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     

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.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Self-masking in an intact ERM-merlin protein: an active role for the central alpha-helical domain

Ezrin/radixin/moesin (ERM) family members provide a regulated link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and organization. Here, we report the crystal structure of intact insect moesin, revealing that its essential yet previously uncharacterized alpha-helical domain forms extensive interactions with conserved surfaces of the band four-point-one/ezrin/radixin/moesin (FERM) domain. These interdomain contacts provide a functional explanation for how PIP(2) binding and tyrosine phosphorylation of ezrin lead to activation, and provide an understanding of previously enigmatic loss-of-function missense mutations in the tumor suppressor merlin. Sequence conservation and biochemical results indicate that this structure represents a complete model for the closed state of all ERM-merlin proteins, wherein the central alpha-helical domain is an active participant in an extensive set of inhibitory interactions that can be unmasked, in a rheostat-like manner, by coincident regulatory factors that help determine cell polarity and membrane structure.     

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 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.     

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     

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.     

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.     

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.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.     

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.     

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.     

Ezrin Is Highly Expressed in Early Thymocytes,but Dispensable for T Cell Development in Mice

Background

Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.

Methodology/Principal Findings

We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin^−/− mice likely arise as a consequence of nutritional stress.

Conclusions/Significance

We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.     

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.     

Cellular distributions of the ERM proteins in MDCK epithelial cells: regulation by growth and cytoskeletal integrity

The highly homologous ERM (ezrin/radixin/moesin) proteins, molecular cross-linkers which connect the cell membrane with the underlying cytoskeleton, have molecular weights of 81, 80 and 78 kDa respectively. We present data which shows significant variation in the molecular weight and presence of multiple forms of ERM proteins in different cell lines, such that specific antibodies to each protein are essential for unambiguous detection. Biochemical fractionation of MDCK cells demonstrates that although the individual ERM fractionation patterns are unaltered by cell density, the multiple forms of moesin each associate with different subcellular fractions. Since ERM proteins can exist in dormant or active conformations corresponding to their phosphorylation state, we propose that the partitioning of ERM proteins between subcellular compartments may depend on their activation status. In addition, we show that when the co-localization between ezrin and F-actin is disrupted by cytochalasin D, MDCK cells undergo a dramatic morphology change during which long, branching, ezrin-rich protrusions are formed. Consistent with other workers, our data suggest that maintenance of ezrin:F-actin interactions are required for the maintenance of normal cellular morphology.     

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.     

ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling.

The ERM (ezrin, radixin and moesin) family of proteins are linkers that tether actin microfilaments to the plasma membrane. Merlin, the NF2 tumor suppressor gene product, is highly homologous to ERM proteins. In ERM proteins and merlin, interdomain binding promotes auto-inhibition and homo-oligomerization or hetero-oligomerization. Recent studies have revealed that ERM proteins transduce growth signals, and have shed new light on how merlin links cell growth to the cytoskeleton.     

The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation.