DEV016063 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. unc-108 encodes C. elegans Rab GTPase 2. (A) Cloning of unc-108. Above is shown the genetic map of the unc-108 region. Beneath is shown the rescue of unc-108(sm237). Fourfold stage embryos non-transgenic or transgenic for each construct were scored. The rescue activity was calculated by subtracting the average number of cell corpses of transgenic embryos from that of non-transgenic embryos and divided by the mean number of cell corpses of the non-transgenic embryos. The resulting rescue % is indicated as: +/−, 30-44%; +, 45-64%; ++, 65-80%; +++, more than 80%; −, less than 25%. The number of cell corpses scored (transgenic and non-transgenic) for each rescue experiment is the mean number of all transgenic lines obtained as indicated in parentheses. (B) Alignment of amino acid sequence of C. elegans RAB-2 (UNC-108), D. melanogaster (d), mouse (m) and human (h) RAB2. Residues that are identical in all four proteins are shaded in black and residues that are similar are in gray. The conserved GTP-binding motifs are boxed. The mutations identified in the sm237 mutant and in the other two unc-108 alleles isolated previously (n501 and n777) are marked.

• Supplemental Figure S2 -

  Fig. S2. Two other alleles of unc-108 showed a weak engulfment defect. (A) ok1246 has a 2198 bp deletion that removes the gene locus of unc-108. (B) n501 and n777 display a weak engulfment defect. Time-course analysis of cell corpses during embryonic development in the wild type (N2, black), the n501 mutant (white) and the n777 mutant (gray) were performed and analyzed as described in Fig. 1. **P<0.0001, *P<0.05; all other points have P-value>0.05. (C) Overexpression of UNC-108(G18E) causes persistent cell corpse phenotype. Wild-type embryos transgenic for P_hspUNC-108(G18E) were kept at 20°C (−) or heat-shocked at 33°C for 1 hour (+), followed by recovery at 20°C. Cell corpses were scored 7 hours after treatment. Embryos from the wild-type or the sm237 mutant that do not carry any transgene were also treated and scored under the same conditions. Average numbers of cell corpses at the 4-fold stage were determined (at least 15 animals were scored) and data from three independent transgenic lines are shown.

• Supplemental Figure S3 -

  Fig. S3. unc-108 is expressed in the known engulfing cells, coelomocyte and neuronal cells. The expression of UNC-108::GFP (Aa-h) or GFP::UNC-108 (Ba-h) driven by its own promoter was examined in wild-type animals. Both N-terminally and C-terminally GFP-tagged UNC-108 showed expression in the hypodermal cells (a,b, arrow), the gonadal sheath cells (c,d, arrow) and the coelomocytes (e,f) with GFP signal observed on both endosomes and lysosomes as indicated by blue and white arrows, respectively. UNC-108 was also seen in the head, tail and ventral cord neurons in a wild-type larva (g,h). GFP::UNC-108 displayed significantly more vesicular localizations than the C-terminally tagged UNC-108 as indicated by the red arrow. Scale bars: 5 µm in a-d; 2.5 µm in e,f; 20 µm in g,h.

• Supplemental Figure S4 -

  Fig. S4. unc-108(sm237) mutant contains large vacuoles with both endosomal and lysosomal components. (Aa-d) mCHERRY::CUP-5 localizes to lysosome in wild-type coloemocyte (a,b) and associates with the large vacuole in the sm237 mutant (c,d). (Ba-d) mCHERRY::RAB-7 associates with late endosomes and lysosomes in wild-type coelomocyte (a,b) and labels the large vacuole in the coelomocyte of sm237 mutant (c,d). (Ca-l) RME-8::mRFP and LMP-1::GFP localized separately to endosomes and lysosomes in wild-type coloemocyte, as indicated by white and blue arrows, respectively (a-d), but co-localized to the large vacuole in the sm237 mutant (e-h) or unc-108(RNAi) animal (i-l) (arrows). (Da-l) RME-8::GFP and mCHERRY::CUP-5 localized separately to endosomes and lysosomes in wild-type coelomocyte as indicated by white and blue arrows, respectively (a-d), but co-localized to the large vacuole in the sm237 mutant (e-h) or unc-108 (RNAi) animal (i-l) (arrows). Scale bars: 2.5 µm.

• Supplemental Figure S5 -

  Fig. S5. The apical uptake in intestine is blocked in the unc-108(sm237) mutant. (Aa-f) Wild-type (a-c) and sm237 animals (d-f) were soaked in TR-BSA and the apical uptake in the intestine was examined. TR-BSA was efficiently taken up into the intestine cells in wild-type, but accumulated in the intestinal lumen of unc-108(sm237) mutant (arrow). (Ba-f) The apical uptake of lipophilic dye FM4-64 in the intestine was examined similarly to that of TR-BSA. FM4-64 was quickly taken up from the intestinal lumen and transported to the intestine cells in wild-type animals (a-c). The accumulation of FM4-64 in the intestinal lumen in unc-108(sm237) animals (d-f) indicates defective apical uptake. Scale bars: 50 µm.

• Supplemental Figure S6 -

  Fig. S6. Endocytic trafficking is blocked from late endosome to lysosome after unc-108 RNAi treatment. unc-108 RNAi was performed as described in the Materials and methods. TR-BSA was injected into the body cavity and its transport through endocytic compartments was analyzed over time with endosomal marker RME-8::GFP (A) or lysosomal marker LMP-1::GFP (B). TR-BSA was trapped in the endosomes (white arrows). Scale bars: 2.5 µm.

• Supplemental Figure S7 -

  Fig. S7. N-terminally tagged UNC-108 showed similar phagosomal association and recruitment kinetics. (Aa,b) GFP::UNC-108 clusters around cell corpse. DIC (a) and fluorescent confocal (b) images of a wild-type embryo transgenic for P_unc-108 gfp::unc-108. The white arrow points to a cell corpse with typical ‘raised button’ morphology and GFP::UNC-108 clustering, whereas the blue arrow points to a GFP::UNC-108-positive cell corpse that was being degraded. (Ba-o) mCHERRY::UNC-108 is recruited to the phagosome preceded by CED-1::GFP. DIC (a-d), confocal time-lapse images of CED-1::GFP (e-h) and mCHERRY::UNC-108 (i-l) around the same cell corpse in a wild-type embryo. The time point was set as 0 minute when a CED-1::GFP ring was clearly seen. Images of five time points after that are shown. Arrows point to the cell corpse and the corresponding fluorescent signals. The association of mCHERRY::UNC-108 with the phagosome was clearly seen at +21 minutes, when the button-like morphology of the cell corpse was being lost. (Ca-l) GFP::UNC-108 co-localizes with mCHERRY::RAB-5, mCHERRY RAB-7 and LMP-1::mCHERRY to the phagosome. DIC and fluorescent confocal images of a wild-type embryo transgenic for P_unc-108gfp::unc-108 and P_ced-1mcherry::rab-5 (a-d) or P_unc-108gfp::unc-108 and P_ced-1mcherry::rab-7 (e-h) or P_unc-108gfp::unc-108 and P_ced-1lmp-1::mcherry (i-l). Arrows indicate the co-localization of GFP::UNC-108 with mCHERRY::RAB-5, mCHERRY::RAB-7 or LMP-1::mCHERRY on the phagosome. Scale bars: 5 µm.