Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.     

ERM proteins: from cellular architecture to cell signaling

ERM (ezrin/radixin/moesin) proteins, concentrated in actin rich cell-surface structures, cross-link actin filaments with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility and membrane trafficking. Recent analyses reveal that they are not only involved in cytoskeleton organization but also in signaling pathway. They play an important role in the activation of members of the Rho family by recruiting their regulators. The functions of ERM proteins are regulated by their conformational charges: the intramolecular interaction between the N- and C-terminal domains of ERM proteins charges masks several binding sites, leading to a dormant protein. Different activation signals regulate ERM proteins functions by modulating these intramolecular interactions. The involvement of ERM proteins in many signaling pathways has led to study their role during development of different species.     

Mucin MUC1 is seen in cell surface protrusions together with ezrin in immunoelectron tomography and is concentrated at tips of filopodial protrusions in MCF-7 breast carcinoma cells.

MUC1, a transmembrane member of the mucin family, is believed to have anti-adhesive properties because of its highly sialylated, extended, and rigid rod-like conformation. The ERM proteins (ezrin, radixin, and moesin) function as membrane-cytoskeletal linkers. MUC1 and ezrin are enriched in microvilli in MCF-7az breast carcinoma cells. Similar localization was also found in peripheral membrane areas and in filopodium-like protrusions. Whereas ezrin was consistently detected in the cell-cell contact region, MUC1 was less frequently found there. MUC1 was distinctly expressed in long filopodial protrusions and was highly concentrated at their tips, which also contained ezrin, whereas F-actin was found along the stalk. This localization of MUC1 suggests a role for MUC1 in transient cell structures of migrating cells and transient cell adhesion. No direct association has yet been found between MUC1 and ezrin. However, both MUC1 and ezrin had a similar overall distribution pattern in microvilli and filopodium-like protrusions in immunoelectron tomography. In addition, MUC1 and ezrin showed spatial association, because several 10-nm gold particles used to decorate ezrin were seen in the vicinity close to the clusters of 5-nm gold particles decorating MUC1. Therefore, MUC1 appears to be associated with ezrin, but the nature of this association requires further study.     

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.     

Regulation of cell shape and adhesion by CD34

We previously reported that expression of CD43/leukosialin induces cell rounding and microvillus formation via inhibition of cell adhesion. Here, we found that CD34, a cell surface sialomucin and marker for hematopoietic progenitor cells, also inhibited cell adhesion and induced cell rounding and microvillus formation. Forced expression of CD34-induced cell rounding, microvillus formation, and phosphorylation of ezrin/radixin/moesin (ERM) proteins in HEK293T cells, while inhibiting integrin-mediated cell re-attachment. Furthermore, CD34+ blood cells and KG-1 cells, which express endogenous CD34 on their surface, were spherical in shape, surrounded by microvilli, and non-adherent to substrata. In addition, cleavage of O-sialomucin augmented integrin-mediated cell adhesion of KG-1 cells. These results suggest the involvement of CD34 in the inhibition of integrin-mediated cell adhesion and formation of the cell surface structure. The inhibitory function of CD34 in cell adhesion may affect cell shape organization via phosphorylation of ERM proteins. Cellular structures such as the spherical shape and microvilli of CD34+ cells may also contribute to regulation of cell adhesion.     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.     

Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line

The apical microvilli are closely related with the development and the maintenance of cell polarization, and the length of microvilli varies in a regular way among cell types. Ezrin, a member of the ezrin/radixin/moesin (ERM) family, seems to be involved in the formation and stabilization of the apical microvilli. We found that phosphorylation of ezrin caused elongation of microvilli via a p38 MAP-kinase signaling pathway in an immortalized mouse hepatic cell line. When, in the oncogenic Raf-1-transfected mouse hepatic cell line, epithelial to mesenchymal transition (EMT) indicated as down-regulation of E-cadherin and up-regulation of Snail occurred, loss of microvilli and down-regulation of ezrin but not radixin and moesin were also observed. In the Raf-1 transfectants treated with the MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580, the numbers of microvilli and the expression of ezrin, E-cadherin and Snail were recovered. More interestingly, treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin (at Thr-567 and Tyr-353). Phosphorylated ezrin-positive dots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580. These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization.     

Manifold reduction of moesin in fetal Down syndrome brain

Moesin is a member of the ERM family and is involved in plasma membrane-actin cytoskeleton cross-linking, resulting cell adhesion, shape, and motility. Because moesin was shown to be highly expressed in growth cones and moesin/radixin suppression led to impaired structure and function of this key element in brain development, we tested the ERM family, ezrin, radixin, and moesin, in fetal Down syndrome (DS) cortex at the early second trimester. We applied two-dimensional gel electrophoresis with subsequent MALDI detection and identification of protein spots followed by quantification with specific software. Moesin was shown to be significantly and manifold reduced in fetal DS brain, whereas reduction of ezrin and radixin did not reach statistical significance. We therefore propose the involvement of moesin in developmental impairment of DS brain, including deteriorated arborisation, neuritic outgrowth, and neuronal migration. Furthermore, decreased moesin is the second F-actin bundling protein, besides drebrin, that is manifold reduced in fetal DS brain.     

Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ~30 and ~100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH₂-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.     

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca²⁺ signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.     

Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH(2)- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.     

Moesin‐dependent cytoskeleton remodelling is associated with an anaplastic phenotype of pancreatic cancer

Cell motility is controlled by the dynamic cytoskeleton and its related proteins, such as members of the ezrin/radixin/moesin (ERM) family, which act as signalling molecules inducing cytoskeleton remodelling. Although ERM proteins have been identified as important factors in various malignancies, functional redundancy between these proteins has hindered the dissection of their individual contribution. The aim of the present study was to analyse the functional role of moesin in pancreatic malignancies. Cancer cells of different malignant lesions of human and transgenic mice pancreata were evaluated by immunohistochemistry. For functional analysis, cell growth, adhesion and invasion assays were carried out after transient and stable knock‐down of moesin expression in pancreatic cancer cells. In vivo tumourigenicity was determined using orthotopic and metastatic mouse tumour models. We now show that moesin knock‐down increases migration, invasion and metastasis and influences extracellular matrix organization of pancreatic cancer. Moesin‐regulated migratory activities of pancreatic cancer cells were in part promoted through cellular translocation of β‐catenin, and re‐distribution and organization of the cytoskeleton. Analysis of human and different transgenic mouse pancreatic cancers demonstrated that moesin is a phenotypic marker for anaplastic carcinoma, suggesting that this ERM protein plays a specific role in pancreatic carcinogenesis.     

Cloning and expression profile of chicken radixin

Radixin is a member of the ERM (ezrin/radixin/moesin) family of cytoskeletal linkers. We have cloned chicken radixin as a 4.3 kb cDNA, which encodes an 80 kDa protein that is more than 98% identical to radixin from evolutionarily diverse species. High sequence homology (70-80%) also extends into the 3'-untranslated region (UTR) of the radixin gene. The 3'-UTR of moesin, but not ezrin, was also conserved, suggesting an essential, and possibly specific, regulatory function. A distinct pattern of radixin expression is seen in chicken tissues, suggesting a cell-type-specific function.     

Ezrin oligomers are the membrane-bound dormant form in gastric parietal cells

Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH₂- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH₂ terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH₂- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells. fluorescence resonance energy transfer; acid secretion; radixin; moesin; cytoskeleton; ERM family     

Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH₂-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC- inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 µM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin. ezrin/radixin/moesin; protein kinase C; Rho     

Conformational activation of radixin by G13 protein alpha subunit

G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13). Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.     

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.     

Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia

Ezrin/radixin/moesin (ERM) proteins cross-link actin filaments to plasma membranes to integrate the function of cortical layers, especially microvilli. We found that in cochlear and vestibular sensory hair cells of adult wild-type mice, radixin was specifically enriched in stereocilia, specially developed giant microvilli, and that radixin-deficient (Rdx(-)(/)(-)) adult mice exhibited deafness but no obvious vestibular dysfunction. Before the age of hearing onset ( approximately 2 wk), in the cochlea and vestibule of Rdx(-)(/)(-) mice, stereocilia developed normally in which ezrin was concentrated. As these Rdx(-)(/)(-) mice grew, ezrin-based cochlear stereocilia progressively degenerated, causing deafness, whereas ezrin-based vestibular stereocilia were maintained normally in adult Rdx(-)(/)(-) mice. Thus, we concluded that radixin is indispensable for the hearing ability in mice through the maintenance of cochlear stereocilia, once developed. In Rdx(-)(/)(-) mice, ezrin appeared to compensate for radixin deficiency in terms of the development of cochlear stereocilia and the development/maintenance of vestibular stereocilia. These findings indicated the existence of complicate functional redundancy in situ among ERM proteins.     

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.     

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.