Figure 3. ESCRT accumulation at injured cell membrane correlates with ALG-2 accumulation but not MVB fusion.

HeLa cells stably expressing low levels of (A) Chmp4B-GFP and (B) Vps4A-GFP, were injured by the pulsed laser at sites marked by the arrow. Images taken 5s before injury (Pre-injury) and at the peak of the respective protein accumulation (Post-injury) show injury triggered accumulation of these proteins similar to C2C12 cells. (C-F) HeLa cells co-expressing CD63-mCherry with (C, D) ALG2-GFP, or (E, F) Chmp4B-YFP were injured by laser (marked by arrow) and accumulation of ALG2 and Chmp4B punctae (green) was monitored together with CD63 (red) by dual-color TIRF imaging. Plots show intensity of CD63 at the site of injury where individual ALG-2 or Chmp4B punctae (marked by white circles) form. Note lack of co-localization between CD63-mCherry vesicle and the nascent ALG-2 or Chmp4B punctae. (G, H) Images of injury-triggered fusion of CD-mCherry (red) labeled vesicles (V1 and V2) together with accumulation of Vps4B-GFP (green) at the site of injury. Note that injury-triggered fusion of CD63-mCherry vesicles does not correlate spatially (V1) or temporally (V2) with formation of Vps4B puncate. (H) Images showing exocytosis of vesicles V1 and V2 as CD63-mCherry released by them diffuses into the cell membrane. (I) Images and (J) averaged plot (n=5) showing accumulation of ALG-2 mCherry and ALIX-GFP following focal laser injury. (K) Images showing accumulation of ALG-2 and Chmp4B in HeLa cell stably expressing Chmp4B-mCherry and transiently expressing ALG-2 following focal injury (white arrow). Zoom of the site of repair as ALG-2 (green) accumulates followed by Chmp4B (red) is shown and the individual channels (red and green) corresponding to the color combined zoomed images are shown below each image. Scale bar represents 10μm for A, B, I, and K; 2μm for C, E, and G.