MEFs expressing full-length WT talin1 and full-length talin1-17b variant expressing cells were seeded on glass-bottom dishes for 8 h. Hoechst (1 µg/ml) was added for 1 h before imaging. Live-cell imaging was performed at 20X on Leica SP8 live-imaging system. Images were acquired every 5 min for 4 h. The movement of the cell was tracked in each frame in ImageJ using the Track-mate plugin and the velocity of each cell was calculated.
FRAP was performed to assess the dynamics of focal adhesions containing WT talin1 or 17b splice variant. Live-cell imaging was performed at 63X on Leica SP8 live-imaging system. Before photobleaching, three pre-bleach images of GFP-talin were acquired, using a 488 nm laser set at 10% of the maximum power. Photobleaching of GFP was conducted for 10 s at 100% laser power. Fluorescence recovery images were acquired every 30 s for 9 min using a 488 nm laser set at 10% of the maximum power. The mean fluorescence intensity pre-bleach was set to 100%. Photobleaching due to continuous illumination during recording was corrected by normalizing the fluorescence intensity at the FA with total cell fluorescence intensity. Corrected recovery fluorescence intensities were normalized to the pre-bleach intensity. The intensity was considered 100 and 0% for pre-bleach and bleach points. The fractional recovery post-bleach was calculated by normalizing the corrected recovery fluorescence intensities at each time point to pre-bleach intensity.
FRAP was performed to assess the dynamics of focal adhesions containing WT talin1 or 17b splice variant. Live-cell imaging was performed at 63X on Leica SP8 live-imaging system. Before photobleaching, three pre-bleach images of GFP-talin were acquired, using a 488 nm laser set at 10% of the maximum power. Photobleaching of GFP was conducted for 10 s at 100% laser power. Fluorescence recovery images were acquired every 30 s for 9 min using a 488 nm laser set at 10% of the maximum power. The mean fluorescence intensity pre-bleach was set to 100%. Photobleaching due to continuous illumination during recording was corrected by normalizing the fluorescence intensity at the FA with total cell fluorescence intensity. Corrected recovery fluorescence intensities were normalized to the pre-bleach intensity. The intensity was considered 100 and 0% for pre-bleach and bleach points. The fractional recovery post-bleach was calculated by normalizing the corrected recovery fluorescence intensities at each time point to pre-bleach intensity.
Free full text: Click here
