Figure 8.
Polarized deposition of FN-PH requires intact myosin II contractility. (A) Gallery of frames from a TIRFM video of a KO67 FN-PH Halo-β1itg fibroblast (paxillin-mCherry in red, FN-PH in green, and Halo-β1itg labeled with SiR-Halo ligand in blue) in the process of spreading on FN-coated glass before and after addition of 25 μM blebbistatin for 1 h 25 min (Video 9, to compare with DMSO vehicle-treated cell in Video 6). At each time point, the cell outline is marked by a solid line and the outline from the previous time point is superimposed as a dotted line for comparison. (B) Quantification of the FN-PH fluorescence intensity in the entire cell by TIRFM over time and comparison of the rate of change in FN-PH signal intensity before and after treatment with blebbistatin in 10 cells. (C) Gallery of frames from a TIRFM video of a KO67 FN-PH notag-β1itg lamellipodin RA-PH-Halo fibroblast (paxillin-mCherry in red, FN-PH in green, and RA-PH-Halo labeled with SiR-Halo ligand in blue) in the process of migrating out of a monolayer on FN-coated glass before and after addition of 25 μM blebbistatin at 1 h 30 min (Video 10, to compare with DMSO vehicle-treated cells in Video 8). The signal for already deposited FN-PH was photobleached before imaging. At each time point, the migration front is outlined by a solid line and the outline from the previous time point is superimposed as a dotted line for comparison. (D) Quantification of the FN-PH fluorescence intensity in the cells at the outer edge of the monolayer by TIRFM over time and comparison of the rate of change in FN-PH signal intensity before and after treatment with blebbistatin in 12 fields. Data were analyzed by paired t test, ** P < 0.01. Data distribution was assumed to be normal, but this was not formally tested. Scale bar = 10 μm.

Polarized deposition of FN-PH requires intact myosin II contractility. (A) Gallery of frames from a TIRFM video of a KO67 FN-PH Halo-β1itg fibroblast (paxillin-mCherry in red, FN-PH in green, and Halo-β1itg labeled with SiR-Halo ligand in blue) in the process of spreading on FN-coated glass before and after addition of 25 μM blebbistatin for 1 h 25 min (Video 9, to compare with DMSO vehicle-treated cell in Video 6). At each time point, the cell outline is marked by a solid line and the outline from the previous time point is superimposed as a dotted line for comparison. (B) Quantification of the FN-PH fluorescence intensity in the entire cell by TIRFM over time and comparison of the rate of change in FN-PH signal intensity before and after treatment with blebbistatin in 10 cells. (C) Gallery of frames from a TIRFM video of a KO67 FN-PH notag-β1itg lamellipodin RA-PH-Halo fibroblast (paxillin-mCherry in red, FN-PH in green, and RA-PH-Halo labeled with SiR-Halo ligand in blue) in the process of migrating out of a monolayer on FN-coated glass before and after addition of 25 μM blebbistatin at 1 h 30 min (Video 10, to compare with DMSO vehicle-treated cells in Video 8). The signal for already deposited FN-PH was photobleached before imaging. At each time point, the migration front is outlined by a solid line and the outline from the previous time point is superimposed as a dotted line for comparison. (D) Quantification of the FN-PH fluorescence intensity in the cells at the outer edge of the monolayer by TIRFM over time and comparison of the rate of change in FN-PH signal intensity before and after treatment with blebbistatin in 12 fields. Data were analyzed by paired t test, ** P < 0.01. Data distribution was assumed to be normal, but this was not formally tested. Scale bar = 10 μm.

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