Figure 2. Septins regulate the organization of the lamellar actin network and are required for the stabilization of nascent FAs. (A) Confocal images of MDCK cells, which were transfected with mCherry-expressing plasmids that encode for control or SEPT2 shRNAs and stained for paxillin after treatment with HGF. The red broken line highlights the cell edge. Panels on the right show enlarged views of the black boxed region. (B and C) Graphs show FA size and number from MDCK cells (n = 20) treated with control, SEPT2, and SEPT9 shRNAs. (D) Graph shows FA distance from the cell edge of MDCK cells (n = 10) treated with control (n = 903 FAs), SEPT2 #1 (n = 749 FAs), SEPT2 #2 (n = 512 FAs), and SEPT9 shRNAs (n = 840 FAs). (E) The graph shows FA lifetime in three different MDCK cells treated with control (n = 6,066 FAs) and SEPT2 shRNAs (n = 10,528 FAs). (F) The graph shows the rates of FA (n = 45) assembly and disassembly in cells treated with control and SEPT2 shRNAs. (G) Representative profiles of the kinetics of paxillin-GFP fluorescence in MDCK cells treated with control and SEPT2 shRNAs. (H) Confocal images show 786-O cells stained for total paxillin (red) and phosphorylated pY118-paxillin (green). Insets show GFP expression from plasmids encoding for control and SEPT2 shRNAs. Panels on the right show enlarged views from the boxed regions. (I) Bar graphs show the ratio of the fluorescence intensity of pY118-paxillin to total paxillin in MDCK cells (n = 10) treated with control (n = 740 FAs) and SEPT2 shRNAs (n = 962 FAs), and 786-O cells (n = 10) treated with control (n = 757 FAs) and SEPT2 shRNAs (n = 602 FAs). (J) Confocal images of control and SEPT2-depleted MDCK cells stained for F-actin (phalloidin; red) and paxillin (white). Insets show GFP expression from plasmids encoding for control and SEPT2 shRNAs. Panels on the right show enlarged views of the boxed region. (K) The plot shows line scans of phalloidin fluorescence across the leading edges of MDCK cells (n = 13) treated with control (n = 54 line scans) and SEPT2 shRNAs (n = 56 line scans). Fluorescence quantifications were performed with a custom MATLAB code (supplemental code 1). (L and M) Graphs show the mean number of RSFs per lamellar length (L) and mean length of RSFs (M) in MDCK cells treated with control (n = 43) and SEPT2 shRNAs (n = 72). (N) Inverted monochrome frames show ABP140-ABD-mCherry from total internal reflection fluorescence (TIRF) microscopy of MDCK cells treated with control or SEPT2 shRNAs and HGF. Arrows point to RSFs that persist and grow over time in control cells, and stress fibers that dissipate in SEPT2-depleted cells. Error bars indicate SEM. **, P < 0.01; ***, P < 0.001.
Figure 2.

Septins regulate the organization of the lamellar actin network and are required for the stabilization of nascent FAs. (A) Confocal images of MDCK cells, which were transfected with mCherry-expressing plasmids that encode for control or SEPT2 shRNAs and stained for paxillin after treatment with HGF. The red broken line highlights the cell edge. Panels on the right show enlarged views of the black boxed region. (B and C) Graphs show FA size and number from MDCK cells (n = 20) treated with control, SEPT2, and SEPT9 shRNAs. (D) Graph shows FA distance from the cell edge of MDCK cells (n = 10) treated with control (n = 903 FAs), SEPT2 #1 (n = 749 FAs), SEPT2 #2 (n = 512 FAs), and SEPT9 shRNAs (n = 840 FAs). (E) The graph shows FA lifetime in three different MDCK cells treated with control (n = 6,066 FAs) and SEPT2 shRNAs (n = 10,528 FAs). (F) The graph shows the rates of FA (n = 45) assembly and disassembly in cells treated with control and SEPT2 shRNAs. (G) Representative profiles of the kinetics of paxillin-GFP fluorescence in MDCK cells treated with control and SEPT2 shRNAs. (H) Confocal images show 786-O cells stained for total paxillin (red) and phosphorylated pY118-paxillin (green). Insets show GFP expression from plasmids encoding for control and SEPT2 shRNAs. Panels on the right show enlarged views from the boxed regions. (I) Bar graphs show the ratio of the fluorescence intensity of pY118-paxillin to total paxillin in MDCK cells (n = 10) treated with control (n = 740 FAs) and SEPT2 shRNAs (n = 962 FAs), and 786-O cells (n = 10) treated with control (n = 757 FAs) and SEPT2 shRNAs (n = 602 FAs). (J) Confocal images of control and SEPT2-depleted MDCK cells stained for F-actin (phalloidin; red) and paxillin (white). Insets show GFP expression from plasmids encoding for control and SEPT2 shRNAs. Panels on the right show enlarged views of the boxed region. (K) The plot shows line scans of phalloidin fluorescence across the leading edges of MDCK cells (n = 13) treated with control (n = 54 line scans) and SEPT2 shRNAs (n = 56 line scans). Fluorescence quantifications were performed with a custom MATLAB code (supplemental code 1). (L and M) Graphs show the mean number of RSFs per lamellar length (L) and mean length of RSFs (M) in MDCK cells treated with control (n = 43) and SEPT2 shRNAs (n = 72). (N) Inverted monochrome frames show ABP140-ABD-mCherry from total internal reflection fluorescence (TIRF) microscopy of MDCK cells treated with control or SEPT2 shRNAs and HGF. Arrows point to RSFs that persist and grow over time in control cells, and stress fibers that dissipate in SEPT2-depleted cells. Error bars indicate SEM. **, P < 0.01; ***, P < 0.001.

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