Figure 6.

Optogenetic recruitment of talin to the plasma membrane activates integrin αVβ3 in endothelial cells. CRISPR–Cas9 was utilized to “tag” endogenous talin in endothelial cells with CRY2–mCherry, as detailed in Experimental procedures. Cells also stably expressed CIBN–GFP–CAAX. A, Western blots showing the expression of CRY2–mCherry–talin using anti-talin and anti-mCherry antibodies. Parental immortalized mouse lung endothelial cells were used as a negative control and β-actin as a loading control. B, recruitment of CRY2–mCherry–talin to the plasma membrane of endothelial cells maintained in suspension and exposed to 450-nm blue light for 30 min. The scale bar represents 20 μm. C, optogenetic recruitment of talin to endothelial cell plasma membranes induces fibrinogen binding to integrin αVβ3. Specific fibrinogen binding was measured by flow cytometry as described in Experimental procedures. Data represent the means ± SEM of five experiments (asterisk, p < 0.05). D, optogenetic recruitment of talin to the plasma membrane promotes endothelial cell migration. Cell migration across fibrinogen-coated transwells was determined as described in Experimental procedures. Data represent the means ± SEM of four experiments (asterisk, p < 0.05). CRY2, Arabidopsis cryptochrome 2; N.S., not statistically significant.