RAB-6.1 promotes GLR-1 recycling from endosomes and prevents turnover.

<p>GLR-1::GFP fluorescence observed in ventral cord dendrites of animals that express a dominant negative (dn) VPS-4 ESCRT subunit in (A) wild type or (B) <i>rab-6</i>.<i>1(tm2124)</i>. The (C) mean density of GLR-1 puncta and (D) spontaneous reversal frequency for the indicated genotype. (E, F, G) GLR-1::GFP and (E’, F’, G’) mRFP::SYX-7 (Syntaxin-13) fluorescence in neuron cell bodies from (E-E”‘) wild-type animals, (F-F”‘) <i>rab-6</i>.<i>1(tm2124)</i> mutants, and (G-G”‘) <i>rab-6</i>.<i>1(tm2124); vps-4(dn)</i> double mutants. Merged images are shown in E”, F”, and G”. Binary masks (E”‘, F”‘, G”‘) indicate colocalization by highlighting pixels with matching intensity values. (H) The mean percent of GLR-1::GFP, colocalized with endosomal marker mRFP::SYX-7, normalized to total GLR-1::GFP in cell bodies. Bar, 5 μm. Error bars are SEM. N = 20–35 animals. ANOVA with Dunnett’s multiple comparison to wild type (#p<0.05; ###p<0.001) or Bonferroni Multiple Comparison test (*p<0.05; ***p<0.001). “n.s.” indicates non-significance.</p

RAB-6.1 colocalizes with Mannosidase/MANS, RME-8 and RAB-6.2, but not with RAB-5 and RAB-7 in intestinal epithelial cells.

<p>GFP-, tagRFP-, or mCherry-tagged proteins were expressed in intestinal epithelial cells specifically using the <i>vha-6</i> promoter. Fluorescent images are shown for the indicated markers in wild-type animals. (A) GFP::RAB-6.1 is colocalized with (A’) Golgi marker MANS::mCherry. (B,C) GFP::RAB-6.1 is not colocalized with (B’) early endosome marker tagRFP::RAB-5 and (C’) late endosome/lysosome marker tagRFP::RAB-7. (D) GFP::RME-8 is localized near (D’) tagRFP::RAB-6.1. GFP::RME-8 and tagRFP::RAB-6.1 do not overlap, but are adjacent (within 1 μm). (E) GFP::RAB-6.1 is colocalized with (E’) tagRFP::RAB-6.2. (A”-E”) Merged images are shown. Intestinal autofluorescent lysosome-like organelles are shown in the DAPI channel (blue) in all panels. Bar, 5 μm.</p

RAB-6.1 functions downstream of GLR-1 endocytosis.

<p>GLR-1::GFP fluorescence in (A) wild-type animals that express a dominant negative RAB-5(GDP) or (B) <i>rab-6</i>.<i>1(tm2124)</i> mutants that express RAB-5(GDP). The fluorescence of GLR-1(4KR)::GFP in (C) wild-type animals or (D) <i>rab-6</i>.<i>1(tm2124)</i> mutants. (E) The mean density of fluorescent puncta for the given genotype and reporter (blue bars, GLR-1::GFP; red bars, GLR-1(4KR)::GFP). Bar, 5 μm. Error bars are SEM. N = 20–30 animals. ANOVA with Bonferroni Multiple Comparison test. “n.s.” indicates non-significance.</p

RAB-6.1 and RAB-6.2 direct MIG-14 back to Golgi.

<p>Fluorescent images were acquired from (A-A”) wild-type animals, (B-B”) <i>rab-6</i>.<i>1(tm2124)</i> mutants, or (C-C”) <i>rab-6</i>.<i>2(ok2254)</i> mutants expressing (A,B,C) MIG-14::GFP and (A’,B’,C’) MANS::mCherry. (A”,B”,C”) Merged images. Intestinal autofluorescent lysosome-like organelles are shown in the DAPI channel (blue). Bona fide fluorescence from GFP or RFP-tagged marker proteins was identified as fluorescence acquired in the green or red channels that also did not overlap with fluorescence acquired in the DAPI channel. MIG-14::GFP colocalized with Golgi marker MANS::mCherry in wild-type animals (A-A” and enlarged inset). Most MANS::mCherry-labeled Golgi ministacks lack MIG-14::GFP localization in <i>rab-6</i>.<i>1(tm2124)</i> mutants (B-B” and enlarged inset) and <i>rab-6</i>.<i>2(ok2254)</i> mutants (C-C” and enlarged inset), although MANS::mCherry and MIG-14::GFP puncta are typically adjacent (within 1 μm) in <i>rab-6</i>.<i>1</i> mutants. (D) Quantification of the number of MAN::mCherry puncta. (E) Quantification of MANS::mCherry and MIG-14::GFP colocalization as measured through average correlation coefficient. Bar, 5 μm. Error bars are SEM. N = 20. ANOVA with Dunnett’s multiple comparison to wild type (###p<0.001) or Bonferroni Multiple Comparison test (***p<0.001).</p

Gene structure for <i>rab-6</i>.<i>1</i> and <i>rab-6</i>.<i>2</i>.

<p>(A) Sequence alignments based on ClustalW for four human (H.s.) Rab6 isoforms, the yeast (S.c.) isoform Ypt6, and the two <i>C</i>. <i>elegans</i> (C.e.) Rab6 isoforms. Conservation of identical and similar amino acids are indicated by black and gray highlighting, respectively. “RabF” refers to Rab family specific regions. “RabSF” refers to Rab subfamily specific regions. “PM” refers to phosphate/magnesium binding residues. “G” refers to guanine nucleotide binding regions. “!” refers to amino acids that differentiate the Rab6A/RAB-6.1 subfamily from the Rab6B/RAB-6.2 subfamily. Orange asterisks indicate the three residues that differ between RAB6A and RAB6A’. Blue asterisks indicate the sites commonly mutated to generate GTP-locked and GDP-locked mutant proteins. The Switch I and Switch II regions are also indicated. Accession numbers: CAG46781.1 (Rab6A); AAF73841.1 (Rab6A’); AAF61637.1 (Rab6B); CAG38500.1 (Rab6C); CAA77590.1 (RAB-6.1); C<i>CD74453</i>.<i>1 (RAB-6</i>.<i>2); and Q99260</i>.<i>1 (Ypt6)</i>. (B) Genomic organization for <i>rab-6</i>.<i>1</i> and <i>rab-6</i>.<i>2</i>. Gray boxes indicate exons. The arrow indicates the start of transcription based on EST alignments with SL1 leader sequences. The brackets indicate the regions of genomic DNA that encode the indicated protein domains. The blue, red, and green lines indicate the regions of genomic DNA deleted in the <i>tm2124</i>, <i>tm2032</i>, and <i>ok2254</i> mutants, respectively. The chromosomal linkage groups (LG) for each gene are indicated.</p

RAB-6.1 is broadly expressed in multiple tissues.

<p>Fluorescence from <i>P</i>_{<i>rab-6</i>.<i>1</i>}::<i>GFP</i> in (A) body wall muscles, (B) intestinal epithelia, (C) the gonad, (D) the distal tip cells, and (E) the vulva. Fluorescence from (F) <i>P</i>_{<i>rab-6</i>.<i>1</i>}::<i>GFP</i> and (F’) <i>P</i>_<i>glr-1</i>::<i>mRFP</i>, with (E”) images merged. The neuron cell bodies for RMDV, AVA, and AVB are outlined. NR indicated the nerve ring bundle of neurites. Bar, 5 μm.</p

RAB-6.1 and RAB-6.2 drive MIG-14 out of early endosomes.

<p>Fluorescent images were acquired in (A-A”) wild-type animals, (B-B”) <i>rab-6</i>.<i>1(tm2124)</i> mutants, or (C-C”) <i>rab-6</i>.<i>2(ok2254)</i> mutants expressing (A,B,C) MIG-14::GFP and (A’,B’,C’) tagRFP::RAB-5. Intestinal autofluorescent lysosome-like organelles are shown in the DAPI channel (blue). MIG-14::GFP colocalized with tagRFP::RAB-5 labeled early endosomes in wild-type animals (A-A” and enlarged inset), <i>rab-6</i>.<i>1(tm2124)</i> mutants (B-B” and enlarged inset), and <i>rab-6</i>.<i>2(ok2254)</i> mutants (C-C” and enlarged inset). (D) Quantification of tagRFP::RAB-5 and MIG-14::GFP colocalization as measured through average correlation coefficient. Bar, 5 μm. Error bars are SEM. N = 20. ANOVA with Dunnett’s multiple comparison to wild type (###p<0.001) or Bonferroni Multiple Comparison test (**p<0.01).</p

RAB-6.1 regulates GLR-1 synaptic localization.

<p>GLR-1::GFP fluorescence along ventral cord dendrites is indicated in (A) wild-type animals, (B) <i>rab-6</i>.<i>2(ok2254)</i> mutants, (C) <i>rab-6</i>.<i>1(tm2124)</i> mutants, (D) <i>rab-6</i>.<i>1(tm2124)</i> mutants expressing a wild-type <i>rab-6</i>.<i>1</i> cDNA from the <i>glr-1</i> promoter, (E) <i>rab-6</i>.<i>1(ok2254)</i> mutants expressing a <i>rab-6</i>.<i>1</i> hairpin RNA, and (F) <i>rab-6</i>.<i>2(ok2254)</i> mutants expressing a dominant negative RAB-6.1(GDP) protein. Fluorescence from the synaptic vesicle protein SNB-1(synaptobrevin)::GFP is indicated in (G) wild-type animals and (H) <i>rab-6</i>.<i>1(tm2124)</i> mutants. (I) The mean density of fluorescent puncta for the given genotype and fluorescent reporter (blue bars, GLR-1::GFP; red bars, SNB-1::GFP). (J) The mean spontaneous reversal frequency for the indicated genotypes. Bar, 5 μm. Error bars are SEM. N = 15–35 animals. ANOVA with Dunnett’s multiple comparison to wild type (###, p<0.001) or Bonferroni Multiple Comparison test (*p<0.05; **p<0.01; ***p<0.001). “n.s.” indicates non-significance.</p

RAB-6.1 colocalizes with RAB-6.2 at the Golgi in neurons.

<p>(A,B,C) Subcellular localization of the indicated GFP::RAB-6.1 variant: (A) wild type, (B) a GDP-locked mutant, and (C) a GTP-locked mutant. (D) CFP::RAB-6.1 and (D’) MANS::YFP in interneuron cell bodies (PVC shown). (D”) Merged image showing CFP::RAB-6.1 colocalization to puncta with MANS::YFP. (E, F) Cerulean::RAB-6.2 and (E’, F’) Venus::RAB-6.1 in interneuron (E-E”) cell bodies (PVC shown) and (F-F”) ventral cord dendrites. Merged images (E”, F”) show colocalization at specific points. Bar, 5 μm.</p

Recycling of MIG-14::GFP requires RAB-6.1 and RAB-6.2 activity.

<p>Intestinally expressed MIG-14::GFP in (A,B,C,D,E) top and (A’,B’,C’,D’,E’) middle focal planes of intestinal epithelial cells was examined by laser scanning confocal microscopy in (A) wild-type animals, (B) <i>rab-6</i>.<i>1(tm2124)</i> mutants, (C) <i>rab-6</i>.<i>2(ok2254)</i> mutants, (D) <i>rab-6</i>.<i>2(ok2254)</i> mutants treated by <i>rab-6</i>.<i>1</i> feeding RNAi since L1 stage, and (E) <i>rme-8(b1023)</i> mutants. (F,G) Quantified average MIG-14::GFP fluorescent intensity (F) and puncta number (G) for the indicated genotypes. Bar, 5 μm. Error bars are SEM. N = 24. ANOVA with Dunnett’s multiple comparison to wild type (###p<0.001) or Bonferroni Multiple Comparison test (***p<0.001).</p