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First published online 6 February 2008
doi: 10.1242/dev.016063

Development 135, 1069-1080 (2008)
Published by The Company of Biologists 2008
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C. elegans Rab GTPase 2 is required for the degradation of apoptotic cells

Qun Lu1,3,*, Yan Zhang2,3,*, Tianjing Hu3,*, Pengfei Guo3, Weida Li3 and Xiaochen Wang3,{dagger}

1 College of Biological Sciences, China Agricultural University, Beijing 100094, China.
2 Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, China.
3 National Institute of Biological Sciences, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing, 102206, China.


Figure 1
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  		Fig. 1. unc-108 is important for cell corpse clearance in C. elegans. (A) Time-course analysis of cell corpse appearance during development in the wild type (N2, black), the unc-108(sm237) mutant (white) and in wild-type animals treated with unc-108 RNAi (gray). Cell corpses were scored at the following embryonic or larval stages: bean/comma (comma), 1.5-fold (1.5F), 2-fold (2F), 2.5-fold (2.5F), 3-fold (3F), 4-fold (4F) and early L1 larvae (L1). The y-axis represents the mean number of cell corpses scored at the head region of embryos or larvae; at least 15 animals were scored at each stage. Error bars indicate s.e.m. (B) unc-108(sm237) mutant contains persistent germ cell corpses. The number of germ corpses were scored every 12 hours after the L4/adult molt from one gonad arm in wild-type (N2, black), unc-108(sm237) mutant (white) and unc-108(RNAi) animals (gray). The y-axis represents the average number of germ cell corpses. At least 15 animals were scored at each time point. Error bars indicate s.e.m. In A and B, data derived from different genetic backgrounds at multiple developmental stages were compared by two-way analysis of variance. Post-hoc comparisons were by Fisher's PLSD (protected least squares differences). *P<0.05, **P<0.0001. All other points had P values>0.05. (C) Four-dimensional microscopy analysis of cell corpse duration in the unc-108(sm237) mutant. The duration of 33 cell corpses from wild-type (N2) embryos (n=3, black), unc-108(sm237) embryos (n=3, white) and unc-108(RNAi) embryos (n=3, gray) were followed. The numbers in parentheses indicate the average durations of cell corpses (±s.e.m.). The y-axis represents the number of cell corpses within a specific duration range as shown on the x-axis. The durations of four cell divisions in the MS cell lineage from MS cell to MS.aaaa cell were also followed to ensure that the embryos scored had similar development rates. The average duration of four cell divisions is 93±6 minutes in N2 embryos, 96±1 minutes in unc-108(sm237) embryos and 103±3 minutes in RNAi-treated embryos.

 

Figure 2
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  		Fig. 2. The coelomocytes of sm237 mutant C. elegans contain aberrant endosomes. (Aa-f) The coelomocyte uptake is reduced in the unc-108(sm237) mutant. The uptake of ssGFP in the coelomocyte of a wild-type animal (a,b), unc-108(sm237) mutant (c,d) and unc-108(RNAi) animal (e,f) carrying Pmyo-3ssgfp were examined by visualizing the GFP accumulation in the body cavity and coelomocytes. White dashed line indicates the outline of the coelomocyte and arrows point to the body cavity with accumulated GFP. (Ba-Df) sm237 animals contain abnormal endosomes. The coelomocytes of a wild-type animal (a,b), unc-108(sm237) mutant (Bc-Bf, c,d in C,D), unc-108(RNAi) animal (Bg-Bj, e,f in C,D) carrying different endosome and lysosome markers were examined. In wild-type coelomocyte, RME-8::GFP (B) associates with early and late endosomes; LMP-1::GFP (C) mostly stains lysosomes and GFP::RME-1 (D) marks recycling endosomes (arrows). Scale bars: 2.5 µm.

 

Figure 3
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  		Fig. 3. UNC-108 localizes to both endosomes and lysosomes. GFP::UNC-108 was specifically expressed in the coelomocyte driven by unc-122 promoter in cdIs113, which carries integrated pcc1:mCHERRY::RAB-5 (A), or in cdIs97 that contains integrated pcc1:mCHERRY::CUP-5 (B). GFP::UNC-108 was observed on endosomes where it overlapped with mCHERRY::RAB-5 (A, arrows) and on lysosomes marked by mCHERRY::CUP-5 (B, arrows). Scale bars: 2.5 µm.

 

Figure 4
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  		Fig. 4. Endocytic trafficking in the coelomocytes of the unc-108(sm237) mutant is blocked from late endosome to lysosome. (A-D) TR-BSA was injected into the body cavity and its transport through endocytic compartments is shown over time in wild-type (A,C) and unc-108(sm237) mutant (B,D) animals with endosomal marker RME-8::GFP (A,B) or lysosomal marker LMP-1::GFP (C,D). White arrows point to the compartments that contain TR-BSA. The blue arrow in D indicates the normally sized lysosome that lacks TR-BSA. Scale bars: 2.5 µm.

 

Figure 5
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  		Fig. 5. Yolk protein trafficking is blocked in the unc-108(sm237) mutant. (Aa-d) Yolk protein uptake is normal in the developing oocytes of unc-108(sm237) mutant C. elegans. The accumulation of VIT-2::GFP in the developing ooctyes was examined in wild-type (bIs1) (a,b) and in the sm237 mutant [unc-108(sm237); bIs1] (c,d). (Ba-d) The yolk protein is redistributed to the intestine primordium (arrow) in the wild-type 1.5-fold (a,b) and 4-fold (c,d) stage embryos. (Ca-Dd) Yolk protein trafficking is blocked in the unc-108(sm237) mutant (C) or unc-108(RNAi) animal (D). 1.5-(a,b) and 4-fold (c,d) stage embryos were examined for the redistribution of VIT-2::GFP from the anterior region to the gut primordium. Abundant VIT-2::GFP signal could be observed in the anterior region of the embryos (arrow). Scale bars: 1 µm in A; 5 µm in B-D.

 

Figure 6
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  		Fig. 6. The persistent cell corpses in the unc-108(sm237) mutant are labeled by Acridine Orange. AO staining of a 1.5-fold stage embryo of wild type (A,B), and of a 4-fold stage embryo of unc-108(sm237) (C,D), unc-108(RNAi) (E,F) or ced-1(e1735) (G,H) that contain apoptotic cells (A,B) or persistent cell corpses (C-H). Bright AO staining was observed in the dying cell of the wild-type embryo and the persistent cell corpses in the unc-108(sm237) mutant and unc-108(RNAi) embryos, but not in the ced-1(e1735) mutant (arrows). Scale bars: 5 µm.

 

Figure 7
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  		Fig. 7. UNC-108 associates with phagosomes. (Aa,b) UNC-108::GFP clusters around cell corpses (arrow). DIC (a) and fluorescent confocal (b) images of a wild-type C. elegans embryo transgenic for Punc-108unc-108::gfp. (Ba-o) UNC-108::mCHERRY is recruited to the phagosome preceded by CED-1::GFP. DIC (a,d,g,j,m), confocal time-lapse images of CED-1::GFP (b,e,h,k,n) and UNC-108::mCHERRY (c,f,i,l,o) around the same cell corpse in a wild-type embryo. The time point was set as 0 minute when the CED-1::GFP ring was clearly seen. Images from five time points after that are shown. Arrows point to the cell corpse and to the corresponding fluorescent signals. (Ca-l) UNC-108::GFP 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 Punc-108unc-108::gfp and Pced-1mcherry::rab-5 (a-d) or Punc-108unc-108::gfp and Pced-1mcherry::rab-7 (e-h) or Punc-108unc-108::gfp and Pced-1lmp-1::mcherry (i-l). Arrows indicate the co-localization of UNC-108::GFP with mCHERRY::RAB-5, mCHERRY::RAB-7 or LMP-1::mCHERRY on the phagosome. Scale bars: 5 µm.

 

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