Figure 7.
Polarized deposition of FN-PH requires intact microtubules. (A) Gallery of frames from a TIRFM video of a KO6 EB3-RFP FN-PH Halo-β1itg fibroblast (EB3-RFP 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 10 μM Nocodazole at 40 min (Video 7, 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 Nocodazole in multiple cells (N = 9). (C) 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 DMSO vehicle in 15 cells. (D) Gallery of frames from a TIRFM video of KO67 FN-PH notag-β1itg lamellipodin RA-PH-Halo fibroblasts (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 the addition of 10 μM Nocodazole at 2 h 45 min (Video 8, compared with DMSO vehicle-treated cells also 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. (E) Quantification of the FN-PH fluorescence intensity by TIRFM in the cells at the outer edge of the monolayer and comparison of the rate of change in FN-PH signal intensity before and after treatment with Nocodazole in eight fields. (F) Quantification of the FN-PH fluorescence intensity by TIRFM in the cells at the outer edge of the monolayer and comparison of the rate of change in FN-PH signal intensity before and after treatment with DMSO in 12 cells. 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 microtubules. (A) Gallery of frames from a TIRFM video of a KO6 EB3-RFP FN-PH Halo-β1itg fibroblast (EB3-RFP 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 10 μM Nocodazole at 40 min (Video 7, 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 Nocodazole in multiple cells (N = 9). (C) 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 DMSO vehicle in 15 cells. (D) Gallery of frames from a TIRFM video of KO67 FN-PH notag-β1itg lamellipodin RA-PH-Halo fibroblasts (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 the addition of 10 μM Nocodazole at 2 h 45 min (Video 8, compared with DMSO vehicle-treated cells also 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. (E) Quantification of the FN-PH fluorescence intensity by TIRFM in the cells at the outer edge of the monolayer and comparison of the rate of change in FN-PH signal intensity before and after treatment with Nocodazole in eight fields. (F) Quantification of the FN-PH fluorescence intensity by TIRFM in the cells at the outer edge of the monolayer and comparison of the rate of change in FN-PH signal intensity before and after treatment with DMSO in 12 cells. 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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