A molecular switch for the orientation of epithelial cell polarization

Abstract

The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.

Figure 1. β1-Integrins Promote Luminal Polarity and Constrain Long-Range Motility in Cysts

(A) GFP-Podxl, active β1-integrin and nuclei localization during progression from initially inverted (12 hr) to open lumen cysts (48 hr). Arrowheads, basal. In all instances, bottom panels are higher magnification of split color images from boxed regions. Scale bars represent 20 µm (unless otherwise indicated). (B) Parental or β1-integrin-GFP-expressing cysts with control or β1-integrin knockdown (KD) stained for Podxl and β-catenin. Arrows, AP; yellow arrowheads, peripheral. All cysts, 48 hr after plating, unless otherwise indicated. (C and D) Quantitation SLF (C) or peripheral Podxl (D) (see Experimental Procedures) upon β1-integrin manipulation in parental (MDCK) or β1-integrin-GFP cells (mean ± SD, n≥100 cysts/condition/experiment, three independent experiments). Black bars, scramble shRNA; white bars β1-integrin KD. In all instances, the red dotted line is the threshold considered for a positive hit (SLF, <75% of control; peripheral Podxl,≥4-fold change over control). *p < 0.05, **p < 0.001, ***p > 0.0001. (E) Still frames of phase-contrast imaging of control or β1-integrin-inhibited (+AIIB2) cysts (time indicated from time-lapse (every 15 min from 3–169 hr after plating)). Cyst outlines were traced from every 12 hr from 3–135 hr, indicated by alternating color. Arrows, collective front. (F) Active FAK (pY397), total FAK, or GAPDH levels from total cell lysates of cysts without (control) or with (AIIB2) β1-integrin inhibition. (G) Control or FAK-depleted cysts stained for Podxl, β-catenin and nuclei. Arrows, peripheral. (H and I) Quantitation of SLF (H) or peripheral Podxl (I) upon control or FAK depletion, with or without Y-27632 (10 µM) inhibition (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). Black bars, scramble shRNA; white bars, β1-integrin KD. See also Figure S1 and Movie S1.

Figure 2. A RhoA-Ezrin Pathway Is Necessary and Sufficient for Front-Rear, but Not Luminal AP-BL, Polarity

(A and B) Quantitation of cyst SLF (A) or peripheral Podxl (B), upon control or β1-integrin inhibition (+AIIB2), with scramble (black bars) or RhoA (white bars) KD (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). Hash symbol, value approaching zero. (C) Cysts with scramble or RhoA KD stained for Podxl, β-catenin, and nuclei. (D) Control or β1-integrin-inhibited (+AIIB2) cysts coexpressing GFP-RhoA and Cherry-tagged-RBD stained for Podxl. White arrowheads, AP; yellow arrowheads, peripheral; arrows, lateral; asterisks, nuclei. Right panels: higher magnification of split-color images from boxed regions. Yellow arrows, line-scan region for (E) and (F). (E and F) Quantitation of pixel intensity from line-scan regions in control (E) or AIIB2-treated (F) cysts. (G) Cysts with repressed (+Dox) or induced (−Dox) RhoA G14V expression stained for Podxl, pERM, and Scribble. Arrowheads, AP; arrows, BL. (H) RhoA (upper band, myc-tagged RhoA G14V), pERM, total Ezrin, and GAPDH levels from total cell lysates of cysts without (+DOX) or with (−Dox) myc-RhoA G14V expression. (I and J) Quantitation of cysts with SLF (I) or peripheral Podxl (J) upon RhoA G14V repression (+Dox) or expression (−Dox) (mean ± SD, n≥100 cysts/condition/ experiment, three independent experiments). (K) β1-integrin, pERM, total Ezrin, and GAPDH expression total cell lysates of cysts expressing scramble or β1-integrin KD. (L) Cysts without (control) or with (+AIIB2) β1-integrin inhibition stained for Ezrin, pERM and nuclei. Yellow arrows, luminal; arrows, peripheral. (M) Cysts coexpressing Ezrin shRNA and RNAi-resistant GFP-Ezrin T567D stained for Podxl and nuclei. Right panels: higher magnification of split red and green images from numbered, boxed regions. (N and O) Quantitation of SLF (N) or peripheral Podxl (O) upon control (scramble shRNA) or Ezrin shRNA/RNAi-resistant GFP-Ezrin T567D coexpression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). See also Figure S2.

Figure 3. Podxl-NHERF-ERM Tail Interactions Are Required for AP-BL Polarization

(A) Podxl, NHERF1, and nuclei localization during lumen formation (12–48 hr). White arrowheads, peripheral; arrows, vesicular; yellow arrowheads, luminal. AMIS, AP membrane initiation site; PAP, pre-AP patch. (B) Control (scramble KD) or Podxl KD cysts stained for nuclei and either (top) NHERF1 and Scribble, or (bottom) Ezrin and pERM. Arrows, luminal; arrowheads, lateral. (C) Cartoon of GFP-Podxl domains and motifs. WT, wild-type; FBM*, FERM-binding motif mutant; PBM, PDZ-binding motif mutant. (D) Quantitation of SLF in control (white bars) or Podxl KD (black bars) cysts without or with indicated RNAi-resistant GFP-Podxl domain mutant expression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). (E) Cysts coexpressing Podxl KD and GFP-Podxl (green) wild-type (WT) or domain mutants stained for either (left two panels) F-actin and nuclei, or (right two panels) Ezrin and β-catenin. Arrows, luminal; yellow arrowheads, vesicular; white arrowheads, lateral.

Figure 4. Formation of a Cortical Podxl-NHERF1-Ezrin Complex Regulates AP Domain Formation

(A) Cysts expressing either scramble (control), or single or dual NHERF1/2 shRNAs were stained for Podxl and β-catenin. Arrowheads, intracellular Podxl. (B) Quantitation of cysts with SLF upon expression of scramble, NHERF1 or NHERF2 shRNAs, alone or in combination (mean ± SD, n ≥ 100 cysts/condition/ experiment, three independent experiments). (C) Cysts coexpressing GFP-NHERF1 or GFP-NHERF2 (both green) and either Scramble, NHERF1, or NHERF2 shRNA stained for Podxl and β-catenin. White arrowheads, luminal; yellow arrowheads, vesicular. (D) Lysates of control or GFP-NHERF1 wild-type and domain mutants immunoprecipitated with anti-GFP antibodies, were western blotted for total Podxl and GFP (input), and immunoprecipitated Podxl and GFP (IP: GFP). Band intensities are a ratio of immunoprecipitated Podxl/NHERF1, normalized to WT NHERF1. Values are mean ± SD from three experiments. (E) Cartoon of GFP-NHERF1 domains and motifs. WT, wild-type; PDZ1*, PDZ domain 1 mutant; PDZ2*, PDZ domain 2 mutant; PDZ1/2*, PDZ domains 1 + 2 mutant; F355R, FERM-binding region mutant. (F) Quantitation of SLF in control (black bars) or NHERF1 KD cysts (white bars), without or with indicated RNAi-resistant GFP-NHERF1 mutant expression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). (G) Cysts coexpressing NHERF1 KD and RNAi-resistant GFP-NHERF1 wild-type (WT) or domain mutants (all green) stained for Podxl and β-catenin. White arrowheads, luminal; yellow arrowheads, vesicular. (H) Cysts expressing either scramble or Ezrin KD and either RNAi-resistant GFP-Ezrin WT or a phosphoinositide binding-deficit (PIP*) mutant stained for Podxl and either (left two panels) F-actin and nuclei, or (right two panels) β-catenin. Arrows, luminal; arrowheads, vesicular. (I) Quantitation of cysts with SLF upon control (black bars) or Ezrin (white bars), KD without or with RNAi-resistant GFP-Ezrin WT or PIP* expression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). See also Figure S3.

Figure 5. PKCβII Regulates Polarity Orientation

(A) Control or classical PKC-inhibited cysts (+Gö-6976, 0.5 µM) stained for Podxl and active β1-integrin. Arrowheads, Podxl; arrows, integrin. (B) Time-lapse dual-color confocal imaging of cysts expressing RFP-H2B (nuclei) and GFP-Podxl without (control, top) or with PKC inhibition (+Gö-6976, 0.5 µM, bottom). Images were taken every 30 min from 16–76.5 hr after plating. Stills are presented from ~24–60 hr. Cyst outlines were traced from still frames every 2 hr (using alternating color), from 24–52 hr. Yellow arrowheads, luminal; White arrowheads, peripheral. (C and D) Quantitation of SLF (C) or peripheral Podxl (D) in cysts upon control (scramble), or single or dual PKCα/β KD (mean ± SD, n ≥ 100 cysts/condition/ experiment, three independent experiments). N.S., not significant. (E) Cysts expressing either scramble or PKCβ KD without (top panels) or with (bottom panels) coexpression of RNAi-resistant GFP-PKCβII (green) stained for Podxl (red) and β-catenin (blue). Insets show Podxl alone (left), GFP-PKCβII alone (middle), and merge (right). Arrows, BL; white arrowheads, vesicular; yellow arrows, AP. (F and G) Quantitation of SLF (F) or peripheral Podxl (G) in cysts upon control (black bars) or PKCβ KD (white bars) without (MDCK) or with GFP-PKCβII coexpression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). Hash symbol, abolished front-rear polarity. (H) Cysts expressing GFP-PKCβII without (top, control) or with (bottom, +AIIB2) β1-integrin inhibition stained for pS660-PKCβII and Podxl. Yellow arrowheads, luminal; arrows, BL, white arrowheads, vesicular. Right panels: higher magnification of split color images from boxed regions. (I and J) β1-integrin, PKCβII (pS660, pT641, total [anti-GFP]) and GAPDH from total cell lysates of parental (MDCK) or GFP-PKCβII-expressing cysts and either (I) scramble or β1-integrin shRNA, or (J) co-overexpressing β1A-integrin (WT, V737N). See also Figures S4 and S5 and Movies S2 and S3.

Figure 6. Podxl Complex Phosphorylation Regulates the Switch between Front-Rear and AP-BL AP Polarization

(A) Cysts expressing either control or RhoA KD grown in the presence of classical PKC inhibitor (+Gö-6976, 0.5 µM) stained for Podxl, β-catenin, and nuclei. (B and C) Quantitation of SLF (B) or peripheral Podxl (C) in control cysts (scramble, black bars) or upon classical PKC inhibition (+Gö-6976, 0.5 µM), and with or without RhoA KD (white bars) (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). (D and E) Cartoons of GFP-Podxl (D) and GFP-NHERF1 (E) phosphorylation mutants used in subsequent experiments. 3×SA/SD, triple Ser-to-Ala/Asp mutants. (F) Podxl, ppS347/348-NHERF1, total NHERF1, PKCα, and GAPDH expression from total cell lysates of cells expressing scramble, PKCα or PKCβ shRNAs that were serum-starved overnight, and treated with either DMSO or PMA (100 nM) for 30 min. (G) Cysts coexpressing Podxl shRNAs and RNAi-resistant GFP-Podxl FBM phosphorylation site mutants (S481A/D) stained for Ezrin and β-catenin. Arrows, AP; arrowheads, vesicular. Smaller panels are higher magnification of split color images from boxed regions. (H and I) Quantitation of SLF (H) or peripheral Podxl (I) in cysts upon control (scramble, black bars) or Podxl (white bars) KD without or with RNAi-resistant GFP-Podxl (WT or PKC phosphorylation mutant) coexpression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). (J) Cysts coexpressing NHERF1 KD and RNAi-resistant GFP-NHERF1 PKC phosphorylation-site mutants stained for Podxl and β-catenin. Arrows, AP; arrowheads, vesicular. (K and L) Quantitation of SLF (K) or peripheral Podxl (L) in cysts upon control (scramble, black bars) or NHERF1 (white bars) KD without or with RNAi-resistant GFP-NHERF1 (WT or PKC phosphorylation mutant) coexpression (mean ± SD, n ≥ 100 cysts/condition/experiment, three independent experiments). See also Figure S6.

Figure 7. PP2A Controls AP-BL Polarization and Model of Polarity Orientation

(A) HA and GAPDH expression from total cell lysates of parental (MDCK) or 3×HA-tagged PP2A-Bα-expressing MDCK. (B) HA, Podxl, and Par3 expression in 3×HA-PP2A-Bα MDCK cells during lumen formation (12–48 hr). White arrowheads, vesicular; arrows, AP; yellow arrowheads, Par3 at AMIS and tight junctions. (C) PP2A-Bα and vinculin expression from total cell lysates of upon scramble or two different PP2A-Bα shRNAs. (D) Cysts expressing PP2A-Bα KD stained for Podxl, β-catenin and nuclei. Arrowheads, vesicular. (E and F) Quantitation of SLF (E) or peripheral Podxl (F) in cysts upon control (scramble) or PP2A-Bα KD (mean ± SD, n≥100 cysts/condition/experiment, three independent experiments). (G) Model of interactions that control AP-BL versus front-rear polarization: (1) the Podxl-NHERF1-pEzrin complex is stabilized at the cell periphery via a feedback loop between RhoA-ROCKI-Ezrin, (2) an α2/α3/β1-integrin/FAK/p190ARhoGAP module breaks this feedback loop by downregulating RhoA-GTP at the periphery, (3) PKCβII phosphorylates and dissociates the Podxl-NHERF1-Ezrin complex, triggering peripheral Podxl endocytosis, (4) Podxl and NHERF2 transcytose to the AMIS, and (5) AP polarity is reestablished at the AMIS, dependent on PP2A and the reassociation of the Podxl-NHERF1-Ezrin complex.