Figure S2.

CYRI proteins localize to macropinocytic structures prior to RAB5 arrival. (See Video 2.) (A and B) Representative images of live COS-7 cells expressing P17-GFP-CYRI-B (scale bar = 10 µm). Tubular and vesicular structures are highlighted in zoomed panels, and quantification of their sizes is shown in B (vesicles, n = 16 events in 3 cells; tubules, n = 22 events in 4 cells; scale bar = 5 µm). (C–E) Time sequence of live HEK293T cells (scale bar = 10 µm) coexpressing P16-GFP-CYRI-A (cyan) and mCherry-RAB5A WT (magenta). Arrowhead points to vesicular structures (C; scale bar = 5 µm). The dynamics of each protein is reported by its normalized intensity plot (D) and lifetime (n = 84 events in 7 cells; E). (F and G) COS-7 cells (scale bar = 10 µm) coexpressing P17-GFP-CYRI-B (cyan) and mCherry-RAB5A WT (magenta). Time sequence corresponding to the white dotted square area is shown in the bottom panel (scale bar = 5 µm). Arrowhead points to tubular and vesicular structures, and intensity profile along the yellow line is plotted in G. (H–M) Time sequence images HEK293T (H) and CHL-1 cells (K) expressing P16-GFP-CYRI-A and incubated with dextran 70 kD (scale bar = 10 µm). Yellow arrowheads indicate macropinocytic events positive for both CYRI-A and dextran signals (scale bar = 5 µm). Quantification showing the majority of CYRI-A–positive vesicles are also dextran-positive in HEK293T (I; 88%, n = 6 cells) and CHL-1 (L; 100%, n = 6 cells) and their sizes (J and M; n = 53 events in 6 cells in HEK293T; n = 57 events in 6 cells in CHL-1). Red line indicates the average value. (N and O) HEK293T cells expressing either GFP control or P16-GFP-CYRI-A and incubated with dextran 70 kD. The size of dextran-positive vesicles in GFP (n = 163 events in 4 cells) is the same as that of CYRI-A–positive vesicles (n = 75 events in 7 cells). Events from each cell are color-coded. Unpaired t test. Mean ± SD.