Zoncu et al. 10.1073/pnas.0611733104. |
Movie 1. Ionomycin-induced phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] depletion causes rapid disappearance of clathrin-coated pits (CCPs) from the plasma membrane. Shown is a total internal reflection fluorescence microscopy (TIRFM) movie of a COS-7 cell expressing clathrin light chain (LCa)-GFP. Images were acquired at 4-s intervals. Playback speed is 45 frames per second (fps). The red square indicates addition of 5 mM ionomycin.
Movie 2. iRAP-induced PI(4,5)P2 depletion causes rapid disappearance of CCPs from the plasma membrane and dimming and immobilization of remaining clathrin spots. Shown is a TIRFM movie of a COS-7 cell expressing LCa-GFP. Images were acquired at 4-s intervals. Playback speed is 30 fps. Blue circles highlight examples of dynamic CCPs. The red square indicates addition of 5 mM iRAP, an indole-modified analog of rapamycin.
Movie 3. iRAP-induced PI(4,5)P2 depletion does not affect Golgi- or endosome-derived CCVs. Shown is a TIRFM movie of a COS-7 cell expressing LCa-GFP. The penetration depth was increased to reveal Golgi-derived clathrin spots. Images were acquired at 4-s intervals. Playback speed is 30 fps. The red square indicates addition of 5 mM iRAP.
Movie 4. iRAP-induced PI(4,5)P2 depletion causes rapid disappearance of m2-GFP spots from the plasma membrane. Shown is a TIRFM movie of a COS-7 cell expressing m2-GFP. Images were acquired at 4-s intervals. Playback speed is 45 fps. The red square indicates addition of 5 mM iRAP.
Movie 5. iRAP-induced PI(4,5)P2 depletion causes the rapid disappearance of dynamin 2 (Dyn2)-GFP spots from the plasma membrane. Shown is a TIRFM movie of a COS-7 cell expressing Dyn2-GFP. Images were acquired at 4-s intervals. Playback speed is 30 fps. Blue circles highlight examples of transient dynamin spots. The red square indicates addition of 5 mM iRAP.
Movie 6. iRAP-induced PI(4,5)P2 depletion blocks motility of Dyn2-positive membrane ruffles. Shown is a TIRFM movie of a COS-7 cell expressing Dyn2-GFP. Note the presence of Dyn2-GFP spots in lamellipodia and the collapse of lamellipodia together with the disappearance of Dyn2-GFP after addition of iRAP. Images were acquired at 4-s intervals. Playback speed is 45 fps. The red square indicates addition of 5 mM iRAP.
Movie 7. iRAP-induced PI(4,5)P2 depletion blocks motility of actin-positive membrane ruffles. Shown is a spinning disk confocal movie of a COS-7 cell expressing actin-GFP. (Left) Before iRAP. (Right) After iRAP. Images were acquired at 15-s intervals. Playback speed is 20 fps.
SI Methods
ImageJ (National Institutes of Health, Bethesda, MD) and Andor iQ software (Andor Technology, South Windsor, CT) were used to analyze raw images and generate integrated intensity plots of areas of interest, corresponding to CCPs or nearby background. The lifetime of fluorescently tagged proteins was calculated by manually assessing at least 120 fluorescent spots (from three different cells) that appeared (spot/background ratio >1.5) and disappeared (spot/background ratio <1.5) from the TIRFM field during a 20-min observation. Colocalization between two proteins was determined by randomly selecting 100 spots in the GFP channel followed by scoring for colocalization in the RFP channel. Measurement of the number of fluorescent spots over time was performed by generating binarized images and scoring the number of objects in each frame with a particle-counting algorithm. The number of newly generated CCPs per mm2/min was determined as follows: fluorescent spots were Gaussian-fitted, and their maximum intensity profiles were automatically calculated. A newly generated spot was counted if its maximum intensity profile rose from the background at least up to the mean maximum intensity value for the entire sequence. Statistical significance was calculated using Student's t test.
Analysis of membrane motility before vs. after iRAP was performed with a differentiation algorithm in Andor iQ where pixel intensity differences between consecutive frames are encoded by grayscale values and then added up, so that darker pixels represent areas of higher motility.