Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie 1 - Video 1. GFP-2xP4M specificity for PtdIns4P in cells. RAW264.7 cells transiently co-expressing GFP-2xP4M, mCh-FKBP-Sac1 and Lyn11-FRB were imaged before (first frame) and after the addition of rapamycin (1 μM). Upon addition of rapamycin, mCh-FKBP-Sac1 (left) was rapidly recruited to the PM. The recruitment of Sac1 caused the release of GFP-2xP4M (right) from the compartment. The released probe rapidly accumulated in endosomal membranes and Golgi structures. Frames were acquired every 6 s. The video is played at 0.5 fps.
- Movie 2 - Video 2. PtdIns4P dynamics during phagocytosis. RAW264.7 cells transiently co-expressing GFP-2xP4M and the PM marker mRFP-PM were challenged with Dylight 650-labeled IgG-SRBCs immediately before being imaged live at 37 °C by spinning disk confocal microscopy. Frames were acquired every 15 s until 2xP4M disappearance from the phagosome; at that point frames were acquired every 60 s. The real time duration of the movie was ~30 min. The video is played at 3 fps.
- Movie 3 - Video 3. Sac2 and Rab5 accumulation in phagosomes during PtdIns4P disappearance. RAW264.7 cells co-expressing mCherry-2xP4M, GFP-Sac2 and CFP-Rab5 during phagocytosis of IgG-SRBCs imaged live at 37 °C by spinning disk confocal microscopy. Frames were acquired every 15 s. The video is played at 3 fps.
- Movie 4 - Video 4. PLC contribution to the early PtdIns4P disappearance. RAW264.7 cells transiently co-expressing GFP-2xP4M, mRFP-FKBP-PLCβ3 and Lyn11-FRB were imaged before (first frame) and after addition of rapamycin (1 μM). Upon addition of rapamycin, mCh-FKBP-PLCβ3 (left) was rapidly recruited to the PM. The recruitment of PLCβ3 caused the release of GFP-2xP4M (right) from the compartment. The released sensing probe rapidly accumulated in endosomal membranes and Golgi structures. Frames were acquired every 6 s. The video is played at 0.5 fps.
- Movie 5 - Video 5. Impairment of RILP acquisition in PtdIns4P-depleted phagosomes. COS-1-FcγRIIa cells transiently co-expressing GFP-RILP-C33, mCh-FKBP-Sac1 and iRFP-FRB-Rab7 were challenged with IgG-SRBCs, treated with rapamycin (1 μM) immediately after, and phagocytosis allowed to proceed for 20 min before imaging. Acquisition of GFP-RILP-C33 was impaired when mCh-FKBP-Sac1 was recruited to phagosomes. Frames were acquired every 30 s. The video is played at 2 fps.