Characterization of the Ce-synaptojanin locus. A, Genetic and physical maps. unc-26 maps to linkage group IV, between ced-3 and let-320. Below the genetic map is a representation of the physical map of this region, showing YACs, cosmids, and phage clones. A cosmid gap (vertical lines) existed in this region. Phage clones (THø1) extended from the end of JC8, but did not contain the complete ORF. B, Predicted mRNAs. UNC-26A (2/12 cDNAs). UNC-26B (9/12 cDNAs). UNC-26C (1/12 cDNAs). The 5′ end of UNC-26C was incomplete, and its 5′ splice pattern of exons was inferred from the other variants. This variant was generated by readthrough of the splice junction at the end of exon 5. UNC-26A and B splice variants generate transcripts with 3 nt of 5′ untranslated sequence and 311 nt of 3′ untranslated sequence. The UNC-26C variant generates a transcript with 21 bp of 3′ untranslated sequence. C, unc-26 mutations were mapped relative to each other (Charest et al. 1990). The order and position of sequenced mutations corresponded to their genetic map positions. e176, e314, e446, e1196, n1307, and ox1 were not mapped relative to other alleles and are indicated only for reference.

Ce-synaptojanin (Genbank/EMBL/DDBJ accession numbers for UNC-26A, B, and C are AF283322, AF283323, and AF283324, respectively) aligned with rat synaptojanin (Genbank/EMBL/DDBJ accession number U45479). Identities are in black boxes. The single exon difference between the splice variants UNC-26A and B is boxed, and the alternative coding residues and truncation of the UNC-26C transcript is shown above where it diverges from the other splice forms. Also indicated are the putative catalytic site for Sac1-like phosphatases (Guo et al. 1999) and the highly conserved catalytic and/or substrate binding regions of 5-phosphatases (Communi and Erneux 1996; Communi et al. 1996; Jefferson and Majerus 1996; Stolz et al. 1998).

Mutations in the unc-26 gene cause a depletion of synaptic vesicles at neuromuscular synapses. A, Representative electron micrographs of acetylcholine and GABA neuromuscular junctions in a wild-type (left) and an unc-26(s1710) adult hermaphrodite (right). The ventral nerve cord was reconstructed from serial electron micrographs. GABA neurons were identified based on their position within the cord, and because they form synaptic connections exclusively to muscle. Cholinergic neurons were identified based on their position in the cord and because they form synaptic connections to neurons and muscle (White et al. 1976). SV, Synaptic vesicle; AZ, active zone. B, The average number of synaptic vesicles per reconstructed synapse was decreased in unc-26 mutants. Number SV per synapse: ACh, wild-type = 173 ± 25 SV, n = 32 synapses; unc-26(s1710) = 72 ± 5 SV, n = 61 synapses. GABA, wild-type = 242 ± 28 SV, n = 14 synapses; unc-26(s1710) = 78 ± 10 SV, n = 23 synapses. Pooled, wild-type = 194 ± 19 SV, n = 46 synapses; unc-26(s1710) = 74 ± 4 SV, n = 85 synapses.

Coated vesicles accumulated in unc-26 mutants. A, An example of a coated vesicle in a VD GABA motor neuron of an unc-26(s1710) adult hermaphrodite. AZ, Active zone. B, Coated vesicles accumulated near electron-dense active zones in unc-26 mutants. Plotted is the average number of coated vesicles per section found in axons of unc-26(s1710) adult hermaphrodites at given distances from the nearest active zone. C, Quantification of coated vesicles in various tissues of wild-type and unc-26(s1710) adult hermaphrodites. Left, Total coated vesicles scored in muscles, hypodermis, neurons, and gonadal sheath cells in sections of the ventral nerve cord. Scale is to the left. Number of sections: wild-type, 328 sections, from two worms; unc-26(s1710), 433 sections, from four worms. Right, total coated vesicles in each tissue visible in these sections. Scale is to the right. D, A coated vesicle in the cell body of a motor neuron. Such coated vesicles were often associated with Golgi stacks.

Vesicle transport was partially disrupted in unc-26. A, left, Electron micrograph of a motor neuron cell body from a wild-type adult hermaphrodite. Right, Motor neuron cell bodies in an unc-26(s1710) adult hermaphrodite. Note large accumulation of vesicles at cell body #1, but absence of vesicles in cell body #2. 10 of 24 cell bodies examined in the mutant exhibited this congestion of vesicles, whereas 0 of 11 wild-type cell bodies exhibited this accumulation. B, Synaptobrevin-GFP distribution in the dorsal nerve cord of wild-type and unc-26 adult hermaphrodites. Synaptobrevin-GFP was localized to synaptic varicosities (arrowheads) in the wild type and in unc-26(s1710) animals. However, synaptobrevin-GFP was more diffuse at synapses in unc-26(s1710), rather than punctate as in the wild type. C, Synaptobrevin-GFP distribution in the ventral nerve cord of wild-type and unc-26(s1710) adult hermaphrodites. Synaptic varicosities (arrowheads) along the ventral nerve cord exhibited the same diffuse staining pattern as seen along the dorsal cord.

Endocytic pits accumulated in the plasma membrane of unc-26(s1710) mutants. A, Representative electron micrographs of endocytic pits from neuromuscular junctions of unc-26(s1710) adult hermaphrodites. Endocytic pits were either coated (left) or noncoated (right) and were observed with open necks (top) or closed necks (bottom). Pits had faint collars (arrows). No endocytic pits were observed in wild-type sections prepared using the same fixation protocol. B, Endocytic pits accumulated near electron-dense active zones in unc-26 mutants. Plotted is the average number of pits per section at given distances from the nearest active zone (see Materials and Methods). Total number of pits scored, 58. C, Noncoated pits were often seen immediately adjacent to active zones. AZ, Active zone.

Endosome-like compartments accumulated in unc-26. A, Electron micrograph of a synapse in the VB cholinergic motor neuron of an unc-26(s1710) adult hermaphrodite. Endosomes were defined as 50 nm or larger membrane-bound structures extending two or more lateral sections. AZ, Active zone. B, Quantification of endosome-like structures in unc-26(s1710) adult hermaphrodites.

Cytoskeletal defects in an unc-26 animal. A, Representative micrograph showing typical arrangements of synaptic vesicles in the VD GABA neuron (top) and VA cholinergic neuron (bottom) of a wild-type adult hermaphrodite. AZ, Active zone; SV, synaptic vesicle. B, Representative micrograph showing the linear string of pearls arrangement of synaptic vesicles at synapses in the VD GABA neuron (top) and the VA cholinergic neuron (bottom) of an unc-26(s1710) adult hermaphrodite. AZ, Active zone; SV, synaptic vesicle.