Septin depletion decreases macropinocytic fusion events and hinders macropinosome maturation and turnover. (A) Time-lapse frames from wide-field deconvolution microscopy show the dynamics of PM-mCherry–labeled macropinosomes in MDCK cells treated with control and SEPT2 shRNAs. Yellow arrow points to a cluster of macropinosomes that fuse into a single vacuole. (B) Control (n = 41) and SEPT2-depleted MDCK-PM-mCherry cells (n = 31) were imaged live every 5 s for 8 min. Bar graph shows percentage cells with a cluster of three or more macropinosomes formed from a lamellar ruffle. Error bars indicate the maximum and minimum values from three independent experiments. (C and D) Bar graphs show the lifetime (C) and fusion events (D) of nascent macropinosomes (n = 24–27). Error bars represent mean ± SEM. (E) Quantification of PM-mCherry fluorescence along the contacting (1) and noncontacting (2 and 3) segments of two macropinosomes from a SEPT2-depleted cell. AU, arbitrary units. (F and G) MDCKs were incubated with FITC-dextran for the indicated times. Bar graphshow the number of FITC-dextran–containing macropinosomes/endosomes (F) and their size (G) per cell (n = 18). Error bars represent mean ± SEM. (H) Still frames show the formation and detachment of PM-mCherry–labeled membrane tubules (arrows) in live MDCKs.