Figure 5. SEPT2 is required for the... | Rockefeller University Press

Figure 5.

$Figure 5. SEPT2 is required for the morphogenesis of polarized, columnar-shaped epithelia. (A) Non-polarized, “contact-naïve” MDCK cells were transfected with control and SEPT2 siRNAs for 60 h. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies to SEPT2 (red) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; green). (B) Schematic of the experimental setup. (C) Representative dorsal views of 3D-rendered confocal images of MDCK cells stained for SEPT2 and α-tubulin. (D) Representative contour sketches of control and SEPT2-depleted cells. The ratios (mean ± SEM) of lengths of major-to-minor cell axes are shown for control (n = 33) and SEPT2-depleted (n = 30) cells from three independent experiments (P < 0.0001). Cell heights were measured from cross sections (xz) of confocal images and their values (mean ± SEM) are shown for control (n = 20) and SEPT2-depleted (n = 20) cells (P < 0.0001). (E) Representative mid-section confocal images of MDCK cells stained for SEPT2 and polyGlu tubulin; fixation/permeabilization conditions differed from those in F (see Materials and methods). Arrowheads point to SEPT2 and polyGlu microtubules that run parallel to the lateral membrane in the apicobasal axis. (F) Representative confocal cross sections (xz) of MDCK cells stained for SEPT2, Na/K-ATPase, and gp135/podocalyxin. Asterisks outline SEPT2-depleted cells; arrowheads point to lateral cell–cell contacts. (G) High resolution 3D-rendered images of the regions outlined by arrows in F. Yellow arrows point to punctate structures containing gp135/podocalyxin and Na/K-ATPase. (H) Cartoons of Na/K-ATPase (green) and gp135/podocalyxin (red) localizations in control and SEPT2-depleted cells. (I) Apical (gp135/podocalyxin) and basolateral (Na/K-ATPase) membrane and intracellular fluorescence intensities were measured from confocal cross sections (xz) of control (n = 23) and SEPT2-depleted (n = 23) cells. Bar graphs show ratios (mean values ± SEM) of plasma membrane to intracellular fluorescence from three independent experiments (***, P < 0.0001). (J) Post-nuclear homogenates from control (red) and SEPT2-depleted (blue) MDCK monolayers separated in iodixanol (OptiPrep) gradients, and analyzed by SDS-PAGE and blotted with antibodies to gp135/podocalyxin (apical), Na/K-ATPase (basolateral), caveolin (apical and basolateral), and furin convertase (TGN). Protein band intensities were plotted as a percentage of their sum intensity. Vertical lines demarcate the peak fractions of apical, basolateral, and Golgi proteins in gradients from control cells. Data are representative of three independent experiments.$

SEPT2 is required for the morphogenesis of polarized, columnar-shaped epithelia. (A) Non-polarized, “contact-naïve” MDCK cells were transfected with control and SEPT2 siRNAs for 60 h. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies to SEPT2 (red) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; green). (B) Schematic of the experimental setup. (C) Representative dorsal views of 3D-rendered confocal images of MDCK cells stained for SEPT2 and α-tubulin. (D) Representative contour sketches of control and SEPT2-depleted cells. The ratios (mean ± SEM) of lengths of major-to-minor cell axes are shown for control (n = 33) and SEPT2-depleted (n = 30) cells from three independent experiments (P < 0.0001). Cell heights were measured from cross sections (xz) of confocal images and their values (mean ± SEM) are shown for control (n = 20) and SEPT2-depleted (n = 20) cells (P < 0.0001). (E) Representative mid-section confocal images of MDCK cells stained for SEPT2 and polyGlu tubulin; fixation/permeabilization conditions differed from those in F (see Materials and methods). Arrowheads point to SEPT2 and polyGlu microtubules that run parallel to the lateral membrane in the apicobasal axis. (F) Representative confocal cross sections (xz) of MDCK cells stained for SEPT2, Na/K-ATPase, and gp135/podocalyxin. Asterisks outline SEPT2-depleted cells; arrowheads point to lateral cell–cell contacts. (G) High resolution 3D-rendered images of the regions outlined by arrows in F. Yellow arrows point to punctate structures containing gp135/podocalyxin and Na/K-ATPase. (H) Cartoons of Na/K-ATPase (green) and gp135/podocalyxin (red) localizations in control and SEPT2-depleted cells. (I) Apical (gp135/podocalyxin) and basolateral (Na/K-ATPase) membrane and intracellular fluorescence intensities were measured from confocal cross sections (xz) of control (n = 23) and SEPT2-depleted (n = 23) cells. Bar graphs show ratios (mean values ± SEM) of plasma membrane to intracellular fluorescence from three independent experiments (***, P < 0.0001). (J) Post-nuclear homogenates from control (red) and SEPT2-depleted (blue) MDCK monolayers separated in iodixanol (OptiPrep) gradients, and analyzed by SDS-PAGE and blotted with antibodies to gp135/podocalyxin (apical), Na/K-ATPase (basolateral), caveolin (apical and basolateral), and furin convertase (TGN). Protein band intensities were plotted as a percentage of their sum intensity. Vertical lines demarcate the peak fractions of apical, basolateral, and Golgi proteins in gradients from control cells. Data are representative of three independent experiments.

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