Filopodia direct nascent lamellipodia. (A, left) Pseudocolored TIRF images of a NIH 3T3 cell expressing the EGFP-AktPH biosensor, representative of 28 cells in four independent experiments. The montage shows that filopodia template nascent lamellipodia, which typically branch from existing ones during random migration on fibronectin (see also Video 1). (center) The corresponding spatiotemporal map of protrusion/retraction velocity, overlaid on regions of morphological extension (gray; see Materials and methods) indicates the timing and angular locations of these branching events relative to the cell centroid (clockwise from the negative x axis). The colored arrowheads on both the montage and map show these events. (right) The accompanying lamellipod/filopod overlap map indicates pixels associated with putative lamellipodia, filopodia, or overlap of the two (black). (B) Analysis of lamellipod/filopod overlap. Indicated are the percentages of lamellipodial regions that emerged overlapping a preexisting filopod, emerged without a preexisting filopod but overlapped with at least one filopod thereafter, or never overlapped with a filopod. (C) TIRF montage, representative of 67 cells showing that local photoactivation of Rac (red spot) in NIH 3T3 cells induces protrusion and PI3K signaling preferentially over actin bundles (red arrowheads). Frames labeled with red circles show the cell immediately before activation of Rac at the indicated position. The other frames show the cell right before activation was ceased at that location and allowed to recover until the next “activation start” (see also Video 2). (D) Montage showing a time course after microinjection of Arp2/3 complex into Arp2/3-depleted IA32 MEFs (Wu et al., 2012). Red arrowheads indicate filopodia where protrusion initiated. Bars: (A) 10 µm; (C and D) 20 µm.