UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans

Abstract

Small guanosine triphosphatases of the Rab family regulate intracellular vesicular trafficking. Rab2 is highly expressed in the nervous system, yet its function in neurons is unknown. In Caenorhabditis elegans, unc-108/rab-2 mutants have been isolated based on their locomotory defects. We show that the locomotion defects of rab-2 mutants are not caused by defects in synaptic vesicle release but by defects in dense core vesicle (DCV) signaling. DCVs in rab-2 mutants are often enlarged and heterogeneous in size; however, their number and distribution are not affected. This implicates Rab2 in the biogenesis of DCVs at the Golgi complex. We demonstrate that Rab2 is required to prevent DCV cargo from inappropriately entering late endosomal compartments during DCV maturation. Finally, we show that RIC-19, the C. elegans orthologue of the human diabetes autoantigen ICA69, is also involved in DCV maturation and is recruited to Golgi membranes by activated RAB-2. Thus, we propose that RAB-2 and its effector RIC-19 are required for neuronal DCV maturation.

Figure 1.

UNC-108 is a homologue of human Rab2. (A) Sequence alignment of Rab2 from C. elegans, Homo sapiens, and Drosophila. Domains responsible for phosphate and Mg²⁺ binding (orange boxes) and guanine moiety binding (yellow boxes) are shown. Arrowheads indicate point mutations within RAB-2. Mutations rendering RAB-2 constitutively inactive (S20N) and constitutively active (Q65L) are shown (Tisdale, 1999). (B) Western blot of protein extracts from different unc-108 alleles probed with polyclonal RAB-2 antibodies. Tubulin loading control is also shown. (C) Quantification of normalized RAB-2 protein levels. Error bars = SEM (*, P < 0.05; Student's t test; n = 4).

Figure 2.

RAB-2 mutations change the biochemical properties of the protein. (A) Rab2 structure with guanine-binding domain labeled in yellow. The altered amino acids in dominant unc-108 mutants are red. (B) GTP hydrolysis rates were determined in vitro using recombinant RAB-2 (n = 3 per time point). (C) GTP affinity of recombinant RAB-2 using nonhydrolyzable γ-[³⁵S]GTP. Error bars = SEM (n = 9 per time point).

Figure 3.

unc-108 mutants have reduced locomotion and aldicarb sensitivity but not SV release at the NMJ. (A) The locomotory defects of unc-108 mutants are synthetically enhanced in unc-108; egl-3 double mutants to the level of unc-31/CAPS. Error bars = SEM (***, P < 0.005; Student's t test; all strains were compared with wild type; double mutants are compared with egl-3 and unc-31 single mutants). (B and C) Dominant unc-108 mutants are resistant to the ACh esterase inhibitor aldicarb (B), similar to rab-3, and exhibit a wild type–like sensitivity to the postsynaptic nicotinic receptor agonist levamisole (C). The weakly levamisole-resistant lev-10 mutant is shown as control. Error bars = SEM (n = 30). (D) The evoked release of SVs at the NMJ is similar between wild type and dominant unc-108 and ric-19 mutants. Error bars = SEM.

Figure 4.

unc-108 mutants have normal synaptic morphology and SV distribution. (A) The morphology of motor neuron synapses in the DNC in different C. elegans strains is shown in 40-nm HPF EM cross sections, with electron micrographs of a single DCV section shown in the insets. (B) SV distribution relative to the presynaptic density was analyzed for unc-108 and ric-19 mutants, and a tethering-defective rab-3 mutant is shown as a control. unc-108 and ric-19 SV distributions are not significantly different compared with wild type (one way analysis of variance with Dunnett's posttest). Error bars = SEM.

Figure 5.

unc-108 mutant DCVs contain less soluble cargo. (A) Animals carrying integrated array nuIs183 are expressing NLP-21–YFP in motor neurons projecting axons into the DNC. Representative images of the synaptic accumulation of DCVs with YFP-tagged NLP-21 neuropeptide in the DNC in the respective genetic backgrounds are shown. Mean fluorescence was normalized to wild type (graph below). The absence of EGL-3 leads to strong accumulation of neuropeptides at the synapse and restores wild-type levels in both dominant and recessive unc-108 alleles. Error bars = SEM (**, P < 0.01; ***, P < 0.005; Student's t test). (B) Once released from axons, NLP-21–derived YFP is taken up by coelomocytes. Mean coelomocyte fluorescence in various mutants was normalized to wild type (graph below). Error bars = SEM (***, P < 0.005). (C and D) NLP-21–YFP fluorescence in motor neuron somas in the VNC are shown, and size distribution of NLP-21–YFP-positive vesicles in neuronal cell bodies of dominant unc-108 mutants is compared with wild type. Arrowheads point to vesicles with surface area >0.8 µm². Error bars = SEM (n = 10). Bars: (A) 10 µm; (B) 5 µm; (C) 2 µm.

Figure 6.

RAB-2 localizes to the Golgi apparatus and is not present at the synapses. (A) Confocal images of motor neuron cell bodies expressing fluorescently tagged RAB-2 along with different intracellular markers are shown. RAB-2 extensively colocalizes with the Golgi marker mannosidase II (MANS II) and COPI-positive structures. RAB-2 shows no or only partial colocalization with the ER (cytochrome b5) or early endosomes (FYVE domain of EEA-1). (B) Immunogold labeling (white circles) of endogenous RAB-2 in motor neuron cell bodies showed staining at the Golgi complex and vesicular structures around the Golgi. (C) mYFP–RAB-2 (red) does not accumulate at the synapses in the DNC-like mCherry–RAB-3 (green). The dashed boxes represent the magnified synaptic puncta demonstrating no accumulation of RAB-2 at the synapses. In HSN neurons, mCherry–RAB-2 is present only in cytoplasmic puncta in the neuronal cell body (closed arrowheads) but not at the synapse with vulval muscles (open arrowheads). HSN synapses are labeled by GFP–RAB-3. Bars: (A) 5 µm; (C) 10 µm.

Figure 7.

In unc-108, NLP-21–YFP enters the late endosomal compartment. (A and B) To identify NLP-21–YFP-positive vesicular structures in wild type (A) and unc-108(n501) strains (B) carrying the NLP-21–YFP integrated array, different intracellular markers were expressed panneuronally: RAB-2, syn-6 (iDCVs), RAB-5 (early endosome), RAB-7 (late endosome), and LMP-1 (lysosome). The closed and open arrowheads represent larger and smaller vesicular structures, respectively. NLP-21–YFP-positive vesicles in the unc-108(n501) strain mostly colocalize with the early endosomal marker RAB-5. Bars, 2 µm.

Figure 8.

Overexpression of constitutively GTP-bound RAB-5 rescues the DCV maturation defects of unc-108 mutants. (A and B) GTP-restricted RAB-5(Q78L)DA was expressed panneuronally in the NLP-21–YFP-expressing strain nuIs183 in wild type and unc-108 mutants. RAB-5(Q78L)DA expression rescues the axonal NLP-21–YFP levels in unc-108(n501) mutants, whereas wild-type levels are not changed. Mean fluorescence was normalized to wild type. (C and D) Overexpression of RAB-5(Q78L)DA leads to the accumulation of NLP-21–YFP in the neuronal cell bodies in the VNC. (B and D) Error bars = SEM (***, P < 0.005; Student's t test). Bars: (A) 10 µm; (C) 2 µm.

Figure 9.

ric-19 mutants show similar phenotypes to unc-108 mutants. (A) ric-19 mutants show movement defects, which can be rescued by neuronal expression of ric-19. Knockdown of RIC-19 in unc-108 has no additional affect on locomotion. (B) ric-19(ok833) mutants are resistant to the ACh esterase inhibitor aldicarb, similarly to unc-108(n501). Aldicarb resistance can be rescued by neuronal expression of ric-19 (n = 30). (C and D) ric-19 deletion or ric-19 RNAi decreases axonal NLP-21–YFP fluorescence (nuIs183) similar to unc-108 mutants. ric-19 RNAi in unc-108 mutants shows epistasis. Neuronal expression of RIC-19 rescues DCV defects of ric-19 mutants. (E) Western blot showing the efficiency of RIC-19 knockdown by RNAi in unc-108(n501) mutants. (F and G) ric-19 mutants secrete less NLP-21–derived YFP (nuIs183), as measured by coelomocyte fluorescence, which is in agreement with the reduced axonal YFP levels in DCVs. This defect can be rescued by neuronal ric-19 expression. (A, B, D, and G) Error bars = SEM (**, P < 0.01; ***, P < 0.005; Student's t test). Bars: (C) 10 µm; (F) 5 µm.

Figure 10.

The RAB-2 GTP-bound form recruits RIC-19 to the Golgi. (A) When expressed in HeLa cells, RIC-19 coimmunoprecipitates with wild-type (WT) RAB-2 and its GTP-bound RAB-2(Q65L)DA form but not its inactive GDP-bound RAB-2(S20N)DN form. As a control, the amount of affinity-purified RAB-2 is shown below. IP, immunoprecipitation. (B) Endogenous RIC-19 levels are not altered in unc-108 mutants, as determined by Western blotting total extracts. (C) RIC-19 localization is mostly cytoplasmic in cells expressing the inactive RAB-2(S20N)DN, but RIC-19 is recruited to Golgi membranes by expression of either wild-type RAB-2 or, to a greater extent, GTP-bound RAB-2(Q65L)DA. Arrowheads show the recruitment of RIC-19 by overexpression of GTP-restricted (DA) RAB-2. (D) As compared with wild-type motor neurons, RIC-19–mYFP shows increased Golgi recruitment in the dominant unc-108 mutants (marked by arrowheads). Bars, 4 µm.