Fig. 4. ESCRT machinery preferentially responds to small membrane disruptions.

(A) HeLa cells producing CHMP4B-GFP were additionally transfected with a plasmid encoding mCherry-GAL3, and imaged live before and after adding LLOME. Colored arrows in magnified views of boxed areas designate structures containing only CHMP4B-GFP (green), only mCherry-GAL3 (magenta), or both (white). (B) Cells imaged as in (A). Boxed areas are magnified at right, with the leftmost two columns showing each signal in grayscale; these are merged and colored in the third column. Arrowhead marks the initial appearance of GAL3. Note that this compartment rotates over time, as denoted by the curved arrows. (C and D) U2OS cells were transfected with plasmids encoding CHMP3-mCherry (C) or mCherry-GAL3 (D) and loaded with 40-kDa pH-sensitive FITC-dextran chased into endolysosomes. Cells were imaged live before and after addition of LLOME. Boxed areas are magnified as in (B). Arrowheads in (D) indicate a dextran-laden compartment that rapidly acquires GAL3 after sudden loss of dextran. (E) Interpretive illustration of the different events seen in (C) and (D). In all panels, single planes of a representative cell are shown at the times indicated from each recording. Scale bars equal 10 µm (2 µm in magnified views).