Figure 1.
Clathrin and the AP2 complex are essential for apoptotic cell clearance.
(A) Quantification of germ cell corpses in N2, chc-1(b1025ts), and chc-1(RNAi) animals. Error bars represent SEM. * p<0.05, ** p<0.001. All other points had p>0.05. (B) Quantification of germ cell corpses in animals treated with RNAi of individual genes of the AP2 complex. Comparisons were performed between control (Ctrl) RNAi - and other RNAi treatments using unpaired t-tests. ** p<0.001. All other points had p>0.05. (C) Four-dimensional microscopy analysis of germ cell corpse duration in animals treated with Ctrl RNAi, chc-1 RNAi and apb-1 RNAi. 30 germ cell corpses were recorded for each RNAi treatment. Numbers in parenthesis indicate the average time of corpse duration (mean±SEM). (D) Representative transmission electron micrographs of an engulfed (top row) and an unengulfed (middle row) germ cell corpse in chc-1(RNAi) animals. Traces of membranes are shown in the left panels and boxed regions in the middle panels are magnified and shown in the right panels. Black arrows indicate gonadal sheath cell membranes. Black and white arrowheads indicate cell corpse membranes and germline syncytium membranes, respectively. Total numbers of germ cell corpses analyzed for each RNAi treatment are shown in the table (bottom row). * Data cited from our previous work [22].
Figure 2.
Clathrin and AP2 likely act in the same genetic pathway as CED-1 and CED-6.
(A and B) Quantification of germ cell corpses in ced-1(e1735) and ced-6(n2095) (A) or ced-2(n1994) and ced-5(n1812) (B) mutants treated with Ctrl RNAi and RNAi of chc-1, dpy-23 and apb-1. Cell corpses were scored in animals at 24 and 36 h after the L4 molt. Error bars represent SEM. Comparisons were made between control RNAi and RNAi of chc-1, dpy-23 and apb-1 using unpaired t-tests. ** p<0.001; all other points had p>0.05. (C) Representative images of cell corpse labeling by APA-2::GFP in N2, ced-1(e1735), chc-1(RNAi) and ced-6(RNAi) germ lines. (D) Representative images of cell corpse labeling by mCherry::CHC-1 in N2, apb-1(RNAi), ced-1(RNAi) and ced-6(RNAi) germ lines. In (C) and (D) arrows indicate cell corpses labeled by APA-2::GFP or mCherry::CHC-1 while arrowheads indicate unstained corpses. Bars, 10 µm.
Figure 3.
CHC-1 and AP2 are required for the rearrangement of the actin cytoskeleton necessary for cell corpse engulfment.
(A) Representative images of cell corpse labeling by GFP::ACT-1 in Ctrl(RNAi), ced-1(e1735), ced-6(RNAi), chc-1(RNAi) and apb-1(RNAi) germ lines. Arrows indicate cell corpses encircled by GFP-ACT-1 and arrowheads indicate unlabelled corpses. Bars, 10 µm. (B) Quantification of the labeling of germ cell corpses by GFP::ACT-1 as shown in (A). (C and D) Representative images of co-localization of mCherry::ACT-1 with CED-1::GFP (C) and GFP::CED-6 (D) on phagosomes. Arrows indicate phagosomes stained by both mCherry::ACT-1 and CED-1::GFP or GFP::CED-6 and arrowheads indicate phagosomes only positive for CED-1::GFP or GFP::CED-6. Bars, 10 µm. (E and F) Quantification of mCherry::ACT-1 labeling of CED-1::GFP-positive (E) and GFP::CED-6-positive phagosomes (F). In B, E and F, ≥100 corpses were scored for each genotype.
Figure 4.
CHC-1 and AP2 components interact with CED-1 or CED-6.
(A) 35S-labeled APA-2, APB-1, DPY-23 and His6-tagged CHC-1C (amino acids 825–1682) were incubated with immobilized GST, GST-CED-1C (amino acids 933–1111) and GST-CED-6. After extensive washing, bound proteins were viewed by autoradiography or detected using immunoblotting with His6 antibody. (B) Quantification of mCherry::CHC-1(yqIs98) labeling of CED-1::GFP (smIs34)- or CED-1ΔC::GFP (smIs110)-positive cell corpses in ced-1(e1735) adult germ lines. ≥50 GFP-positive cell corpses were scored from germ lines 48 h post the L4 molt. (C) CED-6 associated with CED-1 immunoprecipitated from N2 but not ced-1(e1735) cell lysates. CED-1C antibody was used for immunoprecipitations (IPs) and precipitated proteins were detected with CED-1C and CED-6 antibodies. (D and E) APA-2::GFP associated with CED-1 (D) and CED-6 (E) immunoprecipitated from lysates of APA-2::GFP-expressing animals but not the same animals treated with RNAi of ced-1 or ced-6. CED-1C or CED-6 antibodies were used for IPs and precipitates were detected using antibodies against GFP, CED-1C and CED-6. The asterisk indicates a non-specific band. (F and G) DPY-23::GFP and GFP::CHC-1 associated with CED-6 immunopreciptated from lysates of animals expressing DPY-23::GFP (F) or GFP::CHC-1 (G) but not the same animals treated with ced-6 RNAi. CED-6 antibody was used for IP and precipitates were detected with CED-6 and GFP antibodies. (H and I) LST-4::GFP and DYN-1::GFP did not associate with CED-6 immunoprecipitated from lysates of animals expressing LST-4::GFP (H) or DYN-1::GFP (I). CED-6 antibody was used for IP and precipitated proteins were detected with CED-6 and GFP antibodies.
Figure 5.
CHC-1 and AP2 are required for phagosome maturation.
(A) Representative images of germ cell corpse staining by LysoSensor Green DND-189 in gla-3(RNAi), chc-1(RNAi) and apb-1(RNAi) animals. Quantification of corpse staining is shown on the right. ≥100 corpses were analyzed for each RNAi treatment. (B–D) Representative DIC and fluorescence images of germ cell corpse labeling by GFP::RAB-5 (B), mCherry::RAB-14 (C), and NUC-1::mCherry (D) in Ctrl(RNAi), apb-1(RNAi), and chc-1(RNAi) animals. (E) Quantification of cell corpse labeling as shown in (B–D). ≥100 corpses were analyzed for each genotype. In (A–D), arrows indicate cell corpses labeled by phagosomal markers; arrowheads indicate unlabeled corpses. Bars, 10 µm.
Figure 6.
(A) Representative time-lapse images of phagosomal association of DYN-1::GFP and LST-4::mCherry. Images were taken with a spinning disk confocal microscope. The time point when DYN-1 formed a weak ring was defined as 0 min. Arrowheads indicate a newly formed cell corpse. Bars, 5 µm. (B) Representative time-lapse images of phagosomal association of DYN-1::GFP in N2 and lst-4(tm2423) animals. Images were taken and analyzed as in (A). Bars, 5 µm. (C) Representative time-lapse images of germ corpses in yqIs114 (Plst-4lst-4(cDNA)::gfp) animals treated with Ctrl RNAi and dyn-1 RNAi. Adult animals (24 h after the L4 molt) were observed and images were taken and analyzed as above. Bars, 5 µm. In (A–C), ≥15 germ cell corpses were recorded. (D) Quantification of germ cell corpses in lst-4(tm2423) and lst-4(tm2423);qxIs139 (Pced-1dyn-1::gfp) animals at 36 and 48 h after the L4 molt. A total of 50 gonad arms from 50 animals were examined for each strain at every time point. The x-axis represents the number of cell corpses and the y-axis represents the % of animals. The average number of germ cell corpses per gonad arm (mean±SEM) is shown in parenthesis. (E) Quantification of germ cell corpses in N2 and yqIs114 (Plst-4lst-4(cDNA)::gfp) animals treated with dyn-1 RNAi at 24 and 36 h after the L4 molt, respectively. Cell corpses were scored and analyzed as in (D).
Figure 7.
Clathrin and AP-2 function upstream of LST-4 and DYN-1 for apoptotic cell clearance.
(A) Quantification of germ cell corpses in lst-4(tm2423) mutants treated with Ctrl RNAi and RNAi of chc-1, dpy-23 and apb-1. Error bars represent SEM. Comparisons were performed between Ctrl RNAi and RNAi of chc-1, dpy-23 and apb-1 using unpaired t-tests. All points had p>0.05. (B and C) Representative images of phagosomal association of LST-4::GFP (B) and DYN-1::GFP (C) in Ctrl(RNAi), chc-1(RNAi) and apb-1(RNAi) animals. Arrows point to germ cell corpses labeled by LST-4::GFP or DYN-1::GFP and arrowheads indicate unlabeled corpses. Bars, 10 µm. (D) On the left, 35S-labeled APA-2, APB-1, DPY-23 and His6-tagged CHC-1C were incubated with immobilized GST, GST-CED-9, GST-LST-4 and GST-DYN-1 (3 ug of each). Bound proteins were resolved on sodium dodecyl sulfate polyacrylamide gels and viewed by autoradiography or detected by immunoblotting with His6 antibody. GST and GST-fused proteins used for binding are shown in the right panel. (E and F) mCherry-tagged CHC-1 associated with DYN-1::GFP (E) and LST-4::GFP (F) in animals. IPs were performed with GFP antibody on lysates of animals expressing mCherry::CHC-1 (yqIs98) alone, animals co-expressing mCherry::CHC-1 and DYN-1::GFP (yqIs98;qxIs139), and animals co-expressing mCherry::CHC-1 and LST-4::GFP (yqIs98;yqIs114). Precipitates were detected by immunoblotting with antibodies for mCherry and GFP, respectively. (G and H) mCherry-tagged LST-4 associated with APA-2::GFP (G) and DPY-23 (H) in animals. IPs were performed with GFP antibody on lysates of animals expressing mCherry::LST-4 alone (yqIs119), animals co-expressing mCherry::LST-4 and APA-2::GFP (yqIs119;yqIs99), and animals co-expressing mCherry::LST-4 and DPY-23::GFP (yqIs119;yqIs120). Precipitates were detected by immunoblotting with mCherry and GFP antibodies.
Figure 8.
Schematic summary of the role of clathrin and the AP2 complex in both corpse engulfment and phagosome maturation during phagocytosis of apoptotic cells.
In the cell corpse engulfment phase, clathrin and AP2 act downstream of CED-1 and CED-6 to promote actin rearrangement, which is required for phagocytosis. In the phagosome maturation phase, clathrin and AP2 promote phagosomal association of LST-4 and DYN-1, which initiates the maturation process.