The targeting of ALK3 to focal adhesions upon BMP2 treatment leads to decreased ALK3 lateral mobility. Opto-CAAX-RFP/opto-β3 integrin-Venus, opto-ALK3-RFP/opto-β3 integrin-Venus, or opto-BMPRII-RFP/opto-β3 integrin-Venus were coexpressed in REF52 cells. (A) Representative image of fluorescent recovery after photobleaching (FRAP) experimental setup of opto-ALK3-RFP immobilized within FAs upon sBMP2 treatment in the dark or without BMP2 but under blue light stimulation. A region of interest (ROI) close to FA (white circle) is bleached and monitored for fluorescence recovery. (B) The FRAP data are presented as the fitted curve of a single exponential equation, with background and bleaching correction, under basal conditions and sBMP2 and blue light stimulation. FRAP experiments were performed with total photobleaching at t = 0. The mean ± SD is shown for 10 cells. (C) The characteristic time of recovery (1/τ) was plotted for all conditions. Only the ALK3 recovery time was reduced after sBMP2 or blue light stimulation. (D) FRAP analysis by limiting the ROI within and outside FAs shows a decrease in ALK3 lateral mobility within FA sites. Scale bar, 15 µm. Unpaired t test: *0.05 > P > 0.01, **0.01 > P > 0.001 ***0.001 > P < 0.0001.