Interaction Of Ezrin Research Articles

The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

BackgroundEzrin/radixin/moesin (ERM) proteins are members of the protein 4.1 superfamily and function as linkers that connect the actin cytoskeleton to the plasma membrane of cells. ERM also play critical role in the Lipopolysaccharide (LPS)-induced inflammatory response. However, the signaling mechanisms involved in this process remain unclear. In this study, we aimed to investigate the potential role of the rho-associated coiled-coil containing protein kinase (ROCK) pathway in LPS-induced ezrin phosphorylation and cytokine production in pulmonary alveolar epithelial cells.MethodsCultured A549 and HPAEpiC cells were treated with LPS. The expression and localization of ezrin in A549 and HPAEpiC cells were then analyzed by western blotting and immunoflurescence. Activation of RhoA/ROCK was assessed by western blotting and RhoA activity assays. The interaction of ezrin with Syk and myeloid differentiation factor 88 (MyD88)/IL-1R-associated kinase 1 (IRAK-1) was investigated by co-immunoprecipitation. The activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) was measured with electrophoretic mobility shift assays and by western blotting. ELISA and western blotting were performed to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and high mobility group box 1 protein (HMGB1) release into the culture supernatant, and cellular HMGB1 levels.ResultsLPS induced ezrin phosphorylation in a concentration- and time-dependent manner. The blockade of RhoA/ROCK inhibited LPS-induced ezrin phosphorylation and its translocation from the cytoplasm to the cell membrane. Co-immunoprecipitation assays further revealed that ezrin associated with Syk constitutively, but only associated with MyD88/IRAK-1 upon LPS challenge. Moreover, LPS-induced p38 and nuclear NF-κB activation was found to be ezrin dependent. The suppression of ezrin by siRNA or the blockade of ROCK activation with Y-27632 reduced the production of TNF-α, IL-1β, and HMGB1 in response to LPS.ConclusionsOur findings reveal a novel regulatory mechanism involving ezrin in the LPS-induced production of pro-inflammatory cytokines, and highlight the importance of the RhoA/ROCK-ezrin/Syk-MyD88/IRAK1 axis. Data presented in this manuscript provide novel insights into the signaling pathways activated in pulmonary alveolar epithelial cells by LPS.3MUhuZjAzSev1aZk4ypCqUVideo

Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren's Syndrome.

Sjögren’s syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients

Lipid Rafts Interaction of the ARID3A Transcription Factor with EZRIN and G-Actin Regulates B-Cell Receptor Signaling.

Several diseases originate via dysregulation of the actin cytoskeleton. The ARID3A/Bright transcription factor has also been implicated in malignancies, primarily those derived from hematopoietic lineages. Previously, we demonstrated that ARID3A shuttles between the nucleus and the plasma membrane, where it localizes within lipid rafts. There it interacts with components of the B-cell receptor (BCR) to reduce its ability to transmit downstream signaling. We demonstrate here that a direct component of ARID3A-regulated BCR signal strength is cortical actin. ARID3A interacts with actin exclusively within lipid rafts via the actin-binding protein EZRIN, which confines unstimulated BCRs within lipid rafts. BCR ligation discharges the ARID3A–EZRIN complex from lipid rafts, allowing the BCR to initiate downstream signaling events. The ARID3A–EZRIN interaction occurs almost exclusively within unpolymerized G-actin, where EZRIN interacts with the multifunctional ARID3A REKLES domain. These observations provide a mechanism by which a transcription factor directly regulates BCR signaling via linkage to the actin cytoskeleton with consequences for B-cell-related neoplasia.

Ezrin interacts with L-periaxin by the “head to head and tail to tail” mode and influences the location of L-periaxin in Schwann cell RSC96

In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.

Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression

Although Merlin's function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.

The SGK3-triggered ubiquitin\u2013proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex

Podocyte damage is commonly accompanied by destabilization of the podocalyxin (PC)/ezrin complex. Serum- and glucocorticoid-inducible kinase 3 (SGK3) plays a role in the maintenance of podocyte function, but the details of this role are poorly understood. Herein we demonstrated that SGK3 and its downstream target protein neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2) triggered PC and ezrin interaction. In adriamycin (ADR)-induced nephritic mice, and after puromycin aminonucleoside (PAN)-induced podocyte damage in vitro, PC and ezrin protein expression levels decreased significantly, while Nedd4-2 activity increased. Moreover, PAN treatment increased PC and ezrin ubiquitination and decreased PC/ezrin interaction in cultured mouse podocytes. The downregulation of SGK3 activity in mouse podocytes resulted in decreased PC and ezrin protein expression and increased the ubiquitin–proteasome degradation of PC and ezrin. Furthermore, upregulation of SGK3 activity mostly reversed the PAN-induced decrease in PC and ezrin protein expression. Overexpression of Nedd4-2 led to decreased ezrin protein expression via the upregulation of ezrin ubiquitination. In contrast, Nedd4-2 knockdown resulted in increased ezrin protein expression but decreased ezrin ubiquitination. In PC-transfected human embryonic kidney (HEK293T) cells, SGK3 activity downregulation and Nedd4-2 overexpression resulted in decreased PC/ezrin interaction. These results suggested that SGK3 triggers the ubiquitin–proteasome degradation of PC and ezrin, while the SGK3/Nedd4-2 signaling pathway regulates ezrin, but not PC, ubiquitination. Thus SGK3 helps to regulate podocyte function by maintaining the stability of the PC/ezrin complex.

SLC12A7 alters adrenocortical carcinoma cell adhesion properties to promote an aggressive invasive behavior

BackgroundAltered expression of Solute Carrier Family 12 Member 7 (SLC12A7) is implicated to promote malignant behavior in multiple cancer types through an incompletely understood mechanism. Recent studies have shown recurrent gene amplifications and overexpression of SLC12A7 in adrenocortical carcinoma (ACC). The potential mechanistic effect(s) of SLC12A7 amplifications in portending an aggressive behavior in ACC has not been previously studied and is investigated here using two established ACC cell lines, SW-13 and NCI-H295R.MethodsSW-13 cells, which express negligible amounts of SLC12A7, were enforced to express SLC12A7 constitutively, while RNAi gene silencing was performed in NCI-H295R cells, which have robust endogenous expression of SLC12A7. In vitro studies tested the outcomes of experimental alterations in SLC12A7 expression on malignant characteristics, including cell viability, growth, colony formation potential, motility, invasive capacity, adhesion and detachment kinetics, and cell membrane organization. Further, potential alterations in transcription regulation downstream to induced SLC12A7 overexpression was explored using targeted transcription factor expression arrays.ResultsEnforced SLC12A7 overexpression in SW-13 cells robustly promoted motility and invasive characteristics (p < 0.05) without significantly altering cell viability, growth, or colony formation potential. SLC12A7 overexpression also significantly increased rates of cellular attachment and detachment turnover (p < 0.05), potentially propelled by increased filopodia formation and/or Ezrin interaction. In contrast, RNAi gene silencing of SLC12A7 stymied cell attachment strength as well as migration and invasion capacity in NCI-H295R cells. Transcription factor expression analysis identified multiple signally pathways potentially affected by SLC12A7 overexpression, including osmotic stress, bone morphogenetic protein, and Hippo signaling pathways.ConclusionsAmplification of SLC12A7 observed in ACCs is shown here, in vitro, to exacerbate the malignant behavior of ACC cells by promoting invasive capacities, possibly mediated by alterations in multiple signaling pathways, including the osmotic stress pathway.

A decision tree–based combination of ezrin-interacting proteins to estimate the prognostic risk of patients with esophageal squamous cell carcinoma

Our previous studies have highlighted the importance of ezrin in esophageal squamous cell carcinoma (ESCC). Here our objective was to explore the clinical significance of ezrin-interacting proteins, which would provide a theoretical basis for understanding the function of ezrin and potential therapeutic targets for ESCC. We used affinity purification and mass spectrometry to identify PDIA3, CNPY2, and STMN1 as potential ezrin-interacting proteins. Confocal microscopy and coimmunoprecipitation analysis further confirmed the colocalization and interaction of ezrin with PDIA3, CNPY2, and STMN1. Tissue microarray data of ESCC samples (n=263) showed that the 5-year overall survival (OS) and disease-free survival (DFS) were significantly lower for the CNPY2 (OS, P=.003; DFS, P=.011) and STMN1 (OS, P=.010; DFS, P=.002) high-expression groups compared with the low-expression groups. By contrast, overexpression of PDIA3 was significantly correlated with favorable survival (OS, P<.001; DFS, P=.001). Cox regression demonstrated the prognostic value of PDIA3, CNPY2, and STMN1 in ESCC. Furthermore, decision tree analysis revealed that the resulting classifier of both ezrin and its interacting proteins could be used to better predict OS and DFS of patients with ESCC. In conclusion, a signature of ezrin-interacting proteins accurately predicts ESCC patient survival or tumor recurrence.

Ezrin directly interacts with AQP2 and promotes its endocytosis.

The water channel aquaporin-2 (AQP2) is a major regulator of water homeostasis in response to vasopressin (VP). Dynamic trafficking of AQP2 relies on its close interaction with trafficking machinery proteins and the actin cytoskeleton. Here, we report the identification of ezrin, an actin-binding protein from the ezrin/radixin/moesin (ERM) family as an AQP2-interacting protein. Ezrin was first detected in a co-immunoprecipitation (co-IP) complex using an anti-AQP2 antibody in a proteomic analysis. Immunofluorescence staining revealed the co-expression of ezrin and AQP2 in collecting duct principal cells, and VP treatment caused redistribution of both proteins to the apical membrane. The ezrin-AQP2 interaction was confirmed by co-IP experiments with an anti-ezrin antibody, and by pulldown assays using purified full-length and FERM domain-containing recombinant ezrin. By using purified recombinant proteins, we showed that ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain. Knocking down ezrin expression with shRNA resulted in increased membrane accumulation of AQP2 and reduced AQP2 endocytosis. Therefore, through direct interaction with AQP2, ezrin facilitates AQP2 endocytosis, thus linking the dynamic actin cytoskeleton network with AQP2 trafficking.

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss.

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na+/H+ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP₃) receptor-binding protein released with IP₃ (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2-dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.

Ezrin is a Potent Modulator of Alpha-Synuclein Oligomerization

Alpha-synuclein oligomers are associated with toxicity in Parkinson's disease. Different forms of alpha-synuclein (aS) have been described, some of which can destabilize lipid bilayers, and seed the formation of fibrillar assemblies. Therapeutic interventions such as molecular chaperones and small molecule inhibitors are sought to depopulate these transient intermediates and thus diminish their toxicity.We have identified ezrin, a microtubule-associated protein that belongs to the ezrin radixin moesin family, as a potent inhibitor of aS aggregation at sub-stoichiometric concentrations. Evidence showed that binding of ezrin to aS causes conformational rearrangements and sequesters the protein in amorphous aggregates thus preventing fibril formation and rescuing toxicity in primary neurons and yeast cells, thereby acting as a non-conventional chaperone.We have produced and characterized alternate aS oligomers by size exclusion chromatography, circular dichroism, TEM, dot blotting and ThT fluorescence. One group of oligomers is associated with a higher β-sheet content and can be internalized in human neuronal model cells. Following treatment with these oligomers prepared with incorporation of fluorescently labeled aS, we observed their uptake into human SH-EP cells. The other group of oligomers is A11 antibody reactive, mainly unstructured and result in significant cell death.We hypothesize that binding of ezrin may alter the biophysical, biological characteristics and aggregation properties of aS oligomers. Current efforts are concentrating on investigating the interaction of ezrin with these alternate oligomeric species.

Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients.

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.

Direct Interaction of 14-3-3ζ with Ezrin Promotes Cell Migration by Regulating the Formation of Membrane Ruffle

14-3-3 proteins have been shown to regulate the actin cytoskeleton remodeling, cell adhesion and migration. In this study, we identified ezrin, a cross-linker between plasma membrane and actin cytoskeleton, as a novel 14-3-3ζ interacting partner. The direct interaction between 14-3-3ζ and ezrin was validated in the cells and by in vitro assays. We showed that the 14-3-3ζ binding region in ezrin was located within the N-terminal and central α-helical domains and that the αG-to-αI helices of 14-3-3ζ are responsible for the binding to ezrin. Functional analyses revealed that the regulation of cell migration and membrane ruffling by 14-3-3ζ is ezrin dependent, for which the integrity of ezrin protein was required. Conversely, the knockdown of 14-3-3ζ abrogates also the stimulatory effect of ezrin on cell migration and membrane ruffling. Moreover, we found that the phosphorylation of Thr567 in ezrin facilitates the 14-3-3ζ–ezrin interaction and the formation of membrane ruffles. Taken together, these results suggest strongly that the functions of these two proteins in cell migration are linked and might be mediated by their direct physical interaction, which is important for the formation of membrane ruffles.

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: A comparative study of the affinity constants and conformational changes

The plasma membrane–cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP2-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP2-containing large unilamellar vesicles or PIP2-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP2-containing large unilamellar vesicles or PIP2-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP2-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.

PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.

Ezrin Ubiquitylation by the E3 Ubiquitin Ligase, WWP1, and Consequent Regulation of Hepatocyte Growth Factor Receptor Activity

The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY477 present in ezrin’s C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.

Generation and characterization of a novel, permanently active S100P mutant

S100 proteins function as Ca 2+ signal transducers by regulating cellular targets in their Ca 2+ bound conformation. S100P is a member of the S100 protein family that can activate the membrane and F-actin binding protein ezrin in a Ca 2+ dependent manner at least in vitro. Here we generated a novel tool to elucidate directly the S100P–ezrin interaction in vivo. This was achieved by constructing a S100P derivative that contained mutations in the two EF hand loops predicted to lock the protein in a permanently active state. The resulting S100P mutant, termed here S100P pa, could be purified as a soluble protein and showed biochemical properties displayed by wild-type S100P only in the presence of Ca 2+. Importantly, S100P pa bound to the N-terminal domain of ezrin in the absence of Ca 2+ showing an affinity only slightly reduced as compared to that of Ca 2+-bound WT S100P. In line with this permanent complex formation, S100P pa colocalized with ezrin to plasma membrane protrusions of epithelial cells even in the absence of intracellular Ca 2+ transients. Thus, S100P pa is a novel type of S100 protein mutant locked in a permanently active state that shows an unregulated complex formation with its cellular target ezrin.

Giant Unilamellar Vesicles Containing Phosphatidylinositol(4,5)bisphosphate: Characterization and Functionality

Biomimetic systems such as giant unilamellar vesicles (GUVs) are increasingly used for studying protein/lipid interactions due to their size (similar to that of cells) and to their ease of observation by light microscopy techniques. Biophysicists have begun to complexify GUVs to investigate lipid/protein interactions. In particular, composite GUVs have been designed that incorporate lipids that play important physiological roles in cellulo, such as phosphoinositides and among those the most abundant one, phosphatidylinositol(4,5)bisphosphate (PIP 2). Fluorescent lipids are often used as tracers to observe GUV membranes by microscopy but they can not bring quantitative information about the insertion of unlabeled lipids. In this study, we carried out ζ-potential measurements to prove the effective incorporation of PIP 2 as well as that of phosphatidylserine in the membrane of GUVs prepared by electroformation and to follow the stability of PIP 2-containing GUVs. Using confocal microscopy, we found that long-chain (C16) fluorescent PIP 2 analogs used as tracers (0.1% of total lipids) show a uniform distribution in the membrane whereas PIP 2 antibodies show PIP 2 clustering. However, the clustering effect, which is emphasized when tertiary antibodies are used in addition to secondary ones to enhance the size of the detection complex, is artifactual. We showed that divalent ions (Ca 2+ and Mg 2+) can induce aggregation of PIP 2 in the membrane depending on their concentration. Finally, the interaction of ezrin with PIP 2-containing GUVs was investigated. Using either labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs, we showed that clusters of PIP 2 and proteins are formed.

S1890 A Novel Interaction of Ezrin with Cortactin in Podosomal Rosette Assembly in Pancreatic Cancer Cells

S1890 A Novel Interaction of Ezrin with Cortactin in Podosomal Rosette Assembly in Pancreatic Cancer Cells

Quantitative Analysis of the Binding of Ezrin to Large Unilamellar Vesicles Containing Phosphatidylinositol 4,5 Bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP2) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP2. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5μM for PIP2-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP2-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30nm2) binding to a single PIP2 can block access to neighboring PIP2 molecules and thus contributes to lower the accessible PIP2 concentration. In addition, no evidence exists for a clustering of PIP2 induced by ezrin addition.