Structure of ERM (ezrin-radixin-moesin) proteins

The ERM (ezrin-radixin-moesin) protein family links actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks the these domains' binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin (Pearson, 2000).

Subcellular distribution of ERM proteins

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behavior of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction (Amieva, 1999).

The highly conserved ERM (ezrin-radixin-moesin) family of proteins function as molecular linkers between the actin cytoskeleton and transmembrane receptors. Unequivocal evidence is provided that full-length endogenous ezrin and moesin also localise to the nucleus in two independent mammalian cell lines. All three ERM family members can localise to the nucleus upon exogenous expression of their GFP-tagged counterparts, suggesting a common family trend. Furthermore, Drosophila Moesin, the Drosophila ERM homolog, is present in the nucleus of an insect cell line and can localise to the nucleus when exogenously expressed in MDCK cells. The nuclear localisation of endogenous ezrin and moesin is regulated by cell density and is resistant to detergent extraction, suggesting tight association with nuclear structures. Furthermore, phosphorylation in the actin-binding domain is not a prerequisite for nuclear localisation. A specific nuclear localisation sequence has been identified, that is conserved and functional in all ERM family members, implying specific regulated nuclear import. Although the precise nuclear function of the ERM proteins is unknown, these data provide further evidence that an increasing number of cytoskeletal components directly link the plasma membrane with nuclear events (Batchelor, 2004).

Protein interactions of ERM proteins

The ERM proteins are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) were produced by means of recombinant baculovirus infection, and an in vitro assay was constructed for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bind to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bind with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, the immunoprecipitated CD44/ERM complex from BHK cells was reexamined and it was found to contain Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. The involvement of Rho in the regulation of the CD44/ERM complex formation was evaluated. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism (Hirao, 1996).

The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system (Takahashi, 1997).

The plasma membrane consists of a lipid bilayer with integral membrane proteins stabilized by regulated linkages to the cortical actin cytoskeleton. The regulation is necessary for cells to change shape or migrate. The ERM proteins are believed to provide such links, with the N-terminal halves associating with integral membrane proteins, either directly or indirectly through adapter molecules like EBP50 (ERM binding phosphoprotein, 50 kDa), and their C-terminal halves associating with F-actin. However, isolated ERM proteins largely exist in a dormant state by virtue of an intramolecular interaction between amino- and carboxyl-terminal domains, thereby masking membrane and cytoskeletal association sites. C-terminal threonine phosphorylation of a fragment of radixin has been found to destroy its ability to bind the amino-terminal domain without affecting the C-terminal F-actin binding site. C-terminal phosphorylation of full-length, dormant ezrin and moesin by protein kinase C-theta simultaneously unmasks both the F-actin and EBP50 binding sites. Increased phosphorylation of moesin in cells correlated with increased association of moesin with the cortical actin cytoskeleton. These results show that activation of ERM proteins can be accomplished by phosphorylation of a single C-terminal threonine residue (Simons, 1998).

ERM proteins recognize the cytoplasmic domains of adhesion molecules in the formation of the membrane-associated cytoskeleton. The crystal structure of the radixin FERM (4.1 and ERM) domain complexed with the ICAM-2 cytoplasmic peptide is reported. The non-polar region of the ICAM-2 peptide contains the RxxTYxVxxA sequence motif to form a beta-strand followed by a short 3(10)-helix. It binds the groove of the phosphotyrosine-binding (PTB)-like subdomain C mediated by a beta-beta association and several side-chain interactions. The binding mode of the ICAM-2 peptide to the FERM domain is distinct from that of the NPxY motif-containing peptide binding to the canonical PTB domain. Mutation analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insights into the physical link between adhesion molecules and ERM proteins (Hamada, 2003).

Members of ERM protein family serve as regulated microfilament-membrane crosslinking proteins that, upon activation, bind the scaffolding protein ERM-phosphoprotein of 50 kDa (EBP50). A 3.5 Å resolution diffraction analysis of a complex between the active moesin N-terminal FERM domain and a 38 residue peptide from the C terminus of EBP50 is reported. This crystallographic result, combined with sequence and structural comparisons, suggests that the C-terminal 11 residues of EBP50 binds as an alpha-helix at the same site occupied in the dormant monomer by the last 11 residues of the inhibitory moesin C-terminal tail. Biochemical support for this interpretation derives from in vitro studies showing that appropriate mutations in both the EBP50 tail peptide and the FERM domain reduce binding, and that a peptide representing just the C-terminal 14 residues of EBP50 also binds to moesin. Combined with the recent identification of the I-CAM-2 binding site on the ERM FERM domain, this study reveals that the FERM domain contains two distinct binding sites for membrane-associated proteins. The contribution of each ligand to ERM function can now be dissected by making structure-based mutations that specifically affect the binding of each ligand (Finnerty, 2004).

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. Ezrin and moesin have been identified as binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Two basic amino acid residues within the L-selectin tail have been identified that are required for binding to ezrin-radixin-moesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding (Ivetic, 2004).

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. Two previously unrecognized actions of NIK are reported in this study: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology (Baumgartner, 2006; full text of article).

Because activation of ERM proteins promotes F-actin anchoring to the plasma membrane, their phosphorylation by NIK likely stabilizes extending lamellipodia. However, it is predicted that NIK also regulates membrane dynamics through mechanisms independent of ERM proteins. MTLn3 cells expressing NIK-D152N, but not ezrin T567A, had constitutive, albeit small, ruffles. Substrates, including NHE1, and possibly gelsolin or cofilin, which are phosphorylated by the closely related kinases TNIK and NRK, respectively, might contribute to NIK-dependent membrane protrusion. Additionally, NIK phosphorylation of ERM proteins or other substrates might act coordinately with Nck to promote or stabilize membrane protrusions. The finding that NIK activity is necessary to phosphorylate ERM proteins in response to EGF and PDGF, but not to thrombin, is consistent with NIK binding to the Src homology 3 domain of Nck, an adaptor protein associated with receptor tyrosine kinases, and with Msn binding to DOCK, the Drosophila ortholog of Nck. Nck also binds and activates the Wiskott-Aldrich syndrome protein WASP and the WASP family verprolin homologous protein WAVE, which promote actin assembly by the Arp2/3 complex and membrane protrusion. Although NIK may act coordinately with Nck to regulate membrane dynamics, its phosphorylation of ERM proteins can occur independently of Nck because truncated NIK 1-321 lacking the C-terminal Nck-binding domain was sufficient to increase phosphorylation of ERM proteins in quiescent cells. Additionally, kinase inactive NIK-D152N did not block activation of ERK1/ERK2 by PDGF, suggesting that NIK regulates ERM protein phosphorylation downstream or independently of an ERK-mediated pathway, the latter possibility being consistent with NIK acting independently of Nck (Baumgartner, 2006).

These findings indicate that activation of ERM proteins by NIK is a cellular mechanism to promote local alterations in cell morphology in response to growth factors. This mechanism is likely important in migrating cells because NIK activity is necessary for growth factor-induced phosphorylation of ERM proteins in lamellipodia. Because activation of NIK and ezrin is implicated in processes related to tumor cell dissemination with aberrant growth factor signaling, a functional interaction between NIK and ERM proteins might play a previously unrecognized role in tumor cell metastasis (Baumgartner, 2006).

MIG-15 and ERM-1 promote growth cone directional migration in parallel to UNC-116 and WVE-1

Neurons require precise targeting of their axons to form a connected network and a functional nervous system. Although many guidance receptors have been identified, much less is known about how these receptors signal to direct growth cone migration. This study used C. elegans motoneurons to study growth cone directional migration in response to a repellent UNC-6 (netrin homolog) guidance cue. The evolutionarily conserved kinase MIG-15 (NIK; Nck-interacting kinase - Drosophila homolog Misshapen) regulates motoneuron UNC-6-dependent repulsion through unknown mechanisms. Using genetics and live imaging techniques, it was shown that motoneuron commissural axon morphology defects in mig-15 mutants result from impaired growth cone motility and subsequent failure to migrate across longitudinal obstacles or retract extra processes. To identify new genes acting with mig-15, a screen was performed for genetic enhancers of the mig-15 commissural phenotype, and the ezrin/radixin/moesin ortholog ERM-1, the kinesin-1 motor UNC-116 and the actin regulator WVE-1 complex, were identified. Genetic analysis indicates that mig-15 and erm-1 act in the same genetic pathway to regulate growth cone migration and that this pathway functions in parallel to the UNC-116/WVE-1 pathway. Further, time-lapse imaging of growth cones in mutants suggests that UNC-116 might be required to stimulate protrusive activity at the leading edge, whereas MIG-15 and ERM-1 maintain low activity at the rear edge. Together, these results support a model in which the MIG-15 kinase and the UNC-116-WVE-1 complex act on opposite sides of the growth cone to promote robust directional migration (Teuliere, 2011).

Cell signaling upstream of ERM proteins

The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. A biological assay is described that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of GTPgammaS induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTPgammaS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. A 10,000-fold purification of the activity present in pig brain cytosol was achieved and protein sequence analysis showed it to contain moesin. Using recombinant proteins, moesin and its close relatives ezrin and radixin were shown to be able to reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac (Mackay, 1997).

The ERM proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. This study 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 approximately 30 and approximately 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, a mAb was raised 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 (Maeda, 1998).

RhoA is implicted in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. Lysophosphatidic acid stimulation of serum-starved NIH3T3 cells is shown to result in relocalization of radixin into apical membrane/actin protrusions; relocation is blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, is sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment leads 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 (Shaw, 1998).

The small G protein RhoA and its GDP/GTP exchange factors (GEFs) Net and Dbl can transform NIH 3T3 fibroblasts, dependent on the activity of the RhoA effector kinase ROCK. The role of the cytoskeletal linker protein ezrin in this process was investigated. RhoA effector loop mutants which can bind ROCK induce relocalization of ezrin to dorsal actin-containing cell surface protrusions, as do Net and Dbl. Both processes are inhibited by the ROCK inhibitor Y27632, which also inhibits association of ezrin with the cytoskeleton, and phosphorylation of T567, conserved between ezrin and its relatives radixin and moesin. ROCK can phosphorylate the ezrin C-terminus in vitro. The ezrin mutant T567A cannot be relocalized by activated RhoA, Net or Dbl or by ROCK itself, and also inhibits RhoA-mediated contractility and focal adhesion formation. Moreover, ezrin T567A, but not wild-type ezrin, restores contact inhibition to Net- and Dbl-transformed cells, and inhibits the activity of Net and Ras in focus formation assays. These results implicate ROCK-mediated ezrin C-terminal phosphorylation in transformation by RhoGEFs (Tran Quang, 2000).

Cell migration is a well-organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signalling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under the investigation. Crk associates with ERM family proteins including ezrin, radixin, and moesin, activates RhoA, and promotes cell motility towards hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs (pERMs) to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli is suppressed by inhibitors of Rho-associated kinase (ROCK), and the activity of RhoA is elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhances cell motility, while the sole expression of c-Crk-II or either of the ERMs decreases the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs (Tsuda, 2004).

Cell signaling downstream of ERM proteins

ERM proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. Ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1, has been studied. Cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, ezrin was found to interact with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway (Gautreau, 1999).

Mutation of ERM proteins

ERM proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. In contrast, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level (Doi, 1999).

ERM proteins and the anchoring of the cortical cytoskeleton to the cell membrane

ERM proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, a new fixation protocol was developed using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy show that TCA effectively inactivates soluble phosphatases, which maintains the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples has revealed that CPERMs are exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins are distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs have been shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo (Hayashi, 1999).

During activation, T cells associate with antigen-presenting cells, a dynamic process that involves the formation of a broad area of intimate membrane contact known as the immunological synapse. The molecular intermediates that link initial antigen recognition to the cytoskeletal changes involved in this phenomenon have not yet been defined. Ezrin-radixin-moesin proteins are rapidly inactivated after antigen recognition through a Vav1-Rac1 pathway. The resulting disanchoring of the cortical actin cytoskeleton from the plasma membrane decreases cellular rigidity, leading to more efficient T cell-antigen-presenting cell conjugate formation. These findings identify an antigen-dependent molecular pathway that favors immunological synapse formation and the subsequent development of an effective immune response (Faure, 2004).

ERM proteins and the formation of nodes of Ranvier

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using a myelinating explant culture system, it has been observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone-like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component-enriched "caps" and influences the efficiency of node formation (Gotto, 2003).

Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. A combined pharmacological-proteomic approach was undertaken to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). Phospholipid synthesis was perturbed by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulates the overall rate of membrane trafficking toward the ROS. Ppl stimulates budding of RTCs, but blocks membrane delivery to the ROS. Ppl causes accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induces redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalizes with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution is lost upon Ppl treatment. These data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle (Deretic, 2004).

ERM proteins and intestinal development

The C. elegans intestine is a simple and accessible model system to analyze the mechanism of junction assembly. In comparison to Drosophila and vertebrates, the C. elegans apical junction is remarkable because a single electron-dense structure is implicated in complex processes such as epithelial tightness, vectorial transport and cell adhesion. Evidence is presented in support of a heterogeneous molecular assembly of junctional proteins found in Drosophila and vertebrate epithelia associated with different junctions or regions of the plasma membrane. In addition, molecularly diverse complexes participate in different aspects of epithelial maturation in the C. elegans intestine. DLG-1 (Discs large) acts synergistically with the catenin-cadherin complex (HMP-1-HMP-2-HMR-1) and the Ezrin-Radixin-Moesin homolog (ERM-1) to ensure tissue integrity of the intestinal tube. The correct localization of DLG-1 itself depends on AJM-1, a coiled-coil protein. Double depletion of HMP-1 (alpha-catenin) and LET-413 (C. elegans homolog of Drosophila Scribble) suggests that the catenin-cadherin complex is epistatic to LET-413, while additional depletion of subapically expressed CRB-1 (Crumbs) emphasizes a role of CRB-1 concerning apical junction formation in the C. elegans intestine (Segbert, 2004).

The ERM proteins supply regulated linkage between membrane proteins and the actin cytoskeleton. The study of mammalian ERM proteins has been hampered by presumed functional overlap. Ezrin, the only ERM detected in epithelial cells of the developing intestine, provides an essential role in configuring the mouse intestinal epithelium. Surprisingly, Ezrin is not absolutely required for the formation of brush border microvilli or for the establishment or maintenance of epithelial polarity. Instead, Ezrin organizes the apical terminal web region, which is critical for the poorly understood process of de novo lumen formation and expansion during villus morphogenesis. These data also suggest that Ezrin controls the localization and/or function of certain apical membrane proteins that support normal intestinal function. These in vivo studies highlight the critical function of Ezrin in the formation of a multicellular epithelium rather than an individual epithelial cell (Saotome, 2004).

The RNA interference (RNAi) approach was used to assay ERM proteins of the C. elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. erm-1 was identified as the only ERM protein required for development; additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. The initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin-cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction (Van Furden, 2004).

ERM proteins and apoptosis

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses reveal that, at the early phase of Fas ligand (FasL)-induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins is suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins is blocked. The interleukin-1beta-converting enzyme (ICE) protease inhibitors suppress the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade is first activated, and then ERM proteins are dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, DiO-labeled single-layered plasma membranes were prepared with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments are densely detected, whereas those from FasL-treated cells are free from ERM proteins or actin filaments. It is thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes (Kondo, 1997).

CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Human T cells that are susceptible to CD95-mediated apoptosis exhibit a constitutive polarized morphology, and CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4(+) T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides specifically protects from the CD95-mediated apoptosis. Moreover, the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis (Parlato, 2000).

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. Moesin was not involved in the binding to CD95. This study further supports the specificity of the ezrin/CD95 binding, showing that radixin does not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 is located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60%-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induces a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin does not co-localize or co-immunoprecipitate with CD95. Further analysis shows that the mutated ezrin, while unable to bind CD95, is fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, these results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway (Lozupone, 2004).

Moesin : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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