Figure 2

Abnormal distribution of ezrin, elongated actin structure and hyperactive Arf6 in resting ASAP3-deficient cells. (a) Immunofluorescence using specific antibodies for ezrin (red) in gastric parietal cells of ASAP3-deficient mice and control wild-type (WT) mice. Note the staining of ezrin displayed sac-like pattern, with the center occupied by a negatively stained region (white arrows). Scale bars are labeled within immunofluorescent images. (b) Transmission electron microscopy (TEM) analysis of resting parietal cells in WT mice (top) and ASAP3-deficient mice (bottom). While WT parietal cells showed short microvilli structure (MV), ASAP3-deficient parietal cells exhibited broader regions that were occupied by elongated and tightly stacked microvilli. MT, mitochondria; TV, tubulovesicle. Scale bars indicate 2 μm within TEM images. (c) Statistical analysis of longitudinal microvilli lengths (n=25 per genotype) in resting parietal cells measured by ImageJ in WT and ASAP3-deficient mice as determined by TEM (*P<0.01, Student’s t-test). (d) Representative immunofluorescent image of F-actinin resting parietal cells (left) and statistical analysis of F-actin intensity (right). Note the discontinuous staining pattern of F-actin (red) in ASAP3-defecient mice but not WT mice. Statistical analysis of F-actin intensity (n=18 per genotype) in multiple resting parietal cells based on immunostaining in ASAP3-defecient and WT mice (*P<0.01, Student’s t-test). (e) The expression levels of active Arf6-GTP-bound pattern, pan Arf6 in ASAP3-defecient and WT mice without stimulation were detected by Western Blot. The GAPDH level was used as loading control. The data are representative of immunoblots from nine littermates per loading group. (f) Representative immunofluorescent image of Arf6 (green) and F-actin (red) in resting parietal cells. Cell nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI) in blue. Zoomed sections are shown on the right panel. Scale bars indicate 2 μm in all panels.