Figure 6.
FN-PH deposition follows cell polarization. (A) Gallery of frames from a TIRFM video of live KO67 Halo-β1itg FN-PH fibroblasts in the process of spreading on FN-coated glass or PLL-coated glass in a medium depleted of FN. FN-PH is shown in green and paxillin-mCherry in magenta. Note the difference in time scale between the two substrata. The white rectangle indicates the area zoomed in the inset on the right. (B) Gallery of frames from a TIRM video of KO67 untagged-β1itg FN-PH Golgi-Halo fibroblasts, 0 or 4 h after plating on FN-coated glass. FN-PH is shown in green, paxillin-mCherry in red, Golgi-Halo in magenta, and nucleus stained with Hoechst 33342 in blue. The signal for already deposited FN-PH was photobleached before imaging, FN-PH deposition was measured for a duration of 1 h, and the net change in FN-PH signal was determined by subtracting the mean green fluorescence before acquisition to the mean green fluorescence 1 h later. For each cell, a Golgi side (solid gray outline) and an opposite side (dashed gray outline) were delineated by a line passing through the center of the nucleus and running parallel to the crescent-shaped perinuclear Golgi apparatus. Data in the graph represent the ratio of the net change in FN-PH fluorescence on each side in 18–23 cells. Data were analyzed by unpaired t test, **** P < 0.0001. (C) Gallery of frames from a TIRM video of KO67 notag-β1itg FN-PH RA-PH-Halo (paxillin-mCherry in red, FN-PH in green, and lamellipodin RA-PH-Halo labeled with SiR-Halo ligand in blue) migrating out of a monolayer on FN-coated glass. 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. The fluorescence intensity in the three fluorescent channels was measured along the while arrow at each time point. Black arrows point to a paxillin-mCherry-rich area that becomes a hotspot for FN-PH deposition 30 min later. Scale bar = 10 μm.

FN-PH deposition follows cell polarization. (A) Gallery of frames from a TIRFM video of live KO67 Halo-β1itg FN-PH fibroblasts in the process of spreading on FN-coated glass or PLL-coated glass in a medium depleted of FN. FN-PH is shown in green and paxillin-mCherry in magenta. Note the difference in time scale between the two substrata. The white rectangle indicates the area zoomed in the inset on the right. (B) Gallery of frames from a TIRM video of KO67 untagged-β1itg FN-PH Golgi-Halo fibroblasts, 0 or 4 h after plating on FN-coated glass. FN-PH is shown in green, paxillin-mCherry in red, Golgi-Halo in magenta, and nucleus stained with Hoechst 33342 in blue. The signal for already deposited FN-PH was photobleached before imaging, FN-PH deposition was measured for a duration of 1 h, and the net change in FN-PH signal was determined by subtracting the mean green fluorescence before acquisition to the mean green fluorescence 1 h later. For each cell, a Golgi side (solid gray outline) and an opposite side (dashed gray outline) were delineated by a line passing through the center of the nucleus and running parallel to the crescent-shaped perinuclear Golgi apparatus. Data in the graph represent the ratio of the net change in FN-PH fluorescence on each side in 18–23 cells. Data were analyzed by unpaired t test, **** P < 0.0001. (C) Gallery of frames from a TIRM video of KO67 notag-β1itg FN-PH RA-PH-Halo (paxillin-mCherry in red, FN-PH in green, and lamellipodin RA-PH-Halo labeled with SiR-Halo ligand in blue) migrating out of a monolayer on FN-coated glass. 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. The fluorescence intensity in the three fluorescent channels was measured along the while arrow at each time point. Black arrows point to a paxillin-mCherry-rich area that becomes a hotspot for FN-PH deposition 30 min later. Scale bar = 10 μm.

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