PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Abstract

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.

Fig 1. PtdIns(4,5)P2 and PTEN localize to the AP PM in cysts

(A) PtdIns(4,5)P2 is enriched in the AP PM. MDCK PHD-GFP cysts were stained for nuclei (blue) and gp135 (red, upper-right panel merged with DIC). In all micrographs single confocal sections through the middle of cysts are shown and nuclei are stained blue unless otherwise indicated. Bottom-right panel shows a magnification of the indicated region of the merged panel (bottom left). Arrowhead indicates the colocalization of PHD-GFP and gp135 at the AP PM. All scale bars are 5 μm unless otherwise indicated. (B) PtdIns(4,5)P2 segregates from PtdIns(3,4,5)P3 during cystogenesis. MDCK PHD-GFP (top row) and PH-Akt-GFP (middle row, merged with DIC in bottom row) were plated to form cysts for 4 days. Live cells were analyzed by confocal microscopy at 1, 2, 3 or 4d. In this and all other micrographs “L” indicates lumen. (C) GFP-PTEN localizes to the AP PM. MDCK GFP-PTEN cells forming cysts at 1, 2, 3 and 4d. Cells were stained for ZO-1 (red). Arrowheads indicate GFP-PTEN at the AP PM. (D) PTEN localizes to the AP PM in T84 human colon cysts. Endogenous PTEN could not be localized in MDCK cysts, but could be localized in T84 cysts. T84 human colon cells formed cysts for 8d; stained for PTEN (green), actin (red) and nuclei, and visualized. Arrowheads indicate PTEN at the AP PM.

Fig 2. Disruption of PTEN with siRNA or a specific inhibitor blocks central lumen formation

(A) Down-regulation of PTEN by siRNA. Total lysates of MDCK cells transfected with siRNAs PTEN-1 and PTEN-2 or with control siRNA formed cysts for 72h; then western blotted for PTEN and GAPDH. (B) Effect of siRNA PTEN-2 on lumen formation. Cells were transfected with PTEN-2 (bottom row), or control (top row) siRNA and plated to form cysts for 72h. Cells were stained to detect gp135 (red), β-catenin (green), nuclei (left panels) and actin (red, right panels merged with DIC). (C) Quantitation of cysts with normal lumens in cells transfected with control siRNA or specific siRNA PTEN-1, PTEN-2. Values are mean ±SD from 4 different experiments. N=100 cysts/experiment. *P<0.001. (D) The PTEN inhibitor bpV(pic) disrupts lumen formation in a dose-dependent manner. MDCK cells forming cysts for 48h were treated with indicated concentrations of bpV(pic). After 48h cells were stained to detect gp135 (red), β-catenin (green), nuclei (blue)-left panels. Right panel is quantitation of cysts with normal lumens in cells treated with 0 (control), 0.1, 1 or 10μM bpV(pic). The values shown are mean ±SD from 3 different experiments. N=100/experiment. *P<0.005; **P<0.001. (E) BpV(pic) inhibits segregation of phosphoinositides. PHD-GFP (right panels) or PH-Akt-GFP (left panels) cells formed cysts for 4d. The cells were stained to detect ZO-1 (red), nuclei (left panels) and gp135 (red, right panel merged with DIC).

Fig 3. Exogenous PtdIns(4,5)P2 relocalizes AP and TJ proteins to the BL PM. MDCK PHD-GFP cells forming cysts for 2d were incubated with histone (control) or PtdIns(4,5)P2-histone complexes (+PtdIns(4,5)P2) for up to 30min, fixed and visualized

(A) Exogenous PtdIns(4,5)P2 relocalizes PHD-GFP from AP to the basal PM. Arrowheads indicate PHD-GFP enrichment at the AP (control) or basal PM (+PtdIns(4,5)P2). Scale bar 10 μm. (B) Exogenous PtdIns(4,5)P2 relocalizes gp135, actin and ZO-1 from the AP to the basal PM. Top half of figure was treated with PtdIns(4,5)P2 for indicated time, while bottom half was control. Samples were stained for actin (red), ZO-1 (green) and nuclei in top rows; and gp135 (red) merged with DIC in bottom rows. Arrows indicate AP makers and ZO-1. (C) Exogenous PtdIns(4,5)P2 disrupts lateral PM. The samples were stained for actin (red), p58 (green) and nuclei (left panels), or DIC (right panels). Arrowheads indicate p58 localization.

Fig 4. Ax2 localizes to the AP PM and is needed for lumen formation

(A) MDCK Ax2-GFP cells were grown for 5 days to form mature cysts, and stained for actin (red) and nuclei (blue). Actin and DIC (top-right panel). Bottom-right panel shows a magnification of the region indicated with a white square in the merged panel (bottom left). Arrow indicates colocalization of Ax2-GFP and actin at the AP PM. (B) Down-regulation of endogenous Ax2 and p11 by siRNA to Ax2. Total cell lysates of cysts (48 or 72h) treated with siRNAs Ax2-1 and Ax2-2 against canine Ax2, or control siRNA, were analyzed by western blot for Ax2, p11 and caveolin-1 as a control. (C) Effect of siRNA Ax2-2 in lumen formation. MDCK cells were transfected with Ax2-2 (bottom panel), or control (top panel) siRNAs, and plated to form cysts for 48h. Cells were stained for gp135 (red), ZO-1 (green) and nuclei. Arrowheads indicate ZO-1. (D) Quantitation of cysts with normal lumens in cells transfected with scramble (control) siRNA or siRNA Ax2-1 or Ax2-2. Values are mean ±SD from 4 different experiments. N=100/experiment. *P <0.001. (E) Ax2CM disrupts the cortical actin cytoskeleton. Cells were infected with a tet-off regulated adenovirus encoding GFP-Ax2CM, and incubated with a low level of dox (1ng/ml) (GFP-Ax2CM). After 48h cells were fixed, stained for actin (blue) and ezrin (red), and analyzed by confocal microscopy. Arrowheads indicate disruption of actin cytoskeleton in GFP-Ax2 cell. Scale bar 10 μm. (F) Down-regulation of PTEN by siRNA inhibits AP accumulation of Ax2. Cells expressing Ax2-GFP were transfected with siRNAs to PTEN or control siRNAs cysts formed for 48h, fixed and stained for gp135 (red) and nuclei (blue).

Fig 5. Ax2 binds to Cdc42 at the AP PM

(A) Western blot of stable expression of GFP-Rac1 or GFP-Cdc42. Extracts from MDCK GFP-Cdc42, GFP-Rac1 or control cells were inmunoblotted with anti-Rac1 (left panel) and anti-Cdc42 (right panel) to detect endogenous and transfected proteins. (B) GFP-Cdc42 distribution in mature cysts. GFP-Cdc42 cells were grown for 5d and stained for nuclei (blue) and actin (red). Bottom-right panel shows the magnification of the indicated region of the merged panel (bottom left). Arrowheads indicate colocalization of GFP-Cdc42 and actin at the AP PM. Scale bar, 10 μm in upper-right panel and 2 μm in the bottom-right panel. (C) Distribution of CBD-GFP, GFP-Cdc42 and Rac1-GFP during cystogenesis. MDCK CBD-GFP (top row), GFP-Cdc42 (middle row) or GFP-Rac1 (bottom row) cells were grown for 1, 2 or 5d and visualized. Arrows indicate fluorescent proteins at cell-cell or cell-ECM junctions. Arrowheads indicate the localization of CBD-GFP and GFP-Cdc42 at the AP surface. Scale bars are 5 μm or 10 μm as indicated. (D) Ax2-GFP interacts with endogenous Cdc42 in a GTP-dependent manner. MDCK Ax2-GFP or control cells in mature cysts were lysed and extracts were loaded with GDP or GTPγS. Extracts were immunoprecipitated with antibody against GFP and immunoblotted to analyze Cdc42 and p11. 3% of input or 30% of immunoprecipitated material were loaded on the gel. A band corresponding to the light chain (LC) of IgG was detected with the anti-Cdc42 antibodies and served as a loading control. (E) Cdc42-GFP interacts with endogenous Ax2 in a GTP dependent manner. Cdc42-GFP or control cells in mature cysts were lysed and the extracts loaded with GDP or GTPγS. Extracts were immunoprecipitated using antibodies against GFP and immunoblotted to analyze Ax2 and IQGAP1. 3% of input and 30% of immunoprecipitated material were loaded in the gel. (F–H) Effect of Tet-off inducible adenovirus-mediated expression of Ax2X on Ax2-GFP or Cdc42-GFP localization in cysts. MDCK Ax2-GFP (G) or Cdc42-GFP (H) cells formied cysts for 5d were infected with a tet off regulated adenovirus encoding Ax2XM and maintained in the presence (control) or absence (Ax2XM) of 20ng/ml of dox for 16h. Cells were lysed and extracts analyzed by western blot to detect Ax2XM (F). The cysts were and stained for actin (red) and nuclei (blue). Arrowheads indicate aggregates of Ax2-GFP or Cdc42-GFP. Arrows indicate disruption of the AP actin cytoskeleton. Scale bar 10 μm.

Fig 6. Cdc42 siRNA inhibits formation of central lumen

(A) Down-regulation of Cdc42 by siRNA. Cells were transfected with siRNAs Cdc42-1 and Cdc42-2 against canine Cdc42 or with control siRNA, allowed to form cysts for 48 or 72h, and then total cell lysates western blotted for Cdc42 and β-tubulin (control). (B) Effect of Cdc42-1 siRNA on lumen formation. MDCK cells were transfected with Cdc42 siRNA (bottom panels) or siRNA control (top panels) and plated to form cysts for 48h. Cells were stained to detect actin (red), ezrin (blue), β-catenin (green, left panels) and nuclei (right panel merged with DIC). Single confocal sections through the middle of cysts are shown (C) Quantitation of cysts with normal lumens in cells transfected with control siRNA (black bars), specific siRNA Cdc42-1 (white bars) or Cdc42-2 (grey bars). Values shown are mean ±SD from 4 different experiments. *P<0.001. (D) Time course analysis of gp135 localization during lumen formation in control and Cdc42 depleted cells. Cells were transfected with Cdc42 siRNA-1 (bottom panels) or siRNA control (top panels) and plated to form cysts for 8, 16, 24 and 48h. Cells were stained to detect gp135 (red), β-catenin (green) and nuclei. Arrows indicate small intracellular vesicles at 16h and intercellular lumens at 48h. Arrowheads indicate intracellular lumens. (E) siRNA-mediated reduction of PTEN and Ax2 inhibits activation of Cdc42. Cells were transfected with siRNAs Cdc42-1, Ax2-2, or PTEN2 or with control siRNAs; or infected with adenovirus expressing myc-tagged N17Cdc42. Total cell lysates of cysts at 48h were immunoblotted for Cdc42, Ax2 and PTEN, (top-left panel); or for Cdc42 (bottom-left panel). Extracts from these cells were pulled down with PBD-PAK1-GST. Total and GTP–bound Cdc42 was detected by immunoblotting with specific antibodies, and the ratios of GTP-bound to total protein quantified. Values shown are mean ±SD from 3 different experiments. (F) Exogenous PtdIns(4,5)P2 delocalizes Cdc42-GFP from the AP PM. Cdc42-GFP cysts at 48h were incubated with PtdIns(4,5)P2-histone complexes for 30min (+PtdIns(4,5)p2) or histone alone (control), fixed and analyzed by confocal microscopy. Arrowheads indicate Cdc42-GFP enrichment at the basal PM.

Fig 7. Cdc42 targets aPKC to the AP plasma PM to form the central lumen

(A) aPKC and Par3 distribute differently in nature cysts. Cysts were stained for aPKCλ (top panels) or Par3 (bottom panels) (red, left panels), β-catenin or ZO-1 (green, middle panels), and for nuclei (merged with DIC, right panels). Arrowheads indicate aPKC at the AP PM, and colocalization of Par3 and ZO-1 at TJ. (B) Reduction of Cdc42 induces intracellular accumulation of aPKC and Ax2. Ax2-GFP cells were transfected with Cdc42 or control siRNAs and plated to form cysts for 48h. Cells were stained for aPKC (red) and merged with Ax2-GFP and nuclei (bottom-right panels); and actin (red, left-bottom panel merged with DIC). Arrowheads indicate aPKC at the AP PM in controls cells (left panels), or to the intracellular vesicles in Cdc42 depleted cells (right panels). (C) aPKC-PS disrupts lumen formation. MDCK cysts were treated with aPKC-PS (40μM) or not (control). Cells were stained to detect gp135 (red), β-catenin (green) and nuclei in left panels; or ZO-1 (red), β-catenin (green) and merged with DIC in right panels. (D) The aPKC inhibitor aPKC-PS disrupts lumen formation in a dose-dependent manner. MDCK cysts were treated with indicated concentrations of aPKC-PS for 48h. Cells were then fixed, stained and quantified for lumen formation. Values shown are mean ±SD from 3 different experiments. N=100/experiment. (E) aPKC-PS inhibits aPKC phosphorylation in a dose-dependent manner. (F) Model: PtdIns(3,4,5)p3 (red) and PtdIns(4,5)p2 (green) colocalize in unpolarized MDCK cells (yellow). AP recruitment of PTEN induces the accumulation of PtdIns(4,5)p2 at the AP domain. PtdIns(4,5)p2 recruits Ax2, Cdc42 and Par6/aPKC to form the AP PM and lumen.