Two Rab2 interactors regulate dense-core vesicle maturation

Abstract

Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network.

Figure 1. rund-1 and cccp-1 encode conserved proteins

(A) Gene structures of rund-1 and cccp-1. Colored boxes show coding segments. White boxes show untranslated regions. cccp-1 has two transcripts. (B) Domain structures of RUND-1 and CCCP-1. RUND-1 is a 549 amino acid protein with two coiled-coil (CC) domains and a RUN domain. ox328 is a missense mutation in the second coiled-coil domain. ox281 is a splice site mutation. The tm3622 deletion is marked, but because this deletion starts in an intron (Figure 1A), its effect on the protein is unknown. CCCP-1b is a 743 amino acid protein with multiple coiled-coil (CC) domains. The ox334 and e1122 mutations lead to premature stop codons. (C) Alignment of the second coiled-coil domain of RUND-1 and its orthologs from Trichoplax adhaerens (Trichoplax), C. elegans (worm), Drosophila melanogaster (fly) and Homo sapiens (human). The position of the ox328 T237P mutation is shown by an asterisk. (D) Alignment of the RUN domain of RUND-1. The six conserved blocks A-F of the RUN domain (Callebaut et al., 2001) are marked with black bars. The tm3622 deletion removes the A block, but ends before the beginning of the B block. See also Figures S1 and S2.

Figure 2. rund-1 mutants exhibit unmotivated locomotion, but respond to stimulation

(A and B) Locomotion of wild-type and rund-1 mutants. The graphs plot mean speed during the 30 minutes after transfer to a new plate. Wild-type locomotion is stimulated by transfer and decays to baseline in twenty minutes. rund-1 mutants are stimulated by transfer, but less than wild type, and have a reduced baseline locomotion rate. n=21–24 animals. (C) Left, mean crawling speed during the first two minutes after transfer to a new plate. All three rund-1 mutants exhibit reduced speed (*** P<0.001 vs wild type) and the rund-1(tm3622) mutant is fully rescued by a rund-1(+) single-copy transgene. Right, expression of rund-1(+) in the nervous system rescues the rund-1 mutant. The rund-1 cDNA was expressed in either the nervous system (Prab-3) or the intestine (Pvha-6). Expression in the nervous system rescued (** P<0.01), but expression in the intestine did not (P>0.05). Error bars=SEM; n=21–27 (left) and n=9 (right). (D) The rund-1 locomotion defect is rescued by expression of a rund-1(+) transgene in mature animals. The frequency of body bends was measured during the first two minutes after transfer to a new plate. Heatshock-induced expression of rund-1(+) fully rescued the rund-1 mutant defect (*** P<0.001 vs no heatshock). Heatshock did not affect rund-1 (P>0.05). +hs: heatshock, -hs: no heatshock. Error bars=SEM; n=10. (E) rund-1 and rab-2 act in the same genetic pathway to regulate locomotion. Mean speed was measured for two minutes after transfer to a new plate. The rab-2 rund-1 double mutant is similar to the rab-2 single mutant. Error bars=SEM; n=24–31. (F) rund-1 and cccp-1 act in the same genetic pathway to regulate locomotion. Frequency of body bends (“thrashes”) in liquid was reduced for rund-1(ox281), cccp-1(ox334) and cccp-1(ox334); rund-1(ox281) mutants compared to wild type (P<0.001). The cccp-1 rund-1 double mutant is similar to the single mutants (P>0.05). Error bars=SEM; n=8–9. (G) rund-1 and ric-19 act in parallel to regulate locomotion. Frequency of body bends was measured for two minutes after transfer to a new plate. ric-19(pk690) rund-1(tm3622) animals show a more severe defect than either single mutant (*** P<0.001; ** P<0.01). ric-19 is not significantly different from wild type (P>0.05). The wild type and rund-1 data are identical to Figure 2D; these experiments were performed together. Error bars=SEM; n =10. See also Figures S3 and S7, and Table S2.

Figure 3. RUND-1 is not required to make DCVs

(A) Electron microscopy of synapses in the ventral nerve cord of wild type and rund-1(tm3622). White arrowheads point to DCVs. Black arrowheads point to the presynaptic density. Scale bar: 200 nm. (B) Quantification of synaptic vesicles (SVs) and dense-core vesicles (DCVs) at synapses in the ventral cord (V) or dorsal cord (D).

Figure 4. rund-1 and cccp-1 mutants have defects in trafficking DCV cargos

(A) Images of NLP-21::Venus fluorescence in motor neuron axons of the dorsal nerve cord. rab-2, rund-1, and cccp-1 mutants have decreased levels of fluorescence in the dorsal cord, indicative of an NLP-21::Venus trafficking defect. The final two panels (WT and cccp-1) were from a separate experiment. Scale bar: 5 µm. (B) NLP-21::Venus fluorescence levels are decreased in the dorsal cord in rab-2, rund-1, and cccp-1 mutants. The mean fluorescence intensity per µm is given in arbitrary units. All single mutants are defective in trafficking NLP-21::Venus to the dorsal cord (*** P<0.001 ; * P<0.05 vs wild type). The rab-2 rund-1 double mutant is not significantly different from the single mutants (P>0.05). Error bars=SEM; n=9–22. (C) Trafficking of NLP-21::Venus and FLP-3::Venus is rescued by egl-3 mutants. The peptide processing mutant egl-3 suppresses NLP-21::Venus and FLP-3::Venus trafficking defects of the rund-1(tm3622) mutant (** P<0.01, *** P<0.001 respectively). The rund-1 NLP-21::Venus defect is rescued by expression of the wild-type rund-1 cDNA in the dorsal motor neurons (*** P<0.001). Error bars=SEM; n=6–11. (D) Secreted NLP-21::Venus fluorescence levels in coelomocytes. cccp-1(ox334) has decreased coelomocyte fluorescence (*** P<0.001) but rund-1(tm3622) has no defect (P>0.05). Neither rund-1 nor cccp-1 has decreased fluorescence in the ventral cord (P>0.05). Error bars=SEM; n=13–21. (E) rab-2, rund-1, and cccp-1 act in the same genetic pathway. Double mutants of rab-2(nu415), rund-1(tm3622) and cccp-1(ox334) are similar to single mutants in trafficking NLP-21::Venus to the dorsal cord (P>0.05 for all pairwise comparisons except cccp-1; rund-1 vs. cccp-1, P<0.05). All mutants exhibit significant defects compared to wild type (P<0.001). Error bars=SEM; n=10–13. (F) IDA-1::GFP fluorescence levels in the dorsal cord. The rab-2(nu415), rund-1(tm3622), and cccp-1(ox334) mutants are defective in trafficking IDA-1::GFP (*** P<0.001 and ** P<0.01 vs WT). Error bars=SEM; n=9–13.

Figure 5. RUND-1 colocalizes with RAB-2 at the trans-Golgi network

(A) RUND-1 colocalizes with RAB-2 and trans-Golgi markers. Each panel shows a single slice of a confocal image of motor neuron cell bodies in the ventral cord of young adult animals. The top boxes show the localization of a single-copy rescuing RUND-1::tagRFP-T fusion protein (oxIs590). RUND-1 localizes almost exclusively to two or three perinuclear puncta per cell. The middle boxes show the localization of single-copy GFP-tagged compartment markers. The bottom boxes show the merged images. Scale bar: 5 µm, applies to all panels. RUND-1 shows tightest colocalization with RAB-6.2, a trans-Golgi Rab protein, and with SYX-6, the ortholog of the trans-Golgi SNARE syntaxin 6. RUND-1 puncta also colocalize well with RAB-2 puncta. RUND-1 partially overlaps with the medial-Golgi marker mannosidase II (AMAN-2) and the cis-Golgi marker εCOP. RUND-1 is not colocalized with the rough ER marker TRAM-1 nor with the endosomal markers RAB-5, RAB-11.1, RAB-7 and SYN-13. (B) RUND-1 localizes to the Golgi. The left panel shows a backscatter scanning electron micrograph of the cell body of a neuron in the nerve ring. The right panel shows the same image overlaid with the corresponding fluorescence PALM image of RUND-1::tdEos. Scale bar: 1 µm. (C) CCCP-1 colocalizes with RUND-1 and RAB-2. CCCP-1 colocalizes with RUND-1 and RAB-2(GTP). CCCP-1 is still punctate when coexpressed with RAB-2(GDP), which is diffusely expressed. (D) The RUN domain of RUND-1 mediates its localization. Full length RUND-1, the coiled-coil domain, and the RUN domain were tagged at their C-termini with tagRFP-T and integrated in the genome. The truncated proteins were expressed at lower levels. See also Figure S8.

Figure 6. RUND-1 and CCCP-1 interact physically with activated RAB-2

(A) RUND-1 interacts specifically with GTP-bound RAB-2 (RAB-2 GTP) in a yeast two-hybrid assay. RUND-1 did not show an interaction with wild-type RAB-2 (RAB-2 WT) or inactive GDP-bound RAB-2 (RAB-2 GDP). CCCP-1 interacts with RAB-2 GTP and RAB-2 WT, but not with RAB-2 GDP. Growth without histidine (- his) indicates a physical interaction. (B) The RUND-1 and CCCP-1 interactions with RAB-2 are specific. The interactions of RUND-1 and CCCP-1 with C. elegans RAB proteins were examined by yeast two-hybrid. Numbers indicate the number of the RAB gene in C. elegans (e.g. 1 = RAB-1). RUND-1 and CCCP-1 interact only with RAB-2. RAB-27 could not be tested because of self-activation. (C) RUND-1 interacts with RAB-2 via the RUN domain. Two truncations of RUND-1 were used: RUND-1 (CC) consists of amino acids 1–261. RUND-1 (RUN) consists of amino acids 262–549. (D) RUND-1 interacts with RIC-19 and TBC-8. V5-tagged RUND-1 was coexpressed with GFP, GFP::RIC-19 or GFP::TBC-8 in HEK293 cells. Immunoprecipitation of GFP::RIC-19 or GFP::TBC-8 pulled down RUND-1. Immunoprecipitation of untagged GFP did not pull down RUND-1. IN: input, IP: immunoprecipitation, IB: immunoblotting. (E) RUND-1 interacts with TBC-8 outside of its TBC domain. Truncations of TBC-8 were examined for interactions with RUND-1 by yeast two-hybrid. (F) RUND-1 interacts with TBC-8 outside of its TBC domain. V5-tagged TBC-8 (1–597 aa) was coexpressed with GFP or GFP::RUND-1 in HEK293 cells. Immunoprecipitation of GFP::RUND-1 pulled down TBC-8 (1–597). IN: input, IP: immunoprecipitation, IB: immunoblotting.

Figure 7. Model for RUND-1 and CCCP-1 action in DCV maturation

(A) RUND-1 may be in a complex with activated RAB-2, RIC-19 and TBC-8 since all of these molecules bind each other in pairwise combinations. CCCP-1 binds activated RAB-2, but does not bind RUND-1, RIC-19 or TBC-8, so it may be in a separate complex. (B) RUND-1 and CCCP-1 localize near the trans-Golgi and are involved in regulating cargo sorting during the formation of mature DCVs. Soluble cargo (green dots) are retained in the mature vesicle and released at the plasma membrane. In the absence of rund-1 and cccp-1, immature DCVs may have an improper identity (denoted by ‘?’), causing them to lose soluble cargo to the endolysosomal system. However, insoluble cargo is not lost, including peptides that aggregate and form the characteristic dense-core seen by EM. The process of aggregation is depicted as a gradual graying and condensation of the vesicle center during the maturation process. Though axonally localized DCVs carry reduced amounts of certain cargos in rund-1 mutants, overall release is normal as assayed by coelomocyte uptake, suggesting that the “lost” cargos are still secreted, perhaps as a result of their missorting to the constitutive secretory pathway. In cccp-1 and rab-2 mutants, release of such cargos is reduced, suggesting that they may be misdirected to the lysosome and degraded.