Fig. 2.

Repeated optogenetic activation leads to incremental increases in protrusion length. a Montage of PA-Rac1 membrane ruffles in whole RPE1 cells in response to multiple optogenetic stimulations (Additional file 4: Movie S4). Left to right shows changes in Rac1 with time. Top panel shows a 2D slice through the cell (as marked in the last panel). Bottom panels show top views. The blue number overlays indicate the sequential optogenetic stimulation. The red square highlights a collapse following a photoactivation laser switch off. Scale bars for both panels = 5 μm. b Montage of ruffle evolution in response to multiple optogenetic stimulations of PA-Rac1. With multiple excitations, the ruffles become longer, extruding larger sheet like protrusions. Scale bar = 5 μm. c A temporal zoomed montage of red box in a–c. Formation of highly complex collapsed membrane folds post PA-Rac1 inactivation [3] and unfurling of the complex folds upon re-activation of PA-Rac1 [4]. Frames every 20 s. d Unfurling of complex membrane folds undergoing rotational movements with polymerization at ‘leading edges’ of the ruffles. The magenta and green arrows follow the same protrusions across the montage. Frames every 4 s. e Graph of ruffle height vs the number of times stimulated with 445-nm laser. Error bars indicate standard deviation (n = 5 cells, 34 ruffles). f Cross-sectional slice of RPE1 cell transfected with PA-Rac1 (blue) and EB1 (yellow). Bottom right panel shows a temporal stack MIP over 10 min. Scale bar = 5 μm. g Ruffle formation in nocodazole-treated cells (yellow arrows). Scale bar = 3 μm. h Ruffle lengths and widths show no difference between untreated cells and nocodazole-treated cells (n 25 ruffles, 3 cells)