Figure 3. SEPT9 functions as an actin... | Rockefeller University Press

Figure 3.

$Figure 3. SEPT9 functions as an actin cross-linking protein in FA maturation. (A) Confocal images of paxillin-stained MDCK cells transfected with mCherry-expressing plasmids that encode for control or SEPT2 shRNAs, and RLC-DD-GFP, α-actinin-GFP, or GFP vectors. The boxed regions are enlarged in the panels on the immediate right. Bars, 10 µm. (B and C) Graphs show FA size (B) and number per square micrometer (C) in control cells expressing GFP (n = 1,650 FAs; 25 cells), and SEPT2 knockdown cells expressing GFP (n = 1,567 FAs; 24 cells), α-actinin-1-GFP (n = 1,556 FAs; 20 cells), RLC-DD-GFP (n = 1,962 FAs; 18 cells), and α-actinin-1-ΔABD-GFP (n = 1,962 FAs; 20 cells). Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Confocal images of MDCK cells transfected with mCherry-expressing plasmids encoding for control or SEPT2 shRNAs, and α-actinin-1-GFP or GFP vectors. Cells were stained for F-actin (phalloidin). The boxed regions are enlarged in the panels on the immediate right. Bars, 10 µm. (E) The Coomassie-stained gel shows equal volumes of supernatant (S) and pellet (P) fractions from low-speed sedimentation of prepolymerized actin filaments in the presence of recombinant SEPT2/6/7 and SEPT9. (F and G) Negative stain EM images of actin filaments in the absence and presence of recombinant SEPT6 or SEPT9. Boxed regions (F) are shown at a higher magnification in G. Bars, 0.5 µm.$

SEPT9 functions as an actin cross-linking protein in FA maturation. (A) Confocal images of paxillin-stained MDCK cells transfected with mCherry-expressing plasmids that encode for control or SEPT2 shRNAs, and RLC-DD-GFP, α-actinin-GFP, or GFP vectors. The boxed regions are enlarged in the panels on the immediate right. Bars, 10 µm. (B and C) Graphs show FA size (B) and number per square micrometer (C) in control cells expressing GFP (n = 1,650 FAs; 25 cells), and SEPT2 knockdown cells expressing GFP (n = 1,567 FAs; 24 cells), α-actinin-1-GFP (n = 1,556 FAs; 20 cells), RLC-DD-GFP (n = 1,962 FAs; 18 cells), and α-actinin-1-ΔABD-GFP (n = 1,962 FAs; 20 cells). Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Confocal images of MDCK cells transfected with mCherry-expressing plasmids encoding for control or SEPT2 shRNAs, and α-actinin-1-GFP or GFP vectors. Cells were stained for F-actin (phalloidin). The boxed regions are enlarged in the panels on the immediate right. Bars, 10 µm. (E) The Coomassie-stained gel shows equal volumes of supernatant (S) and pellet (P) fractions from low-speed sedimentation of prepolymerized actin filaments in the presence of recombinant SEPT2/6/7 and SEPT9. (F and G) Negative stain EM images of actin filaments in the absence and presence of recombinant SEPT6 or SEPT9. Boxed regions (F) are shown at a higher magnification in G. Bars, 0.5 µm.

This Feature Is Available To Subscribers Only