Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles - PubMed (original) (raw)

Comparative Study

Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles

M L Nonet et al. J Neurosci. 1997.

Abstract

Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

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Figures

Fig. 1.

Fig. 1.

Similarity among rab3 proteins from metazoa. Alignment of C. elegans, Drosophila, and bovine rab3 proteins. The locations of five conserved domains involved in the binding of guanine nucleotides are labeled G-1 to_G-5_ (Bourne et al., 1991). The C. elegans_protein shares 76% identity with Drosophila rab3, 73% identity with both bovine rab3A and rab3C, and 71% identity to both bovine rab3B and murine rab3D. The positions of amino acid substitutions or stop codons identified in rab-3 mutants are indicated. js49 is a G to A transition at position 2 of codon 76, y250 is a C to T transition at position 2 of codon 165, and y251 is a G to A transition at position 2 of codon 80. Dots represent identity among all proteins. The standard single amino acid code is used.O represents a hydrophobic amino acid, and_X represents any amino acid. Amino acid numbering appears on the right.

Fig. 2.

Fig. 2.

RAB-3 expression in the C. elegans nervous system. Whole worms were fixed and stained with anti-RAB-3 and anti-synaptotagmin primary antibodies and visualized with FITC- or Cy3-conjugated antibodies. A, B, Lateral view of the head region of a wild-type adult hermaphrodite showing RAB-3 (A) and synaptotagmin (B) immunoreactivity in the nerve ring (NR), pharyngeal nervous system (PN), and SAB neuron axonal processes (arrows). C, D, Ventral view of the midsection of an adult hermaphrodite showing RAB-3 (C) and synaptotagmin (D) immunoreactivity in the ventral nerve cord (VC;arrow) and the ventral sublateral processes.E, F, Lateral view of the vulval region of an adult hermaphrodite illustrating the absence of RAB-3 (E) but the presence of synaptotagmin (F) immunoreactivity in the uv1 cells of the somatic gonad (uv1; arrow). The ventral cord is also visible (VC).

Fig. 3.

Fig. 3.

Mislocalization of RAB-3 in unc-104_mutants. Whole unc-104(e1265) nematodes were fixed and stained with anti-RAB-3 primary antibodies and visualized with FITC-conjugated secondary antibodies. The anterior of the animal is to the left. A, Oblique lateral view of a young adult hermaphrodite showing RAB-3 immunoreactivity localized to neuronal cell bodies in the ventral nerve cord (arrow).B, DAPI staining of nuclei of the animal shown in_A. A row of neuronal nuclei located in the ventral nerve cord is visible (arrow).

Fig. 4.

Fig. 4.

The rab-3 locus. A, Genetic map of a portion of chromosome II, illustrating the position of genes and deficiencies used for the mapping and isolation of_rab-3_ mutants. B, Organization of the physical region neighboring rab-3. A series of overlapping yeast artificial chromosome and cosmid clones surrounding the rab-3 gene is shown. The positions of the_clr-1_ and lin-4 genes are delineated on the physical map. rab-3 was positioned in this interval as a result of specific hybridization to the YAC and cosmid clones (in_bold_). C, Restriction map of a portion of cosmid F11G1. Exons of the two transcripts derived from_rab-3_ are illustrated. The trans-spliced leader SL1 is found at the 5′ end of both rab-3 messages.SL1 and AAAA mark the trans-spliced leader attachment and poly(A+) addition sites, respectively. The genomic inserts of plasmid clones used in our experiments are illustrated as solid lines. pMG122 is a translational lacZ fusion to the _rab-3_coding sequence at amino acid 194.

Fig. 5.

Fig. 5.

RAB-3 protein is absent or mislocalized in_rab-3_ mutants. Whole worms were fixed and stained with primary antibodies and visualized with FITC-or cy3-conjugated antibodies. A–C, Lateral view of the head region of a_rab-3(js49)_ adult stained with anti-RAB-3 antibodies (A), anti-synaptotagmin antibodies (B), and DAPI to visualize nuclei (C). D–F, Ventral view of the midsection of rab-3(y251) hermaphrodite stained with anti-RAB-3 antibodies (D), anti-synaptotagmin antibodies (E), and DAPI to visualize nuclei (F).

Fig. 6.

Fig. 6.

rab-3 mutants are resistant to an inhibitor of acetylcholinesterase. Young adults worms were assayed for acute body paralysis after a 5 hr incubation with aldicarb on agar plates containing food. Twenty to twenty-five animals were assayed at each concentration.

Fig. 7.

Fig. 7.

Pharyngeal recordings from wild-type and_rab-3_ mutants. Characteristic recordings of a (A, D) wild-type worm,rab-3(js49) (B, E), and_rab-3(y250)_ (C) mutants.Arrows indicate the MC-induced transients, and the_circles_ indicate M3-induced transients. In wild-type animals (D), MC activity that failed to initiate a pharyngeal pump was characteristically observed as a single transient. However, in rab-3 mutants (E), MC activity that failed to initiate a pharyngeal pump was nonsynchronous, typically consisting of a small burst of transients spaced in a 50–100 msec interval. All traces are mV versus time.

Fig. 8.

Fig. 8.

Synaptic vesicle populations are depleted at neuromuscular junctions in rab-3 mutants. Electron micrographs of wild-type (A) and_rab-3(y251)_ (B) neuromuscular junctions in the ventral cord. Arrows demarcate the dark thickening of an active zone, and arrowheads identify a typical synaptic vesicle in each photograph. Scale bar, 500 nm.

Fig. 9.

Fig. 9.

Distribution of vesicles at neuromuscular junctions. Serial section electron micrographs of the ventral cord of wild-type and rab-3(js49) animals were examined. Synaptic vesicles were counted in each motor neuron axonal profile of wild type (black bars; n = 475) and_rab-3(js49)_ (white bars;n = 369). The average number of vesicles is plotted against the distance from the electron-dense specialization found at_C. elegans_ synaptic contacts. Distance was determined by the number of thin sections to the closest active zone in which section thickness was ∼50 nm. Error bars are ± SEM.

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