CYRI-A localizes to large macropinocytic cup-like structures before RAB5A recruitment. (See Video 1.) (A–C) P16-GFP-CYRI-A in HEK293T (scale bar = 20 µm; n = 204 events in 18 cells for cups/vesicles and n = 24 events in 10 cells for tubules) decorates structures resembling macropinocytic cups (yellow arrowheads, diameter ranging from 0.4 to 2.9 µm; scale bar = 5 µm). Tubule length 0.7–7 µm. Average lifetime of CYRI-A on cups, 50 s (n = 58 events in 5 cells). Red lines represent the average value. (D) Still images of COS-7 cells (scale bar = 20 µm) expressing P16-GFP-CYRI-A showing the diffuse pool of CYRI-A (yellow doubled arrow) near the leading edge. Dotted square denotes time sequence on the right (scale bar = 5 µm). (E) Time-lapse sequence of COS-7 cells expressing P16-GFP-CYRI-A (cyan) and mCherry-RAB5A (magenta). Scale bar = 20 µm (full size) or 5 µm (zoom). (F and G) Time sequence of CYRI-A and RAB5A recruitment to the macropinocytic cups (CYRI-A only, n = 75 events in 8 cells; CYRI-A with RAB5A, n = 68 events in 8 cells). (H–J) Dextran uptake assay (scale bar = 5 µm). Quantification of the percentage of CYRI-A–positive cups/vesicles containing dextran (n = 9 cells) and the size (cups/vesicles, n = 15 events in 7 cells; tubules, n = 10 events in 7 cells). Two-tailed unpaired t test.