PtdIns(4,5)P₂ and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 1 -
  Dynamic changes of PtdIns(4,5)P₂ and PtdIns3P on apoptotic cell–containing phagosomes. A cell corpse in a wild-type C. elegans embryo expressing both mCHERRY::PLCδ1-PH and YFP::2xFYVE is followed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 13 min and are displayed every 1 s. Selected images are shown in Fig. 1 A. Bar, 5 µm.
• Video 6 -
  Loss of MTM-1 causes PtdIns3P accumulation on extending pseudopods and forming phagosomes. The internalization of a germ cell corpse in an mtm-1(qx322);controlRNAi animal expressing both mCHERRY::MTM-1(C378S) and YFP::2xFYVE is followed. 2xFYVE appears on the extending pseudopod and nascent phagosome positive for MTM-1(C378S). Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 2 min for 60 min and are displayed every 1 s. Selected images are shown in Fig. 5 H. Bar, 5 µm.
• Video 5 -
  MTM-1 release coincides with PtdIns3P accumulation on phagosomes. Cell corpses in a gla-3RNAi germline expressing both mCHERRY::MTM-1(C378S) and YFP::2xFYVE are followed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 2 min for 60 min and are displayed every 1 s. Selected images are shown in Fig. 4 F. Bar, 5 µm.
• Video 8 -
  LST-4 release coincides with PtdIns3P accumulation on phagosomes. A germ cell corpse in a gla-3RNAi animal expressing both LST-4::mCHERRY and YFP::2xFYVE is followed. LST-4 release coincides with 2xFYVE enrichment on the phagosome. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 2 min for 38 min and are displayed every 1 s. Selected images are shown in Fig. 6 B. Bar, 5 µm.
• Video 3 -
  MTM-1 and PtdIns(4,5)P₂ display identical dynamics on extending pseudopods and forming phagosomes. A cell corpse in a wild-type C. elegans embryo expressing both GFP::PLCδ1-PH and mCHERRY::MTM-1(C378S) is followed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 8 min and are displayed every 1 s. Selected images are shown in Fig. 1 D. Bar, 5 µm.
• Video 9 -
  MTM-1 release coincides with PtdIns3P accumulation on the C3 phagosome. A C3 corpse in a wild-type C. elegans embryo expressing both mCHERRY::MTM-1(C378S) and YFP::2xFYVE is followed. MTM-1(C378) disappearance coincides with 2xFYVE enrichment on the C3 phagosome, and no reappearance of MTM-1 is observed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 30 min and are displayed every 1 s. Selected images are shown in Fig. S5 H. Bar, 5 µm.
• Video 2 -
  PtdIns3P depletion precedes phagolysosome formation. A cell corpse in a wild-type C. elegans embryo expressing both YFP::2xFYVE and the lysosomal membrane protein LAAT-1::mCHERRY is followed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 28 min and are displayed every 1 s. Selected images are shown in Fig. 1 B. Bar, 5 µm.
• Video 7 -
  LST-4 and MTM-1 display identical phagosomal dynamics. A cell corpse in a wild-type C. elegans embryo expressing both LST-4::GFP and mCHERRY::MTM-1(C378S) is followed. LST-4 and MTM-1(C378S) appear simultaneously on the extending pseudopod and nascent phagosome and disappear together from the phagosome. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 15 min and are displayed every 1 s. Selected images are shown in Fig. 6 A. Bar, 5 µm.
• Video 4 -
  MTM-1 release coincides with PtdIns3P accumulation on phagosomes. Cell corpses in a wild-type C. elegans embryo expressing both mCHERRY::MTM-1(C378S) and GFP::2xFYVE are followed. Images were analyzed with a time-lapse confocal microscope using an inverted fluorescence microscope with a spinning-disk confocal scanner unit. The frames were taken every 1 min for 14 min and are displayed every 1 s. Selected images are shown in Fig. 4 A. Bar, 5 µm.