Fig. 3.

Complete reconstitution of Ca²⁺-triggered endolysosomal membrane repair machinery. (A) Fluorescently labeled ALIX (Cy3; 100 nM), CHMP4B (Atto 488; 10 nM), and VPS4B (LD 655; 100 nM) along with ALG-2 (dark; 200 nM), CHMP2A (dark; 100 nM), and CHMP3 (dark; 100 nM) were mixed with 30% DOPS containing GUVs and imaged. The observance of colocalized puncta of the labeled proteins on the periphery of GUVs confirms the recruitment of the entire endolysosomal membrane repair machinery. (B) In the absence of CHMP4B, labeled VPS4B was not recruited to the ALIX puncta on the membrane. (C) The bar chart depicting the colocalization % of the puncta in two different fluorescently labeled protein channels. At least 100 GUVs from three independent experiments were analyzed for each set of conditions plotted in the bar chart. The circles on the bar plots represent independent data points and the data are shown as mean ± SD (vertical line). P = 0.0003 (***). (Scale bar, 10 μm.)