Figure 5. CDC42EP5 associates with SEPT9... | Rockefeller University Press

Figure 5.

$CDC42EP5 associates with SEPT9 and F-actin to promote F-actin cross-linking and the generation of actomyosin structures. (A) Left panel shows a 690.cl2 cell expressing GFP-Cdc42ep5 (green) and stained for Sept9 (blue) and F-actin (magenta). Scale bars, 10 µm. Middle panels show merged and individual channel magnifications of indicated areas: a, perinuclear; b and c, peripheral. Graph shows Pearson’s correlation coefficient of Cdc42ep5, Sept9, and F-actin in perinuclear and peripheral areas. Bars indicate mean ± SEM (n = 7, 6, and 12 independent regions in individual cells; unpaired t tests: †, not significant; *, P < 0.05; #, P < 0.0001). (B) Image of 690.cl2 cell expressing GFP-Cdc42ep5 (green) on collagen-rich matrices. Staining of F-actin (magenta) and Sept9 (cyan) and individual channel magnifications of indicated area are also shown. Scale bars, 10 µm. (C) Images show F-actin (magenta), Sept9 (green), and DAPI (blue) staining in 690.cl2 cells on glass after transfection with indicated siRNAs. Magnifications of perinuclear areas are shown. Scale bars, 20 µm. Violin plot shows quantifications of cytosolic and perinuclear intensity of Sept9 staining. n, individual cells; Mann–Whitney’s test: †, not significant; ***, P < 0.001; #, P < 0.0001. (D) Images show F-actin (magenta), GFP (green), and Sept9 (cyan) of 690.cl2KO cells expressing GFP or GFP-Cdc42ep5 on collagen-rich matrices. Scale bars, 2.5 µm. Single channels for GFP and Sept9 signals are shown. Panels on the right show magnifications of Sept9 signals in cortical and cytosolic areas. Violin plot at the bottom shows quantification of cortical Sept9 signal. n, independent regions in individual cells; Mann–Whitney’s test. (E) Top panel is a Coomassie-stained gel showing equal volumes of supernatant (S) and pellet (P) fractions from low-speed sedimentation of prepolymerized actin filaments in the presence of recombinant Cdc42ep5 (1 µM), SEPT9 (1 µM), and both Cdc42eep5 (1 µM) and SEPT9 (1 µM). Bottom panel shows an anti-actin Western blot of the same experiment. Graph shows the actin bundling coefficient (ratio of actin in pellet vs. supernatant) in the indicated experimental points. Bars indicate mean ± SEM (n = 3 experiments; one-way ANOVA, Tukey’s test: †, not significant; *, P < 0.05; ***, P < 0.001). AU, arbitrary units.$

CDC42EP5 associates with SEPT9 and F-actin to promote F-actin cross-linking and the generation of actomyosin structures. (A) Left panel shows a 690.cl2 cell expressing GFP-Cdc42ep5 (green) and stained for Sept9 (blue) and F-actin (magenta). Scale bars, 10 µm. Middle panels show merged and individual channel magnifications of indicated areas: a, perinuclear; b and c, peripheral. Graph shows Pearson’s correlation coefficient of Cdc42ep5, Sept9, and F-actin in perinuclear and peripheral areas. Bars indicate mean ± SEM (n = 7, 6, and 12 independent regions in individual cells; unpaired t tests: †, not significant; *, P < 0.05; #, P < 0.0001). (B) Image of 690.cl2 cell expressing GFP-Cdc42ep5 (green) on collagen-rich matrices. Staining of F-actin (magenta) and Sept9 (cyan) and individual channel magnifications of indicated area are also shown. Scale bars, 10 µm. (C) Images show F-actin (magenta), Sept9 (green), and DAPI (blue) staining in 690.cl2 cells on glass after transfection with indicated siRNAs. Magnifications of perinuclear areas are shown. Scale bars, 20 µm. Violin plot shows quantifications of cytosolic and perinuclear intensity of Sept9 staining. n, individual cells; Mann–Whitney’s test: †, not significant; ***, P < 0.001; #, P < 0.0001. (D) Images show F-actin (magenta), GFP (green), and Sept9 (cyan) of 690.cl2^KO cells expressing GFP or GFP-Cdc42ep5 on collagen-rich matrices. Scale bars, 2.5 µm. Single channels for GFP and Sept9 signals are shown. Panels on the right show magnifications of Sept9 signals in cortical and cytosolic areas. Violin plot at the bottom shows quantification of cortical Sept9 signal. n, independent regions in individual cells; Mann–Whitney’s test. (E) Top panel is a Coomassie-stained gel showing equal volumes of supernatant (S) and pellet (P) fractions from low-speed sedimentation of prepolymerized actin filaments in the presence of recombinant Cdc42ep5 (1 µM), SEPT9 (1 µM), and both Cdc42eep5 (1 µM) and SEPT9 (1 µM). Bottom panel shows an anti-actin Western blot of the same experiment. Graph shows the actin bundling coefficient (ratio of actin in pellet vs. supernatant) in the indicated experimental points. Bars indicate mean ± SEM (n = 3 experiments; one-way ANOVA, Tukey’s test: †, not significant; *, P < 0.05; ***, P < 0.001). AU, arbitrary units.