Figure 6. Focal adhesion (FA) proteins assemble into integrin waves in a precise order.

(A) α_V integrin-tagRFP (Int, red) was co-expressed with the following proteins: α_V integrin-EGFP (Int, green), talin-EGFP (Tln, green), vinculin-EGFP (Vcl, green), paxillin-EGFP (Pax, green), VASP-Venus (VASP, green), zyxin-EGFP (Zyx, green), Arp3-GFP (Arp, green) and F-tractin-GFP (Act, green). LEFT: Total internal reflection fluorescence microscopy (TIRFM) images showing an inset (yellow box) of a ventral wave. Still image was taken from a time series at the labeled time. Scale bar = 10 µm. Inset scale bar = 5 µm. Yellow arrow indicates region of kymograph measurement. CENTER: Kymograph along the trajectory of integrin wave propagation. The yellow arrow corresponds to the arrow in (A, left) to demonstrate the direction of wave propagation as well as the time the still image corresponds to in the kymograph. Cells imaged at 20s intervals. Kymograph region = 10 µm. RIGHT: Representative quantification of normalized average fluorescence intensity (“Intensity”) over time for ventral waves in cells imaged at 5s intervals. (B) Average lag time between when fluorescence intensity of FA proteins and α_V integrin rise to half-maximal. In B and C, the graphs represent the mean and standard deviation of n>10 integrin wave measurements per condition. Brackets denote lag-times that do not significantly differ from each other as determined by Student's t-test (detailed statistics found in Table S1). (C) Average lag time between when fluorescence intensity of FA proteins and α_V integrin fall from peak to half-maximal.