Figure 4
NA localization, lamellipodia dynamics and FA stability depend on the type of adhesive coating.
(A) FA assembly rates, (B) disassembly rates and (C) lifetimes were calculated for REFYFP-PAX spreading on FN- and VN-coated glass substrates. (D,E) FRAP experiments on mature FAs (>2 μm2) close to the periphery of REFYFP-PAX cultured on FN or VN were performed to estimate paxillin turnover (1 of 3 independent experiments presented). Half-life of fluorescence recovery after paxillin photobleaching (D) and mobile fraction (E) did not show significant differences in paxillin turnover within FAs (n = 30 FAs from 30 cells). (F) Individual frames from time-lapse TIRF imaging of REFYFP-PAX on FN- or VN-coated glass substrates. The time after cell seeding is indicated. NAs on FN assembled persistently at the protruding edge (which changed location between 150 and 180 minutes), whereas new adhesions formed randomly around the cell periphery on VN (indicated by arrows). (G) Kymograph analysis of REFWT seeded on FN or VN (yellow lines in phase contrast images) revealed smoother lamellipodia and slower protrusion/retraction cycles on FN. (H) Immunofluorescence microscopy against cortactin and pY of REFWT cells cultured for 6 hours on FN or VN. Scale bars: 10 μm. Mean and SEM values are presented in dot plots. Experimental data were compared using the unpaired t-test.
