Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density

Abstract

Rim is a multi-domain, active zone protein that regulates exocytosis and is implicated in vesicle priming and presynaptic plasticity. We recently demonstrated that synaptic defects associated with loss of Caenorhabditis elegans Rim (termed UNC-10) are accompanied by a reduction in docked vesicles adjacent to the presynaptic density. Since Rim is known to interact with the vesicle-associated GTPase Rab3A, here we asked whether UNC-10-dependent recruitment of synaptic vesicles to the presynaptic density was through an UNC-10/Rab-3 interaction. We first established that C. elegans Rab3 (termed RAB-3) in its GTP but not GDP-bound state interacts with UNC-10. We then demonstrated by EM analysis that rab-3 mutant synapses exhibit the same vesicle-targeting defect as unc-10 mutants. Furthermore, unc-10;rab-3 double mutants phenocopy the targeting defects of the single mutants, suggesting UNC-10 and RAB-3 act in the same pathway to target vesicles at the presynaptic density. Endogenous release of unc-10;rab-3 double mutants was similar to that of unc-10 single mutants, but more severe than rab-3 mutants, suggesting the common targeting defects are reflected by the milder rab-3 release defect. Rim has recently been shown to positively regulate calcium influx through direct interactions with calcium channels. Consistent with this notion we found UNC-10 colocalized with the calcium channel, UNC-2 at C. elegans presynaptic densities and synaptic release in unc-10 and rab-3 mutants exhibit reduced calcium-sensitivity. Together these results suggest that vesicles targeted to the presynaptic density by RAB-3/UNC-10 interactions are ideally positioned for efficient calcium-dependent release.

Figure 1. UNC-10 interacts with RAB-3^GTP

A. The Rim-RAB-3 interaction is conserved across species boundaries. X-gal filter lift two-hybrid assays reveal interactions between worm UNC-10 Rim_1–187 and rat Rim _1–345 and both rat and worm GTP-bound mimic mutant RAB-3(Q81L), but not the GDP-bound mimic RAB-3(T36N). B. Interactions between worm UNC-10 Rim_1–187 were highly selective for RAB-3 compared to other RABs. C. Rim interacts with RAB-3 in worm extracts. Whole worm solubilized extracts spiked with purified His6Rim Zinc Finger fusion protein were immunopurified using anti-vSNARE SNB-1, anti-Rim, and preimmune sera, separated by SDS-PAGE and analyzed by Western blot using antibodies against SNB-1, Rim and RAB-3. 2% of the input and 10% of each immunoisolate were loaded on the gels.

Figure 2. Ultrastructural analysis of rab-3, unc-10 and unc-10;rab3 mutant synapses

A, Representative micrographs for wild type, rab-3, unc-10 and unc-10;rab-3 animals. The PD (arrow) and closest membrane-contacting vesicle to the PD is indicated (arrowhead). Scale bar 200 nm. B, The number of SVs per profile was significantly increased in unc-10 and unc-10;rab-3 relative to the wild-type. Bar shades for each genotype are also used in subsequent graphs. C,D,E Distribution of membrane-contacting vesicles relative to the PD in rab-3 vs. wild type animals (C) and unc-10 vs. wild type (D) and unc-10;rab-3 vs. wild type (E). All three mutant genotypes exhibit a decrease in the ratio of SVs contacting the plasma membrane adjacent to presynaptic density. Data expressed as mean ± SEM; data in C–E are grouped into 30 nm bins.

Figure 3. Electrophysiological phenotypes of rab-3, unc-10 and unc-10;rab3

A. Representative evoked synaptic recordings for wild type, rab-3, unc-10 and unc-10;rab-3. B. Average evoked amplitudes were reduced in rab-3, unc-10 and unc-10;rab-3 compared to wild-type animals. C. Representative endogenous miniature postsynaptic event recordings from wild type, rab-3, unc-10 and unc-10;rab-3. D. Average frequencies of miniature postsynaptic currents were reduced in rab-3, unc-10 and unc-10;rab-3 compared to wild-type animals. E. Average miniature event amplitudes were not altered in rab-3, unc-10 and unc-10;rab3 mutants compared to the wild type. Data expressed as mean ± SEM. All recordings obtained in 5mM extracellular calcium.

Figure 4. Distribution of the membrane-associated calcium channel UNC-2 at the NMJ

A. Micrographs of NMJs prepared for immunoEM, 15nm gold beads (arrow) labeling UNC-2. Scale bar 200nm. B. Bar graph showing the spatial distribution of membrane-associated gold beads relative to the PD (position 0 on the x-axis). C. Average evoked amplitudes of rab-3, unc-10 and unc-10;rab-3 and wild-type animals in 1mM extracellular calcium. D. Plots of the % decrease in average evoked responses obtained in 1mM Vs 5mM calcium for rab-3, unc-10 and unc-10;rab-3 and wild-type animals.