ALK3 activation is sufficient to promote the formation of adhesion sites and its recruitment to adhesion sites. (A–C) MEFsv40 cells coexpressing opto-ALK3-RFP/opto-β3 integrin-Venus, opto-ALK3Ca (constitutively active)-RFP/opto-β3 integrin-Venus, or opto-ALK3Ci (constitutively inactive)-RFP/opto-β3 integrin-Venus were seeded onto (A) poly-lysine (PLL), (B) vitronectin (VTN), or (C) fibronectin (FN)-coated substrates, then treated or not with sBMP2, and observed by TIRF-mode imaging. (D) Opto-ALK3Ca colocalized with β3 integrin when cells were spread onto VTN and FN without sBMP2. The presence of sBMP2 led to an increase in the colocalization index of opto-ALK3 and opto-ALK3Ca with β3 integrin at adhesion sites. Of note, the presence of sBMP2 was unable to induce opto-ALK3 or opto-ALK3Ca relocalization if β3 integrin was not engaged (e.g., PLL-coated surfaces). (E) Opto-ALK3Ca, identified by β3 integrin-Venus, was able to induce the formation of FAs on PLL-coated substrates independently of sBMP2. N = 20 cells per condition. Scale bar, 15 µm. Unpaired t test: *0.05 > P > 0.01, **0.01 > P > 0.001, ***0.001 > P < 0.0001, ****0.00001 > P.