Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique/Institut Curie, 75248 Paris, France.
Molecular biology of the cell (Impact Factor: 4.47). 01/2012; 23(6):1080-94. DOI: 10.1091/mbc.E11-07-0588
Source: PubMed


The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.

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    • "Biologically, Eps8 regulates cell protrusions (Welsch et al., 2007), migration of dendritic cells (Frittoli et al., 2011), and morphogenesis of intestinal cells and microvilli (Croce et al., 2004; Zwaenepoel et al., 2012). In addition, Eps8 regulates stereocilia function and length, and Eps8 2/2 mice are deaf (Manor et al., 2011; Olt et al., 2014; Zampini et al., 2011). "
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    ABSTRACT: Eps8 is an actin regulatory scaffold protein increased in Squamous Cell Carcinoma (SCC) cells. It forms a complex with both Focal Adhesion Kinase (FAK) and c-Src in SCC cells derived from the DMBA/TPA model of skin carcinogenesis. Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active c-Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with c-Src and drives c-Src's trafficking to autophagic structures that SCC cells use to cope with high levels of active c-Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, so tethering active c-Src at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a critical mediator of Src/FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine c-Src's spatial localization and Src/FAK functions in invasive migration.
    Journal of Cell Science 10/2014; DOI:10.1242/jcs.157560 · 5.43 Impact Factor
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    • "Actin polymerization and cell surface remodeling in CLIC5A- transfected COS-7 cells It is well established that ezrin phosphorylation can induce the formation of actin-rich apical protrusions (Yonemura et al., 1999; Zwaenepoel et al., 2012). We therefore determined whether ectopic CLIC5A expression in COS-7 cells altered actin polymerization and cell surface architecture. "
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    ABSTRACT: CLIC5A is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction, enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the PI(4,5)P2 reporter RFP-PH-PLC to translocate from cytosol to discrete plasma membrane clusters at the cell surface, where it co-localized with CLIC5A. Transiently expressed HA-PIP5Kα co-localized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Kα abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane failed to co-localize with PIP5Kα, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5 deficient mice glomerular ezrin phosphorylation was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2 generating kinases, in turn facilitating ezrin activation, and actin-dependent cell surface remodeling.
    Journal of Cell Science 10/2014; 127(24). DOI:10.1242/jcs.147744 · 5.43 Impact Factor
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    • "Proteins of the ERM (ezrin–radixin– moesin) family (Bretscher et al., 2002) and scaffold proteins, such as EBP50 and PDKZ1 (Garbett et al., 2010; LaLonde et al., 2010), have been shown to play a crucial role in MV assembly and morphogenesis. Although there has been progress in elucidating the molecular composition of individual MV (Garbett et al., 2010; Garbett and Bretscher, 2012; Zwaenepoel et al., 2012), the basis for the morphological diversity of MV point (Fig. 2 C, blue). Hence, this difference in MV morphology correlated with cell position, domelike shape, and absence of neighbors. "
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    ABSTRACT: Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.
    The Journal of Cell Biology 10/2014; 207(1):107-21. DOI:10.1083/jcb.201402037 · 9.83 Impact Factor
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