Membrane-cytoskeleton Linker Protein Research Articles

O-GlcNAcylation regulates phagocytosis by promoting Ezrin localization at the cell cortex

O-GlcNAcylation is a post-translational modification that serves as a cellular nutrient sensor and participates in multiple physiological and pathological processes. However, it remains uncertain whether O-GlcNAcylation is involved in the regulation of phagocytosis. Here, we demonstrate a rapid increase in protein O-GlcNAcylation in response to phagocytotic stimuli. Knockout of the O-GlcNAc transferase or pharmacological inhibition of O-GlcNAcylation dramatically blocks phagocytosis, resulting in the disruption of retinal structure and function. Mechanistic studies reveal that the O-GlcNAc transferase interacts with Ezrin, a membrane-cytoskeleton linker protein, to catalyze its O-GlcNAcylation. Our data further show that Ezrin O-GlcNAcylation promotes its localization to the cell cortex, thereby stimulating the membrane-cytoskeleton interaction needed for efficient phagocytosis. These findings identify a previously unrecognized role for protein O-GlcNAcylation in phagocytosis with important implications in both health and diseases.

Effects of black cumin seed oil on oxidative stress and expression of membrane-cytoskeleton linker proteins, radixin, and moesin in streptozotocin-induced diabetic rat liver.

Background and Aim:This study examined the effects of black cumin seed oil treatment on oxidative stress and the expression of radixin and moesin in the liver of experimental diabetic rats.Materials and Methods:Eighteen rats were divided into 3 equal groups (control, diabetes, treatment). The control group was not exposed to any experimental treatment. Streptozotocin was administered to the rats in the diabetes and treatment groups. A 2.5 mL/kg dose of black cumin seed oil was administered daily for 56 days to the treatment group. At the conclusion of the experiment, the blood level of malondialdehyde (MDA) and glutathione (GSH) was measured. The expression level and the cellular distribution of radixin and moesin in the liver were analyzed.Results:The plasma MDA (3.05±0.45 nmol/mL) and GSH (78.49±20.45 μmol/L) levels in the diabetes group were significantly different (p<0.01) from the levels observed in the control group (MDA: 1.09±0.31 nmol/mL, GSH: 277.29±17.02 μmol/L) and the treatment group (MDA: 1.40±0.53 nmol/mL, GSH: 132.22±11.81 μmol/L). Immunohistochemistry and western blotting analyses indicated that while the level of radixin was not significantly between the groups (p>0.05) and moesin expression was significantly downregulated (p<0.05) in the experimental group, the treatment was ineffective.Conclusion:The administered dose was sufficient to prevent oxidative stress, but was not sufficient to alleviate the effects of diabetes on moesin expression in hepatic sinusoidal cells.

Ezrin Mediates Invasion and Metastasis in Tumorigenesis: A Review.

Ezrin, as encoded by the EZR gene, is a member of the Ezrin/Radixin/Moesin (ERM) family. The ERM family includes three highly related actin filament binding proteins, Ezrin, Radixin, and Moesin. These three members share similar structural properties containing an N-terminal domain named FERM, a central helical linker region, and a C-terminal domain that mediates the interaction with F-actin. Ezrin protein is highly regulated through the conformational change between a closed, inactivate form and an open, active form. As a membrane-cytoskeleton linker protein, Ezrin facilitates numerous signal transductions in tumorigenesis and mediates diverse essential functions through interactions with a variety of growth factor receptors and adhesion molecules. Emerging evidence has demonstrated that Ezrin is an oncogene protein, as high levels of Ezrin are associated with metastatic behavior in various types of cancer. The diverse functions attributed to Ezrin and the understanding of how Ezrin drives the deadly process of metastasis are complex and often controversial. Here by reviewing recent findings across a wide spectrum of cancer types we will highlight the structures, protein interactions and oncogenic roles of Ezrin as well as the emerging therapeutic agents targeting Ezrin. This review provides a comprehensive framework to guide future studies of Ezrin and other ERM proteins in basic and clinical studies.

Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

Acetylation of ezrin regulates membrane-cytoskeleton interaction underlying CCL18-elicited cell migration.

Ezrin, a membrane–cytoskeleton linker protein, plays an essential role in cell polarity establishment, cell migration, and division. Recent studies show that ezrin phosphorylation regulates breast cancer metastasis by promoting cancer cell survivor and promotes intrahepatic metastasis via cell migration. However, it was less characterized whether there are additional post-translational modifications and/or post-translational crosstalks on ezrin underlying context-dependent breast cancer cell migration and invasion. Here we show that ezrin is acetylated by p300/CBP-associated factor (PCAF) in breast cancer cells in response to CCL18 stimulation. Ezrin physically interacts with PCAF and is a cognate substrate of PCAF. The acetylation site of ezrin was mapped by mass spectrometric analyses, and dynamic acetylation of ezrin is essential for CCL18-induced breast cancer cell migration and invasion. Mechanistically, the acetylation reduced the lipid-binding activity of ezrin to ensure a robust and dynamic cycling between the plasma membrane and cytosol in response to CCL18 stimulation. Biochemical analyses show that ezrin acetylation prevents the phosphorylation of Thr567. Using atomic force microscopic measurements, our study revealed that acetylation of ezrin induced its unfolding into a dominant structure, which prevents ezrin phosphorylation at Thr567. Thus, these results present a previously undefined mechanism by which CCL18-elicited crosstalks between the acetylation and phosphorylation on ezrin control breast cancer cell migration and invasion. This suggests that targeting PCAF signaling could be a potential therapeutic strategy for combating hyperactive ezrin-driven cancer progression.

Cholesterol-Dependent Modulation of Stem Cell Biomechanics: Application to Adipogenesis.

Cell mechanics has been shown to regulate stem cell differentiation. We have previously reported that altered cell stiffness of mesenchymal stem cells can delay or facilitate biochemically directed differentiation. One of the factors that can affect the cell stiffness is cholesterol. However, the effect of cholesterol on differentiation of human mesenchymal stem cells remains elusive. In this paper, we demonstrate that cholesterol is involved in the modulation of the cell stiffness and subsequent adipogenic differentiation. Rapid cytoskeletal actin reorganization was evident and correlated with the cell's Young's modulus measured using atomic force microscopy. In addition, the level of membrane-bound cholesterol was found to increase during adipogenic differentiation and inversely varied with the cell stiffness. Furthermore, cholesterol played a key role in the regulation of the cell morphology and biomechanics, suggesting its crucial involvement in mechanotransduction. To better understand the underlying mechanisms, we investigated the effect of cholesterol on the membrane-cytoskeleton linker proteins (ezrin and moesin). Cholesterol depletion was found to upregulate the ezrin expression which promoted cell spreading, increased Young's modulus, and hindered adipogenesis. In contrast, cholesterol enrichment increased the moesin expression, decreased Young's modulus, and induced cell rounding and facilitated adipogenesis. Taken together, cholesterol appears to regulate the stem cell mechanics and adipogenesis through the membrane-associated linker proteins.

Cutting Edge: Deletion of Ezrin in B Cells of Lyn-Deficient Mice Downregulates Lupus Pathology.

Genetic deletion of the Src family tyrosine kinase Lyn in mice recapitulates human systemic lupus erythematosus, characterized by hyperactive BCR signaling, splenomegaly, autoantibody generation, and glomerulonephritis. However, the molecular regulators of autoimmunity in Lyn-deficient mice and in human lupus remain poorly characterized. In this study, we report that conditional deletion of the membrane-cytoskeleton linker protein ezrin in B cells of Lyn-deficient mice (double knockout [DKO] mice) ameliorates B cell activation and lupus pathogenesis. B cells from DKO mice respond poorly to BCR stimulation, with severe downregulation of major signaling pathways. DKO mice exhibit reduced splenomegaly as well as significantly lower levels of autoantibodies against a variety of autoantigens, including dsDNA, histone, and chromatin. Leukocyte infiltration and deposition of IgG and complement component C3 in the kidney glomeruli of DKO mice are markedly reduced. Our data demonstrate that ezrin is a novel molecular regulator of B cell-associated lupus pathology.

Loss of B cell Ezrin in lupus-prone Lyn-deficient mice downregulates B cell activation and autoimmune pathology

Abstract Systemic lupus erythematosus (SLE) is an aggressive autoimmune disorder that afflicts 1.5 million Americans. Human GWAS studies have reported a strong association between the Src family tyrosine kinase Lyn and incidence of SLE, and B cells from SLE patients exhibit decreased Lyn expression. Genetic deletion of Lyn in mice results in loss of peripheral B cell tolerance and development of human SLE-like autoimmunity, characterized by hyperactive B cell antigen receptor (BCR) signaling, autoantibody production and glomerulonephritis. However, the molecular regulators of B cell pathogenesis in Lyn-deficient mice have not been fully characterized. We have previously shown that the membrane-cytoskeleton linker protein ezrin regulates multiple aspects of B cell function through its dynamic switching between phosphorylated and dephosphorylated states. We observed that ezrin is hyperphosphorylated in Lyn-deficient B cells, suggesting that it may impact B cell activation in this context. To test this hypothesis, we generated conditional deletion of ezrin in the B cells of Lyn-deficient mice (hereafter referred to as DKO mice). Compared to Lyn-deficient mice, the DKO mice exhibited a significant decrease in splenomegaly and reduction in autoantibodies against a variety of autoantigens. Glomerulonephritis and deposition of IgG and complement factor C3 in the kidneys of DKO mice were also markedly reduced. The DKO B cells were hyporesponsive to BCR stimulation and displayed a calcium mobilization pattern reminiscent of that observed in anergic B cells. Our data demonstrate that ezrin regulates B cell hyperactivation and associated pathology in the absence of Lyn, and suggest that ezrin may be a novel molecular target in SLE.

Concomitant expression of ezrin and HER2 predicts distant metastasis and poor prognosis of patients with salivary gland carcinomas

Salivary gland carcinomas (SGCs) exhibit heterogeneous biological behaviors, including the formation of distant metastases, which is a critical event associated with poor prognosis. Ezrin, which is a member of the ezrin-radixin-moesin family of plasma membrane-cytoskeleton linker proteins, may provide a marker for metastasis and poor survival of patients with cancer. The aim of the present study was to investigate the relationship between ezrin expression and the expression of HER2, p53, and Ki-67 as well as clinicopathological factors in SGCs. For this purpose, we used immunohistochemistry to analyze the expression of these proteins in tissue microarrays prepared from formalin-fixed, paraffin-embedded primary tumor tissues of 221 patients with SGCs. Using receiver operating characteristic curves, we determined cut-off values of 30% and 5.0% for high expression of ezrin and Ki-67, respectively. High ezrin expression detected in samples from 63 (28.5%) patients with SGCs significantly correlated with male sex, high-grade histopathology, high Ki-67 labeling index, HER2 overexpression, aberrant expression of p53, and distant metastasis. Multivariate analysis demonstrated that high ezrin expression was an independent prognostic factor for shorter overall survival (hazard ratio, 2.11 [1.09-4.05]; P=.027). Furthermore, concomitant high expression of ezrin and HER2 overexpression correlated significantly with shorter disease-free survival and overall survival as well as a high incidence of distant metastasis (P<.001). These findings indicate that ezrin and HER2 expression in patients with SGCs represents a high-grade histopathological subtype that requires adjuvant therapy, including molecularly targeted therapies, to decrease the risk of subsequent metastasis.

Correlation of Ezrin Expression Pattern and Clinical Outcomes in Ewing Sarcoma.

Background. Ezrin is a membrane-cytoskeleton linker protein that has been associated with metastasis and poor outcomes in osteosarcoma and high-grade soft tissue sarcomas. The prognostic value of ezrin expression in Ewing sarcoma is unknown. Methods. The relationship between ezrin expression and outcome was analyzed in a cohort of 53 newly diagnosed Ewing sarcoma patients treated between 2000 and 2011. The intensity and proportion of cells with ezrin immunoreactivity were assessed in diagnostic tumor tissue using a semiquantitative scoring system to yield intensity and positivity scores for each tumor. Results. Ezrin expression was detected in 72% (38/53) of tumor samples. The proportion of patients with metastatic disease was equal in the positive and negative ezrin expression groups. There was no significant difference in the 5-year event-free survival (EFS) between patients with positive versus negative ezrin expression. Patients whose tumor sample showed high ezrin intensity had significantly better 5-year EFS when compared to patients with low/no ezrin intensity (78% versus 55%; P = 0.03). Conclusions. Ezrin expression can be detected in the majority of Ewing sarcoma tumor samples. Intense ezrin expression may be correlated with a favorable outcome; however further investigation with a larger cohort is needed to validate this finding.

Prognostic impact of ezrin expression in Ewing sarcoma.

10545Background: Ezrin is a membrane-cytoskeleton linker protein that has been associated with metastasis and poor outcomes in a variety of human cancers including osteosarcoma and high-grade soft ...

Ezrin regulates inflammation by limiting B cell interleukin-10 production

Abstract In addition to their essential role in antibody production, B cells regulate immune responses by secreting effector cytokines. Interleukin 10 (IL-10) produced by B cells is important for controlling inflammation and autoimmunity, underscoring the importance of identifying mechanisms regulating B cell IL-10 production. We have previously demonstrated that the membrane-cytoskeleton linker protein ezrin regulates B cell antigen receptor organization and signaling. Further, we have shown that ezrin undergoes rapid dephosphorylation in naïve B cells treated with LPS, suggesting that it may regulate TLR4-mediated IL-10 production. In the present study, we demonstrate that conditional deletion of ezrin in B cells increases IL-10 production induced by TLR4, but not TLR2 or TLR9, ligation. The MyD88-independent TRIF-TBK1-IRF3 pathway is required for ezrin-deficient B cells to produce higher IL-10 upon LPS stimulation. Treatment of B cells with a novel small molecule inhibitor of ezrin induces its dephosphorylation, and increases LPS-induced NF-κB and IRF3 activation and IL-10 secretion, indicating a role for Thr567 phosphorylation of ezrin in limiting IL-10. Loss of ezrin in B cells results in dampened pro-inflammatory response to a sub-lethal dose of LPS in vivo, which is dependent on increased IL-10 production. Taken together, our data yield new insights into molecular and membrane-cytoskeletal regulation of B cell IL-10 production, and reveal ezrin as a potential therapeutic target in inflammatory diseases.

Ezrin interacts with the scaffold protein IQGAP1 and affects its cortical localization

The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane–cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca2+-dependent manner to the EF hand protein S100P and complexes consisting of Ca2+-bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca2+, thus independent of S100P. Direct ezrin–IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Formation and characterization of supported lipid bilayers containing phosphatidylinositol-4,5-bisphosphate and cholesterol as functional surfaces.

Solid-supported lipid bilayers (SLBs) mimicking a biological membrane are commonly used to investigate lipid-lipid or lipid-protein interactions. Simple binary or ternary lipid systems are well established, whereas more complex model membranes containing biologically important signaling lipids such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and cholesterol have not been extensively described yet. Here we report the generation of such bilayers and their relevant biophysical properties and in particular the accessibility of PI(4,5)P2 for protein binding. Ternary mixtures of POPC with 20% cholesterol and either 3 or 5 mol % dioleoyl-phosphatidylinositol-4,5-bisphosphate were probed by employing the quartz crystal microbalance and atomic force microscopy. We show that these mixtures form homogeneous solid-supported bilayers that exhibit no intrinsic phase separation and are characterized by long-term stability (>8 h). Bilayers were formed in a pH-dependent manner and were characterized by the accessibility of PI(4,5)P2 on the SLB surface as shown by the interaction with the PI(4,5)P2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P2 levels in the upper leaflet of SLBs was observed, which could be effectively inhibited by the incorporation of a negatively charged lipid such as phosphatidylserine. Furthermore, quartz crystal microbalance measurements revealed that cholesterol affects bilayer adsorption to the solid support.

Role of Moesin in Murine Hematopoiesis

Abstract 1338 Background:Moesin, a membrane-cytoskeleton linker protein, functions in microtubule formation, in membrane ruffling, and at sites of cell-adhesion. However, its role in hematopoiesis is unclear. Although a previous study demonstrated normal hematopoietic function in moesin knock-out (KO) mice (JBC 1999), the changes in blood cell counts observed in the mice have not been thoroughly analyzed. Moesin-specific antibodies are detected in the serum of approximately 30% of patients with acquired aplastic anemia (AA). Moesin is expressed on the surface of peripheral blood T cells and monocytes, but not on hematopoietic stem cells (HSCs). Our recent study demonstrated the involvement of anti-moesin antibodies in the development of AA by stimulating T cells and monocytes to secrete tumor necrosis factor-α and interferon-γ. Given that hematopoiesis is regulated by T cells and monocytes, the absence of moesin should impact hematopoiesis. The purpose of this study was to examine blood cell counts in moesin KO mice and WT mice transplanted with anti-moesin antibody producing B cells to clarify the role of moesin in hematopoiesis. Methods:Moesin KO mice (F1 heterozygous females (-/X)), which were a hybrid strain between 129/Sv and C57BL/6 (B6), were kindly provided by Professor Tsukita of Osaka University. F1 heterozygous females and B6 F1 wild-type (WT) males were interbred and the littermates were genotyped. Moesin KO mice, F2 hemizygous males (-/Y), and F3 homozygous females (−/−) were analyzed in a previous study (JBC 1999). F1 heterozygous females were backcrossed to B6 males for at least 8 generations to generate moesin KO mice in a B6 background. Moesin KO mice (12 months old) were used for hematopoietic phenotype analysis and to raise anti-moesin antibodies by immunization with moesin protein. Moesin KO male mice (-/Y) were immunized with 2×107 spleen cells containing moesin from WT B6 male mice to produce anti-moesin antibody secreting cells. Anti-moesin antibody titers were determined by Western blot analysis. To analyze the effects of anti-moesin antibodies in hematopoiesis, spleen cells (1.5×107) and bone marrow (BM) cells (1×107) containing anti-moesin antibody secreting cells from moesin KO mice were transplanted to the BM of 8-week-old WT B6 male mice conditioned with irradiation (3 Gy). Results:Moesin KO mice were born at expected Mendelian ratios and developed and grew normally. However, the body weight of moesin KO mice (n=7) was lower than that of WT mice (n=9) (26.63±2.47 g vs. 30.71±4.85 g; P=0.049). Complete blood counts of moesin KO mice (n=4) revealed leukocytopenia (WBC 12000±3096/μL vs. 19750±2582/μL; P=0.03) and macrocytic anemia (Hb 13.8±0.50 g/dL vs. 15.8±0.50 g/dL; P=0.06; MCV 51.7±1.7 fL vs. 47.6±1.3 fL, P=0.002) as compared with WT mice (n=4); platelet counts were comparable. Moesin KO mouse spleens (n=8) were heavier than those of WT mice (0.316±0.355 g vs. 0.091±0.034 g; P=0.0002) and follicle formation in spleens of moesin KO mice were obscure with increased CD3+ lymphocytes and reduced CD19+ lymphocytes as compared with WT mice. BM of moesin KO mice was normocellular and characterized by increased CD3+ lymphocytes and reduced CD19+ lymphocytes as seen in spleens of moesin KO mice. The number of lineage negative, Sca-1+, and c-Kit+ (LSK) mouse HSCs in moesin KO BM (n=4) tended to be lower than that of WT mice (n=3) (0.97±0.29% vs. 1.85±0.20%; P=0.06). Moesin KO spleen/BM cells capable of producing anti-moesin antibodies were infused into BM of WT B6 male mice (n=3), and moesin KO hematopoietic cells were engrafted on day 21. Transplanted mice showed leukocytopenia compared to WT B6 male mice transplanted with WT spleen/BM cells (n=3) (WBC 2667±333/μL vs. 7333±333/μL; P=0.0006), but anemia and thrombocytopenia were not observed. Conclusions:Moesin plays an important role in murine hematopoietic and immune system development. Anti-moesin antibody-induced leukocytopenia may result from excessive secretion of myelosuppressive cytokines from immune cells as observed in AA patients. The mechanistic details of HSC impairment in moesin KO mice are currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Activation of F-Actin Binding Capacity of Ezrin: Synergism of PIP2 Interaction and Phosphorylation

Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP2). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP2 binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP2, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP2 was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP2, which is enhanced by the phosphorylation.

Dynamic Propagation of Long-Range Allosteric Signals by Nanoscale Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

NHERF1 is a multi-domain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model suffices to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

Activation of Nanoscale Allosteric Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE.

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

The survival of osteosarcoma patients is connected to metastasis. The ezrin expression is associated with the development of metastasis and poor outcome in osteosarcoma. Ezrin is present in the cytoplasm and after phosphorylation assumes an active form and links F-actin to the cell membrane. This study evaluated ezrin and phosphorylated ezrin at site Tyr354 and Thr567 expression and its subcellular localization in osteosarcoma. We studied 50 osteosarcoma patients (mean follow-up 9.8 years). Ezrin expression was assessed using immunohistochemical and immunofluorescence analysis on tissue microarray and cultured cells of human osteosarcoma 143B. The western blot analysis was carried out on cultured cells. The majority of osteosarcomas, showing cytoplasmic positivity for ezrin, phosphorylated and unphosphorylated, were associated with membranous and nuclear positivity for phosphorylated ezrin Thr567 and phosphorylated ezrin Tyr354, respectively. Ezrin expression was associated with high-grade osteosarcoma (P=0.04), with metastasis (P=0.04) and with tumors that developed metastasis (P=0.04); phosphorylated ezrin Thr567 expression was present mostly in tumors with metastasis (P=0.01) and in osteosarcomas that did not develop metastasis (P=0.002). The osteosarcoma patients with ezrin expression have a short survival. The cytoplasmic ezrin expression in osteosarcoma matches its role of membrane-cytoskeleton linker protein. The subcellular trafficking of ezrin is not blocked and it is linked to ezrin phosphorylation, also in cancer. The phosphorylated ezrin Tyr354 nuclear localization suggests its possible role as a nuclear factor in osteosarcoma. The phosphorylated ezrin Thr567 phosphorylation may not be necessary in osteosarcoma metastatic progression but it was modulated. The ezrin expression is associated with more aggressive osteosarcomas and with metastasis.

Die Belegzelle des Magens: diametrale Bedeutung für die gastroösophageale Refluxkrankheit (GERD)

BACKGROUND: The gastric parietal cell (also called oxyntic cell) is a remarkable physiological entity in view of its regulation and participation in digestion, but also as an exemplary model for its exaggerated biophysical and cell biological processes. Moreover, the ability to pharmacologically control parietal cell activity has proven enormous value in treating a number of pathological conditions. METHODS: This review summarizes what is known about some of the most important physiological activities of the gastric parietal cell. RESULTS: Clues from differences in phenotype between resting and actively secreting cells provide a morphological basis for the massive biological process of intracellular membrane recycling, and explain the regulation by recruitment of the primary proton pump, H,K-ATPase, to the secretory surface. The large changes in membrane surface area require the sub-structural support of the filamentous actin cytoskeleton, which together with a dynamic membrane-cytoskeleton linker protein, known as ezrin, offer the scaffold for large membrane elaborations as well as required plasticity of the resulting microvillar extensions. Studies on basic protein/glycoprotein structure of H,K-ATPase have offered autoprotective mechanisms for the proton pump, and suggest the possibility that similar features in other gastric apical surface proteins may provide a molecular basis to why the stomach does not digest itself. An adjacent anatomical area, the esophagus, does not have the power to resist autodigestion resulting from backflux of gastric (and duodenal) juices; this is the pathological condition called gastroesophageal reflux disease, GERD. Interestingly, GERD related metaplastic changes in the distal esophageal epithelium ultimately result in one of two epithelial phenotypes: one called cardio-oxyntic mucosa, rarely leading to more serious consequences; and the other intestinal mucosa, characterized by goblet cells and having increased probability for progression to dysplasia and adenocarcinoma. CONCLUSIONS: Factors that promote lower esophageal metaplasia and dysplasia have interesting similarities with atrophic gastritis and glandular metaplasia of the gastric mucosa, i.e., intestinalization indicative of adenocarcinoma. These comparisons are discussed for the parietal cell in an unusual environment.