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A possible mechanism for ezrin to establish epithelial cell polarity.

ABSTRACT: Ezrin is an important membrane/actin cytoskeleton linker protein, especially in epithelia. Ezrin has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin only after a conformational activation by phosphorylation at Thr567 of ezrin. The present experiments were undertaken to investigate the detailed cellular changes in the time course of expression of ezrin-T567 mutants (nonphosphorylatable T567A and permanent phospho-mimic T567D) in parietal cells and to assess ezrin distribution and its influence on the elaborate membrane recruitment processes of these cells. T567A mutant and wild-type (WT) ezrin were consistently localized to the apical plasma membrane, even with overexpression. On the other hand, T567D went first to apical membrane at early times and low expression levels, then accumulated mainly at the basal surface after 24 h. Overexpression of WT or T567A led to incorporation of internal membranes to apical vacuoles, while overexpression of T567D led to large incorporation of apical and intracellular membranes (including H-K-ATPase) to the basal surface. Differences in polar distribution of ezrin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections, forming the basis for a novel theory for ezrin as an organizer and regulator of membrane recruitment. A model simulating the cellular distribution of ezrin and its associated membrane- and F-actin-binding forms is given to predict redistributions observed with phosphorylation and mutant overexpression, and it can easily be modified as more specific information regarding binding constants and specific sites becomes available.

SUBMITTER: Zhu L

PROVIDER: S-EPMC2928635 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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A possible mechanism for ezrin to establish epithelial cell polarity.

Zhu Lixin L Crothers James J Zhou Rihong R Forte John G JG

American journal of physiology. Cell physiology 20100526 2

Ezrin is an important membrane/actin cytoskeleton linker protein, especially in epithelia. Ezrin has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin only after a conformational activation by phosphorylation at Thr567 of ezrin. The present experiments were undertaken to investigate the detailed cellular changes in the time course of expression of ezrin-T567 mutants (nonphosphorylatable T567A and permanent phosp ...[more]

PMID: 20505040

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Project description:Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Project description:Background: Interleukin-22 (IL-22) impacts the integrity of intestinal epithelia and has been associated with the development of colitis-associated cancer and inflammatory bowel diseases (IBD). Previous data suggest that IL-22 protects the mucosal barrier and promotes wound healing and barrier defect. We hypothesized, that IL-22 modulates cell polarity of intestinal epithelial cells (IECs) acting on tight junction assembly. The aim of the study was to investigate IL-22-dependent mechanisms in the reprogramming of intestinal epithelia. Methods: IECs were exposed to IL-22 at various concentrations. IECs in Matrigel® were grown to 3-dimensional cysts in the presence or absence of IL-22 and morphology and expression of polarity proteins were analyzed by confocal microscopy. Epithelial cell barrier (TER and sandwich assay) and TJ assembly analysis (calcium-switch assay) were performed. TJ and cell polarity protein expression were assessed by western blotting and confocal microscopy. Cell migration and invasion assays were performed. Induction of epithelial-mesenchymal transition (EMT) was assessed by RT-qPCR analysis and western blotting. Signaling pathway analyses were performed by phosphoblotting and functional assays after blocking STAT3 and ERK signaling pathways. Using the toxoplasma-model of terminal ileitis, IL-22-knock-out mice were compared to wild-type littermates, analyzed for barrier function using one-path-impedance-analysis and macromolecular flux (H3-mannitol, Ussing-chambers). Results: IECs exhibited a barrier defect after IL-22 exposure. TJ protein distribution and expression were severely impaired. Delayed recovery in the calcium-switch assay was observed suggesting a defect in TJ assembly. Analyzing the 3D-cyst model, IL-22 induced multi-lumen and aberrant cysts, and altered the localization of cell polarity proteins. Cell migration and invasion was caused by IL-22 as well as induction of EMT. Interestingly, only inhibition of the MAPK pathway, rescued the TJal barrier defect, while blocking STAT3 was relevant for cell survival. In addition, ileal mucosa of IL-22 deficient mice was protected from the barrier defect seen in Toxoplasma gondii-induced ileitis in wild type mice shown by significantly higher Re values and correspondingly lower macromolecule fluxes. Conclusion: IL-22 impairs intestinal epithelial cell barrier by inducing EMT, causing defects in epithelial cell polarity and increasing cell motility and cell invasion. IL-22 modulates TJ protein expression and mediates tight junctional (TJal) barrier defects via ERK pathway.

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A possible mechanism for ezrin to establish epithelial cell polarity.

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A possible mechanism for ezrin to establish epithelial cell polarity.

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