RW7097, TR679) derived from BristollSergerac hybrids (Collins et al.. 1987; Mori et al., 1966). Three spontaneous alleles (e2184, rh1016, rhlO17) are known to be caused by insertions of a Tel transposon (Dtsuka et al., 1991). ’ Relative locomotory abilities were ranked by subjective comparison with four strains: 3 = wild-type movement (N2); 2 = mildly uncoordinated (rh1017); 1 = severely uncoordinated (e7265); 0 = paralyzed (rh142). d Mean feeding rates f SD for L4 larvae (n = 10) observed during 100 consecutive pharyngeal pumps or for 1 min if shorter. ’ Mean periods between defecations f SD were determined for L4 larvae (n = 3) from the time elapsed between 11 consecutive defecations. ’ Mean brood sizes f SD for hermaphrodites cultured at 20%. Sample sizes shown in parentheses.

Wild-type larvae mature rapidly and synchronously at 20°C (IV 2 days from hatch to adult). Most 87265 larvae also mature rapidly (-2-3 days), but r/743 larvae mature significantly more slowly and asynchronously (~3-6 days). While some r/7742 larvae arrest at each stage, about lo%-20% eventually reach adult (~10-15 days) and sometimes reproduce (discussed below). Adults of all three uric-704 alleles are significantly smaller than wild type. A survey of adult anatomy, including the nervous system and musculature, by differential interference contrast (DIC) microscopy revealed no abnormalities in cell numbers or positions, suggesting that embryonic and larval lineages are normal (Sulston and Horvitz, 1977; Sulston et al., 1983). Wild-type adults laid 160-310 fertilized eggs each, 87265 laid 120-270 eggs, rh43 laid 30-170 eggs, and rh742 laid only O-l 1 eggs in unpaired studies of eight or more broods (see Table 1 for averages). (The linked dpy-70 mutation present in rh742 strains may exacerbate some defects. However, a rare non-Dpy Uric recombinant also matured slowly [ml 0 days] and produced but 1 egg.) Rareeggsobtained from r/1742 homozygotes were indistinguishable from mutant eggs obtained from rh742/+ heterozygotes, i.e., each egg embryonated and most hatched, but larvae developed extremely slowly. Because most larvae arrest before adult, such occasional small broods are insufficient to maintain a homozygous strain. Finally, fh742/mnDf30 heterozygotes were qualitatively similar to rh742 homozygotes. In particular, 2 out of 10

larvae examined reached adult, and one hermaphrodite produced two viable embryos. Behavioral Defects of uric-104 Mutants We examined two embryonic and three larval/adult motor behaviors to establish the scope of possible neural and muscular defects in uric-704 mutants. Embryonic Muscle Contractions Regular synchronous contractions of the body muscles during embryonic elongation ensure that myofilaments align along the longitudinal axis of the hypodermis (Priess and Hirsh, 1986; Venolia and Waterston, 1990). These muscles begin contracting at 430 min, shortly after the onset of elongation (Sulston et al., 1983). Although their movements are small and localized at first, by 460 min (Bfold elongation) all dorsal or ventral muscles contract synchronously and vigorously. These contractions alternate regularly between dorsal and ventral muscles (up to 5 per minute) and continue throughout elongation (Figure 1). Because the embryo is folded within its rigid eggshell, each longitudinal contraction creates torque, which flips the embryo 180“ about its anteroposterior axis. At the onset of cuticle synthesis, flipping declines abruptly and the larval locomotor pattern emerges. In rh742, embryonic contractions closely resemble wild type. In particular, the rates of flipping are comparable throughout elongation. Hatching About 1 hr before hatching, the gl glands of the pharynx synthesize and transport secretory granules along their

Mutant 939

Transport

of Synaptic

Vesicles

FLIPS 1

0 3 --

PW

2

MINu!rE

3

4

5

6

in

o

wild

type

.

+/+

& rhl42/+

X

rh142/rh142

q

wild

FLIPPING

PUMPING

c

hmc

--

gl

glands

un 0 1. Rates

of Flipping

dies

active

10 PUNPING

Figure

horn&g

type

20

CYCLES

and Pumping

30

PER MINUTE during

Embryogenesis

Flipping and pumping rates were determined for a single wild-type embryo (open circles) by counting events in 5 or 10 min intervals, respectively. Flippingratesweredeterminedforacohortof 23embryos from a hermaphrodite of genotype uric-104 (fb742) dpy-lO/dpy-2 by timing the interval between six consecutive flips. Non-Uric embryos (16 of 23; closed circles) and Uric embryos (7 of 23; “exes”) are plotted separately. Onsets of pumping and hatch are normal in non-Uric embryos, but pumping does not occur and hatch is delayed in Uric embryos (see Results). Marker events are described in Sulston et al. (1963). Neurulation is th: ingression of neuroblasts and concomitant spread of hypodermis over the ventral surface of the embryo.

processes (Singh and Sulston, 1978; Sulston et al., 1983). About 30 min before hatching, pharyngeal muscles begin contracting at 1O-l 5 cpm; each pumping cycle comprises simultaneous contraction of the corpus and terminal bulb, followed by contraction of the isthmus. The intestine, initially collapsed, quickly becomes distended with ingested fluid. This extraembryonic fluid appears to recirculate through the alimentary tract by a cycle of ingestion, and presumably release through the rectum, that continues until hatching (Figure 1). Wild-type eggshells invariably rupture within 20-30 min after the onset of pumping. In most rh742 embryos, the pharynx never contracts (7 of 9 cases) or quivers only briefly (1 of 9) and the intestine remains undistended. In one embryo (1 of 9) however, one or more complete pumping cycles occurred and the

intestine became slightly distended. Generally, hatching is delayed by 30-l 20 min and some 10% of mutant larvae fail to ever rupture their eggshell. Locomotion The instantaneous body posture, a sine wave of slightly over 1 wavelength, is determined by localized, antiphasic contractions of the dorsal and ventral body muscles (Figure 2). During forward and reverse locomotion, bends propagate posteriorly or anteriorly, respectively, along the body at up to 30 cpm. On surfaces without slippage, the body traces a sinusoidal path. In mild alleles, the regular alternation between regions of contraction and relaxation is incomplete. For example, a region of dorsal contraction may be followed by only a shallow relaxation before another region of contraction. During locomotion, bends propagate slowly and decline in amplitude. In severe alleles, both spatial and temporal alternation fail entirely. Animals assume tightly coiled postures, with all dorsal orventral musclescontracted simultaneously, for several minutes at a time (Figure 2). Feeding The pharynx is a muscular organ joining mouth and intestine that ingests suspended bacteria and delivers them to the intestine (Albertson and Thomson, 1976). A feeding cycle comprises a simultaneous contraction and then relaxation of the corpus and terminal bulb (called pumping), followed (optionally) by a peristaltic contraction of the isthmus (Avery and Horvitz, 1989). When bacteria are present, the rate of feeding in wild type is fast and constant (ml20 pumps/min). In severe alleles, e.g., rh43 and rh742, the rate is both slow and irregular (Table 1). Within each feeding cycle, however, muscle contractions appear normal, and some bacteria reach the intestine. Defecation During defecation, posterior body muscles, anterior body muscles, and then expulsion muscles contract in rapid succession (Thomas, 1990). In expulsion, the intestinal muscles contract, forcing material into the rectum via a dilated sphincter, and then the anal depressor muscle contracts, releasing material into the medium. The complete motor sequence of defecation takes less than 2 s. When food is present, defecation occurs regularly at about 50 s intervals. In e7265 and rh43, the sequence of contractions during defecation is usually normal. Defecations occur regularly, but the period between them is abnormally long (Table 1). In rh742, posterior body contractions are often not followed by the rest of the motor sequence. These attempts are infrequent and irregular, and their mean period varies greatly between individuals.

Neuroanatomical Defects of uric-704 Mutants Axon Growth and Guidance A survey of axon tracts by electron microscopy, including longitudinal and circumferential nerves and nerve ring, revealed no gross abnormalities in axon positions or numbers, suggesting that axonal growth and guidance are largely normal (compare White et al., 1986). Additional surveys of some 20 classes of neurons by various light

Cell 840

Figure

2. Larval

DIC micrographs

Body Postures of Ll larvae

illustrating

sinusoidal

([A], wild type) and coiled

microscopic methods for possible defects in axonal growth or guidance revealed only three affected pairs. PVP neurons are positioned on the ventral hypodermis at the posterior limit of the developing nerve cord. In wildtype embryos, PVPL and PVPR axons decussate and grow anteriorly along the ventral hypodermis, pioneering the major (right) and minor (left) fascicles of the ventral nerve cord, respectively (Durbin, 1987). In severe alleles, these axons occasionally fail to separate and instead run anteriorly as a single fascicle (H. Bhatt and E. M. H., unpublished data). AVMlPVM and PDE neurons are positioned on the lateral hypodermis in midbody. In wild-type larvae, AVMl PVM and PDE axons grow directly toward the ventral nerve cord. In rh43, about 50% of AVMlPVM axons and 10% of PDE axons grow longitudinally along the lateral hypodermis rather than ventrally. Axons that reach the ventral midline, however, fasciculate and grow along the ventral nerve cord normally. Chemical Synapses Synapses occur at axonal varicosities (boutons en passant) between parallel axons in the nerve cords and ring (White et al., 1978, 1986; Hall and Russell, 1991). Most boutons, or varicosities, are densely packed with transmitter vesicles clustered near the active zone. Several morphological classes of vesicles with distinctive size, shape, and contents have been distinguished, but small spherical vesicles (~36 nm diameter) with clear cores are most common. The active zone is identified by a dark cytoplasmic tuft attached to the presynaptic membrane (Figure 3). USUally, there are one or two postsynaptic axons. The cleft

postures

([B], uric-704

[rh742]).

Bar = 10 pm.

and postsynaptic membrane generally have no overt specialization. Both the number of vesicles and the size of the active zone vary significantly among synapses. nerve cords and ring, axons have few synapIn uric-104 tic vesicles and no large varicosities. Instead, axons have small constant caliber, resembling bouton-free regions of wild-type axons (Figure 3). Vesicles were counted in a sampling of axon profiles from nerve rings of new adults (see Experimental Procedures). All profiles with ten or more vesicles were arbitrarily classed together, namely, bouton-like, for purposes of quantitation. While 42% (n = 128) of all axon profiles in wild type are bouton-like, only 14% (n = 202) in rh43 reach this criterion. In wild type, the mean number f standard deviation (SD) of vesicles is 41 f 51 in bouton-like profiles and 3.7 f 2.5 elsewhere. In rh43, these means are 17 f 12 and 3.2 f 2.3, respectively. have large surfeits of Neuron cell bodies in uric-704 small vesicles (30-50 nm) resembling synaptic vesicles but with generally denser cores (Figure 4). Although these cytoplasmic vesicles are clustered together as in synaptic boutons, they are not associated with the cell membrane and no synapse is formed. Vesicles were counted in a sampling of soma profiles from the lateral ring ganglia of new adults. The mean number f SD of vesicles per soma profile is only 15 f 22 (n = 98) in wild type but 71 f 49 (n = 106) in rh43. Mutant axons form few synapses, and these have smaller active zones and fewer vesicles than normal. No active zones were observed, however, without some vesicles nearby. Based on sampling new adults, the mean

Mutant 341

Figure

Transport

3. Electron

of Synaptic

Micrographs

Vesicles

of Nerve

Ring

Longitudinal sections of new adults showing axon profiles and muscle arms (arrowheads). (A) Many of the wild-type axons have a bouton en passant, i.e., an axonal varicosity containing neurotransmitter vesicles clustered near a presynaptic active zone (small arrows). Other axons have small caliber. (B) uric-704 (rh43) axons have few vesicles and only occasional small boutons. Although these axons have small caliber, their number is normal

Cdl 842

Figure 4. Electron

Micrographs

of Ventral

Ganglion

Neurons

Transverse sections of adults showing neuron cell bodies. (A) Wild-type cells have only small loose clusters of synaptic vesicles (open arrows) usually near the Golgi apparatus or axon hillocks (curved arrow). (8) uric-104 (e1265) cells contain large clusters of closely packed vesicles (open arrows), while other regions of the cytoplasm are devoid of vesicles.

Figure

5. Muecfe

Arm Homing

Incorrectty

toward

Ventral

Nerve

Cord

DIC micrograph of an urrc-704 (rfr43) c/r-l adult in lateral aspect. Lateral margins of body muscles are visible at the top and bottom. (A) Superficial focus showing nerves and excretory (exe) canal. Small longitudinal nerves run at lateral (L) and ventral lateral (VL) positions. Axons from mechanosensory neurons PDE and PVD run as a single fascicle (double arrowheads) to the ventral nerve cord (not shown). The PVD dendrite joins the lateral nerve, while the ciliated PDE dendrite forms a cuticular sensillum with sheath (sh) and socket (so) cells. Motor axons (double arrowheads) from cell bodies in the ventral nerve cord run to the dorsal nerve cord (not shown). (B) Deeper focus of same individual showing an arm from a dorsal muscle targeting incorrectly to the ventral nerve cord. Bar = 10 pm.

Mutant 843

Transport

of Synaptic

Vesicles

passant onto the muscle arms. The anteriormost muscle cells in each quadrant are exceptional in that their arms extend to nerve ring motor axons (cells l-4) or to nerve ring and a cord (cells 5-6). Muscle arms were examined by DIC microscopy in living animals using the c/r-l (e7 745hs) mutation and by electron microscopy(Hedgecocket al., 1990). Inrh43adults, dorsal and ventral body muscles usually have only l-2 and 3-5 arms per cell, respectively. Arms from dorsal muscle cells occasionally extend incorrectly to the ventral rather than the dorsal nerve cord (Figure 5). At the nerve cords, muscle arms interdigitate with one another, forming muscle-muscle gap junctions, but rarely receive synapses. Arms from the anteriormost cells extend correctly to the nerve ring in these mutants (see Figure 3).

Figure

6. Secretory

Granules

in the gl Pharyngeal

Glands

DIC micrographs of pharynx in wild-type L4 larva. Axon-like processes (arrowheads) travel through the isthmus (glA and glP) and corpus (gl P only) connecting gland cell bodies to their release sites (Albertson and Thomson, 1976). (A) During intermolt, only small secretory granules are present in the gl P process. During lethargus, distinctive larger secretory granules are synthesized (Singh and Sulston, 1976). In gl P only, granules leave the cell body to mill within the proximal process (isthmus segment). The glA cell bodies and glP isthmus segment gradually become packed with granules. (6) Abruptly, about 20-30 min before ecdysis, granules start moving anterograde (averaging -1 pm/s) to their release sites at the anterior (glP) and posterior (gl A) ends of the corpus. Exocytosis is completed within several minutes.

Secretory Granule Translocation Excretory and gl pharyngeal gland cells have axon-like processes connecting their cell bodies to the sites of exocytosis (Albertson and Thomson, 1976; Nelson et al., 1963). In rh43 excretory glands, cell bodies and processes are densely packed with secretory granules as in wild type. During feeding (intermolts), gl glands continuously secrete materials into the alimentary tract, which may aid digestion (Singh and Sulston, 1976). During molts, these same cells synthesize and transport distinctive larger granules that can be easily followed by DIC microscopy (Figure 6). Secretion occurs rapidly just minutes before ecdysis and may help loosen and weaken the old cuticle before it tears apart. In rh43 larvae (n = 4), L4 lethargus is longer than normal and the timing of granule synthesis and transport is poorly regulated. Nonetheless, each molt concludes with a period of rapid, steady granule movement as in wild type. Discussion

number f SD of active zones per nerve ring are: wild type, 1010 -c 200(n = 6); e7265, 500 f 50 (n = 3); and rh43, 220 f 20(n = 3). In contrast, gap junctions (electrotonic synapses) between neurons appear to be unaffected in these mutants. , The appearance and subcellular distribution of other MBOs, e.g., endoplasmic reticulum, Golgi apparatus, and mitochondria are qualitatively normal in uric-704 neurons (Figures 3 and 4). Finally, all axons have smooth tubular membranes, commonly one per profile, aligned along their axes. These organelles appear normal in rh43. Muscle Arms and Neuromuscular Junctions Body muscles receive synapses (neuromuscular junctions) by extending cytoplasmic arms to motor axons in the dorsal and ventral nerve cords. The number of arms, usually 1 per cell at hatching, increases to 5 or more by adult. All arms extend to the nearer nerve cord (e.g., dorsal muscles extend arms to the dorsal nerve cord) and none extend to the opposite cord. At the nerve cords, arms from adjacent cells interdigitate, forming muscle-muscle gap junctions, and spread along the hypodermal basal lamina that covers the motor axons. Like axo-axonal synapses formed between neurons, motor axons make boutons en

uric-104 Is Essential for Most Neural Functions uric-704 mutations affect the function of many or all classes of neurons in C. elegans. The regulation of complex motor behaviors, including hatching, locomotion, feeding, and defecation, is impaired. These motor defects range from mild to severe depending upon the allele (Table 1). In comparisons between alleles, the degree of locomotor paralysis correlates with the slowing of feeding and defecation. However, locomotion is more sensitive to reductions in gene function, e.g., e7265 is nearly paralyzed yet feeds normally. Whereas several classes of interneurons and motor neurons are essential for normal locomotion, only one neuron is required for isthmus peristalsis and none for pharyngeal pumping (Chalfie et al., 1965; Avery and Horvitz, 1969). Finally, severe locomotor defects preclude many behavioral assays, but sensory processing may be equally impaired. Individual muscles are contractile, and interestingly, some coordinated movements persist even in severe alleles. For example, synchronous contractions of body muscles during embryonic elongation are unaffected. These regular contractions could be myogenic rather than neurogenic in origin, spreading via gap junctions between

Cdl 844

muscle arms (White et al., 1986). The contraction rate may be an intrinsic property of the embryonic body muscles. Alternatively, various mesoglia (i.e., GLR, hmc and homolog) that form gap junctions with body muscles in the head could act as pacemakers. The purpose of pharyngeal pumping preceding hatch was hitherto unknown. In severe allefes, larvae fail to pump, and hatching is significantly delayed. We propose that the extraembryonic fluid acquires enzymes for digesting the eggshell while recirculating through the pharynx and intestine. Possible sources for these enzymes include the gl gland cells and the intestine itself (Singh and Sulston, 1978). A protease for digesting the outermost shell has been identified genetically (h&l; Hedgecock et al., 1987). uric-704 mutants closely resemble &a-l (choline acetyltransferase) mutants in motor phenotypes (Rand and Russell, 1984; Rogalski and Riddle, 1988; Rand, 1989; Avery and Horvitz, 1990; Thomas, 1990). In these mutants, the body locks in coiled postures (i.e., a prolonged synchronous contraction that might spread between body muscles via gap junctions), feeding and defecation are slow and irregular, but egg-laying is proficient. While severe alleles are larval lethal% moderate alleles grow slowly and form small adults with low brood sizes. Similarities between uric-704 and cha-7 mutants could reflect their common lack of functional cholinergic synapses, including neuromuscular junctions. By analogy, other mutants with these phenotypes may lead to theidentification of additional molecules required for development or function of cholinergic, and perhaps other, synapses. UNC-104 May Be the Anterograde Motor for Synaptic Vesicles Synapses are identified ultrastructurally in C. elegans as boutons en passant filled with transmitter vesicles that are tethered collectively to the presynaptic active zone. In severe uric-704 alleles, most or all classes of chemical synapses, including neuromuscular junctions, are rare or absent. Two observations suggest that the primary defect is a failure to transport synaptic components, notably, synaptic vesicles, along the axon. First, axons have few transmitter vesicles, while cell bodies have surfeits of similar vesicles. The entire ring neuropil, comprising axons from 182 neurons, has about 350,900 synaptic vesicles in wildtype new adults, but only 50,099 in rh43 (compare White et al., 1988). Moreover, the surfeits in cell bodies (m19992999 vesicles per soma on average in 17743)could account numerically for the deficits in their axons. Interestingly, the untransported synaptic vesicles in uric-704 mutants are not distributed loosely in the cytoplasm but are tethered together as in synaptic boutons (Figures 3 and 4). Synap sins, a family of vesicle-associated phosphoproteins, are believed to cross-bridge synaptic vesicles at boutons (Landis et al., 1988; Hirokawa et al., 1989). We suggest that these proteins associate with synaptic vesicles before their translocation along the axon. Second, even null alleles have some small, but ultrastructurally normal, synapses that could be functional. Thus, the uric-704 gene product facilitates synapse forma-

tion, but it is not a structural component that is absolutely required. Sporadic small synapses may account for the variable penetrance of feeding, defecation, and growth defects observed in severe alleles (Table 1). uric-704 has been molecularly cloned by transposoninsertion mutagenesis and its sequence determined (Otsuka et al., 1991). The predicted product, UNC-194, is a novel protein of 1584 amino acids with a kinesin-like motor domain at its N-terminus. By analogy to kinesin and related proteins, it is likely that UNC-194 also functions as a microtubule-based motor. Based on mutant phenotypes, we propose that UNC-104 is used specifically for anterograde axonal transport of synaptic vesicles or their precursor. As axonal microtubules are believed to be oriented with plus ends away from the cell body, we predict that UNC-104 is a plus end-directed motor (see Black and Baas, 1989). Two aspects of synaptic vesicle transport must be somehow regulated. First, vesicles are sorted and moved along axons but apparently not dendrites. Second, vesicles can halt and tether at boutons en passant spaced along the axon. Any of several membrane and cytoplasmic proteins, and perhaps unusual lipids, associated with synaptic vesicles could provide docking sites for UNC-104 (see De Camilli and Jahn, 1990). Some proteins (e.g., synapsins) are conditionally phosphorylated and could regulate halting and tethering. A small GTP-binding protein (rab3A) associated with synaptic vesicles may regulate exocytosis (Fischer von Mollard et al., 1991). Other such proteins could oversee earlier docking events between synaptic vesicles, motor proteins, axonal microtubules, and cytoskeletal specializations at presynaptic active zones (see Bourne et al., 1990). Anterograde Transport of Other YBOs Is Normal In principle, all MBOscould share acommon motor protein for anterograde axonal transport. uric-704 mutants suggest, however, that separate motors maybe used for different classes of organelles. Two separate pathways of regulated secretion with distinct storage vesicles originate frqm the trans Golgi network, i.e., synaptic vesicles storing classical neurotransmitters and secretory granules storing secretory proteins and peptide neuromodulators (see De Camilli and Jahn, 1998). Whereas UNC-194 is required for transporting synaptic vesicles in neurons, some other motor protein must be used for transporting secretory granules in gland cells. A universal pathway of constitutive secretion delivers new membrane from the trans Golgi network to the plasmalemma for immediate exocytosis without processing or storage. The smooth tubulovesicles found in axoplasm could be the structural correlate of this pathway in neurons. When axons elongate, whether by active migration or passive stretching, they maintain constant caliber(Bray, 1984). This requires anterograde transport and insertion of new membrane at the growth cone or along the entire axolemma (Cheng and Reese, 1988). Because UNC-104 is neither required for axonal growth nor for their tubulovesicles, some other motor protein must be used for constitutive secretion.

Mutant 845

Transport

of Synaptic

Vesicles

Translocation Figure 7. Model of Genesis, Vesicles

Translocation,

Recycling and Recycling

of Synaptic

New synaptic vesicles may have two roles in synaptogenesis: to target specific cell surface and matrix molecules to the synaptic junction and to prime the pool of recycling vesicles. New vesicles are sorted via the trans Golgi network and are translocated along axonal microtubules by UNC-104. These vesicles halt at nascent active zones, secreting proteins and proteoglycans into the developing cleft. Vesicle membranes are rapidly recycled, perhaps via early endosomes (not shown), and locally refilled with neurotransmitter without returning to the Golgi apparatus.

In summary, UNC-104 is required for transporting only one known class of MB0 in vivo, i.e., synaptic vesicles. Kinesin itself is apparently the anterograde motor for lysosomes in macrophages and probably neurons as well (Hollenbeck and Swanson, 1990; Saxton et al., 1991). However, it is unknown whether kinesin has additional in vivo roles. Specific motor proteins for anterograde transport of other MBOs, including mitochondria, are yet unknown, but they may include new members of the kinesin superfamily (Rose, 1991). Developmental Roles of Synaptic Vesicles The physiological role of synaptic vesicles is to store neurotransmitter for regulated secretion into the synaptic cleft upon electrical stimulation. Vesicle membranes are then rapidly recycled and locally refilled with transmitter without return to the Golgi apparatus (Miller and Heuser, 1984; Torri-Tarelli et al., 1990). New vesicles transported from the trans Golgi network are needed to prime the recycling

wild

Figure

type

8. A Chemoattraction

Experimental

Procedures

Genetics Five independent, partial revertants were obtained from e7265or rh43 mutants following ethyl methanesulfonate (EMS) treatment; a sixth revertant arose spontaneously among rh43 mutants (Table 1). By genetic criteria, each revertant is a second-site mutation within the unc704gene that restoressome, butnotall, wild-typefunction.Theoriginal e7265 or rh43 mutation could not be recovered from the revertants upon outcrossing with wild type, indicating that the suppressing mutations are closely linked to uric-704 and are possibly intragenic. Whereas heterozygotes with a revertant chromosome opposite a wildtype allele, e.g., rb43rh726rvl one-704(+), are fully wild type, heterozygotes opposite severe loss-of-function alleles, e.g., rh43 rb726rvlrh43, are intermediate in the Uric phenotype. To obtain possibly lethal alleles, a total of 500 males, heterozygous for a mild uric-704 allele, either rh7077 or rb43 rh128rv, were mated with 500 EMS-mutagenized hermaphrodites homozygous for a linked recessive marker, dpy-70 (e728). Three classes of progeny were observed: Dpy non-Uric self-progeny; non-Dpy non-Uric cross-progeny; plus five exceptional non-Dpy Uric cross-progeny, each carrying a new

uric-104

uric-5

Model of Muscle

pools during synapse assembly and to replace lost materials at functioning synapses (Figure 7). New synaptic vesicles likely have different contents than recycled transmitter vesicles (Figures 4 and 7). Observations of uric-704 mutants suggest that these vesicles have developmental roles beyond simply priming the pools of recycling vesicles. First, they are needed for the formation and enlargement of synaptic junctions, presumably by secreting adhesion molecules into the nascent synaptic cleft (see Schubert, 1991). Second, while most axon trajectories are correct in detail, some neuron classes make occasional errors while pioneering on the hypodermis. Their growth cones could require that some substance be transported and secreted via synaptic vesicles for correct navigation. Finally, because muscle arms can target correctly to ectopic motor axons, we proposed that motor terminals release a chemoattractant (Hedgecock et al., 1990). Here we suggest that this attractant may be transported and secreted via synaptic vesicles (Figure 8).

Arm Targeting

Over 50 motor neurons are arranged in file along the ventral nerve cord, but only two cells are shown. Motor axons run along the ventral and dorsal nerve cords. Wild-type axon terminals transport and secrete a hypothetical substance (small dots) via synaptic vesicles, which attracts arms from nearby muscles. In uric-5 mutants, mispositioned motor axons run along the lateral hypodermis but still recruit arms from dorsal muscles and sometimes from ventral muscles as well (Hedgecock et al., 1990). In uric-704 mutants, the chemoattractant is not transported to the axon terminals but is released, instead, closer to the cell bodies, As a consequence, dorsal body muscles extend fewer arms than normal, and some arms home incorrectly toward cell bodies in the ventral nerve cord while ignoring nearby axon terminals.

Cdl 846

uric-104 allele, e.g., genotype +rh1017le128 rh140, where rh140 is a new mutation (Table 1). Whereas homozygousunc-704 (r/rl47)dpy-70 hermaphrodites are highly fertile, the other four alleles (rh140, rh742, rh743, rb744) are subviable in this genetic background.

Microscopy Methods for examining specific neurons by DIC or fluorescence microscopy are given in Hedgecock et al. (1965,199o) and Herman and Hedgecock (1990). Animals were fixed for electron microscopy using buffered glutaraldehyde and then osmium tetroxide as described by Sulston et al. (1983). Usually three or four animals were aligned within a small agar block, then embedded and sectioned together. Serial sections were collected through the nerve ring and ventral ganglion and in selected regions of the dorsal and ventral nerve cords. Sections were poststained with uranyl acetate and lead citrate. Synaptic vesicles and presynaptic active zones were counted using enlarged micrographs made about every sixth section of transverse series through the nerve ring and ventral ganglion. As active zones generally span from one to five sections, each micrograph sampled distinct synapses. To estimate the total number of synapses, we arbitrarily assumed that one-third of all synapses are counted at this sampling interval. As small synapses are undersampled relative to large synapses, a smaller total may reflect a decrease in the number of synapses, their average size, or both.

Acknowledgments We thank D. Riddle, M. Shen, and the Caenorhabditis Genetics Center for providing strains; H. Bhatt for examining the PVP neurons with MAb44; H. Rubin for photographic services; A. Gtsuka and A. Jeyaprakash for sharing the DNA sequence of uric-104; and P. Meluh, C. Norris, A. Dtsuka, and W. Wadsworth for helpful discussions. This work was supported by NIH grants NS07512 (D. H. H./M. V. L. Bennett) and NS26295 (E. M. H.) and by Hoffmann-LaRoche. The costs of publication of this article were defrayed in par-i by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with I6 USC Section 1734 solely to indicate this fact. Received

January

15, 1991; revised

April 9, 1991.

References Albertson, D. G., and Thomson, J. N. (1976). The pharynx of Caeno. rhabditis elsgans. Phil. Trans. Roy. Sot. (Lond.) B 275, 299-325. Avery, L., and Horvitz, H. R. (1969). after laser killing of the pharyngeal Neuron 3, 473-465. Avery, L., and Horvitz, tive drugs on feeding 263-270.

Pharyngeal pumping continues nervous system of C. elegans.

H. R. (1990). Effects of starvation and neuroacin Caenorhebditis ebgans. J. Exp. Zool. 253,

Black, M. M., and Baas, P. W. (1969). Trends Neurosci. 72, 211-214. Bourne, H. R., Sanders, superfamily: a consewed 125-132.

The basis of polarity

Fischer von Mallard, G., Sudhof, GTP-binding protein dissociates cytosis. Nature 349, 79-61.

e&fans.

PhD thesis,

University

T. C., and Jahn, R. (1991). A small from synaptic vesicles during exo-

Hall, D. H., and Russell, R. L. (1991). The posterior nervous system of the nematode Caenodtabditis elegans: serial reconstruction of identified neurons and complete pattern of synaptic interactions. J. Neurosci. 71, l-22. Hedgecock, E. M., Culotti, J. G.. Thomson, J. N., and Perkins, L. A. (1965). Axonal guidance mutants of Caenorhabdtis e/egans identified by filling sensory neurons with fluorescein dyes. Dev. Biol. 117, 158170. Hedgecock. E. M., Culotti, J. G., Hall, D. H., and Stern, 8. D. (1987). Genetics of cell and axon migrations in Caenorhabditis ebgans. Development 100,365-382. Hedgecock, E. M., Culotti, J. G., and Hall, D. H. (1999). The uric-5, uric-6, and uric-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. ekqans. Neuron 4, 61-65. Herman, R. K.. and Hedgecock, E. M. (199Q. Limitation of the size of the vulva1 primordium of Caenorhabdtis elegans by W-15 expression in surrounding hypodermis. Nature 348, 169-I 71. Hirokawa, N., Sobue, K., Kanda, K., Harada, A., and Yorifuji, H. (1969). The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin I. J. Cell Biol. 708, 111-126. Hollenbeck, macrophage 664-966.

P. J., and Swanson, J. A. (1990). Radial tubular lysosomes supported by kinesin.

extension of Nature 346,

Kosik, K. S., Orecchio, L. D., Schnapp, B., Inouye, H., and Neve, R. L. (1990). The primary structure and analysis of the squid kinesin heavy chain. J. Biol. Chem. 265, 3276-3263. Landis, D. M. D.. Hall, A. K., Weinstein, L. A.. and Reese, T. S. (1986). The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse. Neuron I, 201-209. Miller, T. M., and Heuser, J. E. (1964). membrane at the frog neuromuscular 698.

Endocytosis of synaptic vesicle junction. J. Cell Biol. 98, 665-

Mori, I., Moerman, D. G., and Waterston, R. H. (1966). Analysis of a mutator activity necessary for germline transposition and excision of Tel transposable elements in Caenofiabditis elegans. Genetics 720, 397407. Nelson, F. K., Albert, P. S., and Riddle, D. L. (1983). Fine structure of the Caenotidditis e/egans secretory-excretory system. J. Ultrastruct. Res. 82, 156-171. Otsuka, A. J., Jeyaprakash, A., Garcia-Anoveros, J., Tang, L. Z., Fisk, G., Hartshorne, T., France, R., and Born, T. (1991). The C. elegans uric-704 gene encodes a putative kinesin heavy chain-like protein. Neuron 6, 113-122. Priess, J., and Hirsch, D. (1986). Caenorhsbditisebgans morphogenesis: the role of the cytoskeleton in the elongation of the embryo. Dev. Biol. 117, 156-173.

in neurons.

D. A., and McCormick, F. (I 990). The GTPase switch for diverse cell functions. Nature 348,

Bray, D. (1964). Axonal growth in response to experimentally mechanical tension. Dev. Biol. 102, 379-369.

the nervous system of Caenorhalnlitis of Cambridge, Cambridge England.

applied

Rand, J. B. (1969). in Caenorhabditis.

Genetic analysis of the cha-l-unol7gene Genetics 722,73-80.

Rand, J. B., and Russell, R. L. (1964). Choline deficient mutants of the nematode Caenorhabditis 708,227-246.

complex

acetyltransferaseGenetics

elegans.

Chalfie, M., Sulston, J. E., White, J. G., Southgate, E., Thomson, J. N.. and Brenner, S. (1966). The neural circuit for touch sensitivity in CaenorhsLWtis elegans. J. Neurosci. 5, 956-964.

Rogalski, T. M.. and Riddle, D. L. (1966). A Caenorhabditis e/egsns RNApolymerase II gene, am%t IV, and nearby essentia genes. Genetics 118.61-74.

Cheng, T. P. O., and Reese, T. S. (1966). Compartmentalization of anterogradely and retrogradely transported organelles in axons and growth cones from chick optic tectum. J. Neurosci. 8, 3190-3199.

Rose, M. (1991). The kinesin-related genes. In Guide to Cytoskeletal and Structural Proteins, R. Vale and P. Kreis, eds. (Cambridge, England: Tooze and Sambrook Publishers).

Collins, J., Saari. B., and Anderson, P. (1987). Activation of a transposable element in the germ line but not the soma of Csenofhabditis elegens. Nature 328, 726-726.

Saxton, W. M., Hicks, J., Goldstein, L. S. B., and Raff, E. C. (1991). Kinesin heavy chain is essential for viability and neuromuscular functions in Drosophila, but mutants show no defects in mitosis. Cell 64, 1093-1102.

De Camilli, in neurons. Durbin,

P., and Jahn. R. (1999). Pathways Annu. Rev. Physiol. 52, 625-645.

R. M. (1967).

Studies

to regulated

on the development

exocytosis

and organization

of

Schubert, D. (1991). The possible roleof adhesion tion. Trends Neurosci. 14, 127-130.

in synaptic

modifica-

Mutant 847

Transport

of Synaptic

Vesicles

Sigurdson, D. C., Spanier, G.J., and Herman, R. K. (1984). Caenorhab ditis elegans deficiency mapping. Genetics 108, 331-345. Singh, Ft. N.. and Sulston, in Caenorhabditis elegans.

J. E. (1978). Some observation Nematologica 24, 63-71.

on molting

Sulston. J. E., and Horvitz, H. Ft. (1977). Postembryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56, 11 O-l 56. Sulston, J. E., Schierenberg, E., White, J. G., and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64-119. Thomas, elegans.

J. H. (1996). Genetic analysis Genetics 124, 855-872.

of defecation

in Caenorhabditis

Torri-Tarelli, F., Villa, A., De Camilli. P., Greengard, P., and Ceccarelli, B. (1990). Redistribution of synaptophysin and synapsin I during a-latrotoxin-induced release of neurotransmitter at the neuromuscular junction. J. Cell Biol. 170, 449-459. Vallee, R. B., and Shpetner, H. S. (1990). Motor proteins microtubules. Annu. Rev. Biochem. 59, 909-932.

of cytoplasmic

Venolia, L., and Waterston. R. H. (1990). The uric-45 gene of Caenorhabditis elegans is an essential muscle-affecting gene with maternal expression. Genetics 726, 345-353. White, J. G., Southgate, E., Thomson, J. N., and Brenner, S. (1976). The structure of theventral cord of Caenorhabditis elegans. Phil. Trans. Roy. Sot. (Lond.) B 275, 327-348. White, J. G., Southgate, E., Thomson, J. N., and Brenner, S. (1986). The structure of the nervous system of the nematode Caenofhabditis ekgans. Phil. Trans. Roy. Sot. (Lond.) B 314, l-340. Yang, J. T., Saxton, W. M., Stewart, R. J., Raff, E. C., and Goldstein, L. S. 8. (1990). Evidence that the head of kinesin is sufficient for force generation and motility in vitro. Science 249, 42-47.