Figure 1.
Podocalyxin induces microvillus formation.
MDCK (A, B, E, F) and MCF-7 (C, D, G, H) cells transfected with murine Podocalyxin (B, D, F, H) or empty vector (A, C, E, G) and examined by TEM (A–D; scale bar: 1 µm) and SEM (E–H; scale bar: 2 µm). In TEM images, vertical slices are shown near the apical cell surface with some of the numerous additional microvilli labeled with red arrows. Many microvilli are visible as small circles as they are seen in cross-section. SEM images are of the apical cell surface with microvilli evident as thin surface projections. (I) Microvilli in six 50 µm2 fields were enumerated and graphed. Averages are shown; error bars represent standard deviation. T-tests were used to show statistically significant differences between vector and Podocalyxin-transfected cells, with p<0.002 in both cases. Representative of two independent experiments.
Figure 2.
Podocalyxin-induced microvilli are structurally dependent on intact actin filaments.
MDCK (A) and MCF-7 (B) cells transfected with murine Podocalyxin or empty vector were treated with the actin disrupting agent latrunculin A (LatA) or DMSO (negative control) and then fixed. Podocalyxin was labeled with an anti-Podocalyxin antibody (green); f-actin was labeled with phalloidin (red), and nuclei were labeled with DAPI (blue). Scale bar: 5 µm.
Figure 3.
Podocalyxin induces apical expansion and recruits NHERF-1 to the apical membrane.
3D images of empty vector and Podocalyxin-transfected MCF-7 cells showing ectopic Podocalyxin (red), endogenous NHERF-1 (green), and DAPI (blue) labeling. Dimensions of blue rectangles: 118×26 µm. (A) Podocalyxin-transfected cells labeled with an anti-Podocalyxin antibody. (B) The same cells labeled with an anti-NHERF-1 antibody. (C) Merged images showing Podocalyxin, NHERF-1, and DAPI labeling. Inset in (A): projection of apical surface of a single Podocalyxin-transfected MCF-7 cell, labeled with an anti-Podocalyxin antibody.
Figure 4.
Podocalyxin induces apical recruitment of f-actin and ezrin, and all three molecules colocalize in microvilli.
Projections of apical confocal images of MCF-7 cells transfected with murine Podocalyxin or empty vector showing ectopic Podocalyxin (blue), endogenous ezrin (green), and f-actin (red) labeling. White represents colocalization of all three molecules. Note the long extended microvilli in the Podocalyxin-transfected sample. Scale bars: 5 µm.
Figure 5.
Apical localization of Podocalyxin and NHERF-1 is not dependent on ezrin.
MCF-7 cells stably expressing ectopic murine Podocalyxin were either transfected with VSV-tagged dominant negative (N-terminal) ezrin after formation of monolayers and appropriate localization of Podocalyxin and NHERF-1 (A, B) or transfected with VSV-tagged N'ezrin and then replated before immunostaining (C, D). (A–D) Immunolabeling of VSV-tagged N'ezrin (green), ectopic Podocalyxin (red), and endogenous NHERF-1 (blue). Purple represents apical colocalization of Podocalyxin and NHERF-1 in cells not expressing dominant negative ezrin (internal negative control); white represents apical colocalization of all three molecules in cells expressing dominant negative ezrin. Scale bars: 5 µm. (A, C) Projections of merged confocal stacks taken near the apical cell surface showing individual colors and merged images; (B, D) vertical slices of confocal stacks.
Figure 6.
Schematic of Podocalyxin mutants and FACS expression profiles.
(A) Podocalyxin mutants. Blue: extracellular domain, horizontal bars: carbohydrates/sialic acid residues, red: transmembrane region, purple: cytoplasmic tail (including C-terminal DTHL in wildtype and ΔEC), and green: flag-tag replacing most of Podocalyxin's extracellular domain. (B) FACS profiles demonstrating comparable Podocalyxin expression levels in sorted clones. Dashed lines represent negative controls (vector-transfected cells stained with the same antibodies); solid lines represent Podocalyxin expression. Note: all mutants were detected with an anti-Podocalyxin antibody with the exception of ΔEC, which was detected with an anti-flag antibody.
Figure 7.
Podocalyxin's C-terminal DTHL motif is required for recruitment of NHERF-1.
Projections of apical confocal images of transfected MCF-7 cells showing ectopic Podocalyxin (red), endogenous NHERF-1 (green), and DAPI (blue) labeling. Isotype control sample is Podocalyxin-transfected cells labeled with anti-Podocalyxin (red) and NHERF-1 isotype control (green) to demonstrate lack of non-specific NHERF-1 staining. White numbers represent Pearson's colocalization coefficient. Scale bar: 5 µm.
Figure 8.
Podocalyxin's extracellular domain and the first four amino acids of its cytoplasmic tail are necessary and sufficient for microvillus formation.
(A) TEM images of MCF-7 cells transfected with vector or full-length and mutant Podocalyxin. Vertical slices are shown near the apical cell surface with some of the numerous additional microvilli labeled with red arrows. Scale bar: 1 µm. (B) SEM images at the apical surface of transfected MCF-7 cells with microvilli evident as thin surface projections. Scale bar: 2 µm. (C) Microvilli in six 50 µm2 fields were enumerated and graphed. Averages are shown; error bars represent standard deviation. T-tests were used to show statistically significant differences between vector and Podocalyxin-transfected cells, and between wildtype Podocalyxin and ΔEC-transfected cells with p<0.003 in all cases. Representative of two independent experiments.
Figure 9.
Podocalyxin colocalizes with ezrin in a manner independent of any direct interaction with NHERF-1.
(A) Vertical slices of confocal stacks demonstrate increased apical recruitment of ezrin in Podocalyxin-transfected MCF-7 cells. Green: ectopic Podocalyxin, red: endogenous ezrin, blue: nuclei (DAPI). Scale bar: 5 µm. (B) Projections of merged apical confocal images of MCF-7 cells transfected with various Podocalyxin constructs or empty vector. Green: Podocalyxin, red: ezrin (or isotype control for ezrin), blue: DAPI. White numbers represent Pearson's colocalization coefficient. Scale bar: 5 µm. (C) In vitro pull-down assays with biotinylated Podocalyxin cytoplasmic tail (PCT) peptides and GST-N'ezrin reveal a lack of interaction between a Podocalyxin peptide representing the cytoplasmic tail of the Δtail mutant (HQRF) and ezrin. Biotinylated Podocalyxin peptides bound to streptavidin-sepharose were incubated with GST-N'ezrin or GST alone, and bound recombinant proteins were detected with an anti-GST-antibody by Western blot.
Figure 10.
Podocalyxin colocalizes with f-actin at the apical surface of cells, even in the absence of most of Podocalyxin's cytoplasmic tail.
(A, B) Podocalyxin-transfected MCF-7 cells with f-actin (red), ectopic Podocalyxin (green), and DAPI (blue) labeling. Scale bars: 5 µm. (A) Projections of merged apical confocal images; white numbers represent Pearson's colocalization coefficient. (B) Vertical slices of confocal stacks. (C, D) High magnification TEM images demonstrate the presence of actin filaments (red arrows) running the length of individual microvilli. Although microvilli of different lengths are shown in this figure, there was no consistent difference in microvilli lengths between samples. Scale bars: 0.1 µm. (C) Cross-sections; (D) longitudinal-sections.
Figure 11.
Model of Podocalyxin's functions.
(A) Cells expressing low or no Podocalyxin have well-defined cell-cell junctions and adhere well to the substratum. Overexpression of Podocalyxin leads to recruitment of f-actin to the apical membrane for formation of microvilli along with apical domain expansion and weakening of cell-cell junctions. Higher expression induces further recruitment of f-actin to the apical surface, which may lead to a decrease in basolateral actin and disruption of integrin-mediated cell-substratum adhesion. Two models are proposed to explain Podocalyxin's mechanism of action. (B) In the absence of Podocalyxin expression NHERF-1 is localized throughout the cytoplasm, whereas in cells expressing high levels of Podocalyxin NHERF-1 is recruited to the apical membrane. Thus, Podocalyxin may regulate NHERF's role in many biological processes.