RCP–α5β1 integrin trafficking promotes actin spike formation. (A) A2780 cells transiently transfected with Lifeact-mRFPmars and paGFP-Actin were seeded onto 3D CDM 4 h before imaging and treated with cRGDfV (2.5 µM, 2 h) as indicated. Yellow broken lines mark the center of the photoactivated region. The boxed regions are enlarged in the insets. (B and C) ROI1 was photoactivated and cells were imaged every second for 100 s. Normalized intensity was analyzed for ROI1 (B) and ROI2 (C; n = 15 [−cRGDfV], n = 16 [+cRGDfV]). Blue bars indicate time points at which datasets are significantly different (P < 0.01; Student’s t test). Yellow arrows indicate the direction of protrusion. Error bars represent the SEM. (D and E) A2780 cells transiently transfected with Lifeact-mEGFP were seeded as in A and z stacks were captured (7 sections, 0.2 µm intervals) on a spinning disk confocal microscope every minute for >25 min. Z projections are shown. White arrowheads indicate lamellipodia-like actin veils, red arrowheads indicate actin spikes. The white boxed regions are enlarged on the right, and the kymograph images are taken from the yellow boxed regions. (F and G) Quantification of Lifeact-mEGFP protrusions. Protrusions were identified by overlapping thresholded video frames with a lag of 3 min. Insets show the boxed regions. (H) Normalized protrusion size was measured using image masks as represented in F and G. n = 28 cells per condition (n > 25 frames per cell). (I) A2780 cells were seeded as in A before fixation and Alexa Fluor 488 Phalloidin staining. SIM was used to generate super-resolution images; maximum intensity projections are shown. The number (J) and length (K) of filopodia per protrusion was quantified from SIM images; n = 10 cells per condition. All data represent at least three independent experiments; statistical significance was evaluated using ANOVA/Tukey’s multiple comparison test (H, J, and K). +, mean; *, P < 0.05; **, P < 0.01.