Ezrin Activation Research Articles

Ezrin Contributes to the Damage of Airway Epithelial Barrier Related to Diabetes Mellitus.

Diabetes mellitus predisposes individuals to respiratory infections. The airway epithelial barrier provides defense against inhaled antigens and pathogens. Ezrin, is a component of the membrane-cytoskeleton that maintains the cellular morphology, intercellular adhesion, and barrier function of epithelial cells. This study aimed to explore the role of ezrin in airway epithelial barrier damage and correlate its expression and activation with diabetes mellitus. This study was performed in a murine model of diabetes mellitus and with human bronchial epithelial BEAS-2B cells using real-time PCR, Western blotting, immunohistochemical and immunofluorescence staining. Ezrin was knocked down in BEAS-2B cells using siRNA. Ezrin phosphorylation levels were measured to determine activation status. The integrity of the airway epithelial barrier was assessed in vivo by characterizing morphological structure, and in vitro in BEAS-2B cells by measuring tight junction protein expression, transepithelial electrical resistance (TER) and permeability. We demonstrated that ezrin expression levels were lower in the lung tissue and airway epithelium of diabetic mice than those in control mice. The morphological structure of the airway epithelium was altered in diabetic mice. High glucose levels downregulated the expression and distribution of ezrin and connexin 43, reduced the expression of tight junction proteins, and altered the epithelial barrier characteristics of BEAS-2B cells. Ezrin knockdown had effects similar to those of high glucose levels. Moreover, a specific inhibitor of ezrin Thr567 phosphorylation (NSC305787) inhibited epithelial barrier formation. These results demonstrate that ezrin expression and activation are associated with airway epithelial damage in diabetes mellitus. These findings provide new insights into the molecular pathogenesis of pulmonary infections in diabetes mellitus and may lead to novel therapeutic interventions for airway epithelial barrier damage.

ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structures.

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show that the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly outnumber Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin

Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin’s FERM domain and C408 in PALM2’s CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.

Podoplanin promotes the carcinogenicity of gastric cancer by activating ezrin and mediating the crosstalk between tumour cells and cancer-associated fibroblasts.

What is the central question of this study? To reveal the role and biological mechanism of PDPN in the progression of gastric cancer. What is the main finding and its importance? This study focused on a prognostic predictor, PDPN, which acted as a promoter in the progression of gastric cancer through the activation of Ezrin expression and CAFs. This finding may expand a new route for the gene-targeted therapy in gastric cancer. Gastric cancer (GC) is a frequent malignant disease and the main cause of cancer-related death in the world. Podoplanin (PDPN) has been proved to be involved in the progression of various cancers. However, the role and biological mechanism of PDPN in GC are still vague. In our study, we detected the expression of PDPN in GC tissues and cell lines using RT-qPCR, western blot and datasets. The overall survival of GC patients was analysed with a Kaplan-Meier plot. The effects of PDPN overexpression and silencing on GC cell progression were assessed by Cell Counting Kit-8, flow cytometry and a wound healing assay. Besides, the modulation of PDPN on ezrin activation was investigated. We further explored the role of PDPN in the crosstalk between GC cells and cancer associated fibroblasts (CAFs). Results showed that PDPN was upregulated in GC tissues and cell lines. High expression of PDPN was correlated with poor prognosis of GC patients. PDPN positively regulated the viability, migration and invasion, but inhibited apoptosis, of GC cells by mediating the activation of ezrin. Meanwhile, the change in PDPN in GC cells activated CAFs and promoted the production of cytokines secreted by CAFs, which induced the progression of GC cells. These findings may provide a novel target for GC therapy.

Hypoxia enhances interactions between Na+/H+ exchanger isoform 1 and actin filaments via ezrin in pulmonary vascular smooth muscle.

Exposure to hypoxia, due to high altitude or chronic lung disease, leads to structural changes in the pulmonary vascular wall, including hyperplasia and migration of pulmonary arterial smooth muscle cells (PASMCs). Previous studies showed that hypoxia upregulates the expression of Na+/H+ exchanger isoform 1 (NHE1) and that inhibition or loss of NHE1 prevents hypoxia-induced PASMC migration and proliferation. The exact mechanism by which NHE1 controls PASMC function has not been fully delineated. In fibroblasts, NHE1 has been shown to act as a membrane anchor for actin filaments, via binding of the adaptor protein, ezrin. Thus, in this study, we tested the role of ezrin and NHE1/actin interactions in controlling PASMC function. Using rat PASMCs exposed to in vitro hypoxia (4% O2, 24h) we found that hypoxic exposure increased phosphorylation (activation) of ezrin, and promoted interactions between NHE1, phosphorylated ezrin and smooth muscle specific α-actin (SMA) as measured via immunoprecipitation and co-localization. Overexpression of wild-type human NHE1 in the absence of hypoxia was sufficient to induce PASMC migration and proliferation, whereas inhibiting ezrin phosphorylation with NSC668394 suppressed NHE1/SMA co-localization and migration in hypoxic PASMCs. Finally, overexpressing a version of human NHE1 in which amino acids were mutated to prevent NHE1/ezrin/SMA interactions was unable to increase PASMC migration and proliferation despite exhibiting normal Na+/H+ exchange activity. From these results, we conclude that hypoxic exposure increases ezrin phosphorylation in PASMCs, leading to enhanced ezrin/NHE1/SMA interaction. We further speculate that these interactions promote anchoring of the actin cytoskeleton to the membrane to facilitate the changes in cell movement and shape required for migration and proliferation.

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration.

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.

High glucose induces Nox4 expression and podocyte apoptosis through the Smad3/ezrin/PKA pathway.

ABSTRACTPodocytes are the major target in proteinuric kidney diseases such as diabetic nephropathy. The underlying molecular mechanisms by which high glucose (HG) results in podocyte damage remain unclear. This study investigated the regulatory role of Smad3, ezrin, and protein kinase A (PKA) in NADPH oxidase (Nox4) expression, reactive oxidative species (ROS) production, and apoptosis in HG-treated podocytes. A human podocyte cell line was cultured and differentiated, then treated with 30 mM HG. Apoptosis and intracellular ROS levels were assessed using TUNEL and DCF assays, respectively. Expressions of Nox4, phospho-Smad3Ser423/425, phospho-PKAThr197, and phospho-ezrinThr567 were evaluated using western blotting. ELISA was used to quantify intracellular cAMP concentration and PKA activity. Knockdown assay was used to inhibit the expressions of Smad3, Nox4, and ezrin by lentiviral shRNA. In HG-treated podocytes, the level of phospho-Smad3Ser423/425 and phospho-ezrinThr567 was increased significantly, which was accompanied by the reduction of cAMP and phospho-PKAThr197. HG-induced apoptosis was significantly prevented by the Smad3-inhibitor SIS3 or shRNA-Smad3. In podocytes expressing shRNA-ezrin or shRNA-Nox4, apoptosis was remarkably mitigated following HG treatment. HG-induced upregulation of phospho-ezrinThr567 and downregulation of phospho-PKAThr197 was significantly prevented by SIS3, shRNA-ezrin or shRNA-Smad3. Forskolin, a PKA activator, significantly inhibited HG-mediated upregulation of Nox4 expression, ROS generation, and apoptosis. Additionally, an increase in the ROS level was prohibited in HG-treated podocytes with the knockdown of Nox4, Smad3, or ezrin. Taken together, our findings provided evidence that Smad3-mediated ezrin activation upregulates Nox4 expression and ROS production, by suppressing PKA activity, which may at least in part contribute to HG-induced podocyte apoptosis.

A Direct CLIC5A/Ezrin Interaction Enables CLIC5A‐Dependent Rac1 Activation

Introduction The CLIC (Chloride Intracellular Channel) protein CLIC5A is a component of the ezrin/podocalyxin/actin complex in kidney podocytes, where it stabilizes actin-based projections (foot processes). Similarly, in inner ear hair cells CLIC5A in the radixin complex stabilizes sensory stereocilia, also actin-based projections. ERM (ezrin, radixin, moesin) proteins help organize the cortical actin cytoskeleton, linking membrane-spanning proteins to actin. We have reported that CLIC5A stimulates Rac1-GTP-dependent phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] generation, causing ezrin activation and phosphorylation, thus coupling podocalyxin, via ezrin, to the cytoskeleton (J. Cell Sci. 127:5164, 2014 & Kidney Int. 89:833, 2016). Hypothesis Direct interaction(s) of CLIC5A with Rac1 and/or ezrin enable CLIC5A-dependent Rac1 activation. Methods Rac1 activity was determined by Rac1-GTP specific PAK-PBD pull-down from CLIC5A or vector transfected cells and by Rac1-GTP G-LISA® (Cytoskeleton Inc). Association of CLIC5A with Rac1 was determined by GST-CLIC5A pull-down of exogenously expressed wild-type GFP-Rac1(WT), constitutively active GFP-Rac1 (Q61L) and dominant negative GFP-Rac1 (D17N) from COS7 cell lysates and by in vitro GST-CLIC5A pull-down of GTPγS- or GDP-loaded recombinant His-Rac1. Direct protein-protein interactions were evaluated by Yeast two-Hybrid (Y2H) screening of a mouse kidney protein domain library, and Y2H mapping of interacting domains in the related ERM protein ezrin, using CLIC5A as the bait (Fig 1A). The CLIC5A/ezrin association was studied by co-immunoprecipitation and GST-CLIC5A pull-down. Results Exogenous CLIC5A expression activated Rac1 and CLIC5A was co-precipitated with Rac1-GTP and ezrin by PAK-PBD affinity beads. GST-CLIC5A pulled GFP-Rac1 WT, -Q61L, and -D17N equally from cell lysates, but recombinant GTPγS- or GDP-loaded His-Rac1 did not directly bind GST-CLIC5A, suggesting that the CLIC5A/Rac1 interaction is indirect. The Y2H screen revealed that CLIC5A interacts directly with the C-terminal domain of radixin, and Y2H mapping (Fig. 1A) showed that CLIC5A similarly interacts directly with the ezrin C-terminal domain (432-586). Direct interactions with full-length ezrin (1-586) and the C-terminally deleted ezrin (432-570) domain were not detected. Furthermore, ezrin (432-586), but not full-length ezrin (1-586) or ezrin (432-570) was pulled down by GST-CLIC5A and co-immunoprecipitated with CLIC5A. Finally, siRNA silencing of endogenous ezrin significantly reduced CLIC5A-stimulated Rac1 activation (Fig. 1B). Conclusion Although CLIC5A activates Rac1 and is part of the Rac1-GTP complex, the CLIC5A/Rac1 interaction appears to be indirect. CLIC5A interacts directly with ezrin (and radixin), and Rac1 activation by CLIC5A is ezrin-dependent. The finding that a direct interaction of CLIC5A with full-length ezrin was not detected suggests that only the active, open ezrin conformation, freeing the C-terminus, has a high affinity for CLIC5A. The data suggest that the direct CLIC5A/ezrin interaction may enhance localized Rac1 activation and thus Rac1-GTP-dependent PI(4,5)P2 generation.

Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real time.

Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

[Figure: see text].

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Cell Surface Mechanics Gate Embryonic Stem Cell Differentiation.

SummaryCell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency.

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Progression of hepatocellular carcinoma involves multiple genetic and epigenetic alterations that promote cancer invasion and metastasis. Our recent study revealed that hyperphosphorylation of ezrin promotes intrahepatic metastasis in vivo and cell migration in vitro. Celastrol is a natural product from traditional Chinese medicine which has been used in treating liver cancer. However, the mechanism of action underlying celastrol treatment was less clear. Here we show that ROCK2 is a novel target of celastrol and inhibition of ROCK2 suppresses elicited ezrin activation and liver cancer cell migration. Using cell monolayer wound healing, we carried out a phenotype-based screen of natural products and discovered the efficacy of celastrol in inhibiting cell migration. The molecular target of celastrol was identified as ROCK2 using celastrol affinity pull-down assay. Our molecular docking analyses indicated celastrol binds to the active site of ROCK2 kinase. Mechanistically, celastrol inhibits the ROCK2-mediated phosphorylation of ezrin at Thr567 which harnesses liver cancer cell migration. Our findings suggest that targeting ROCK2-ezrin signaling is a potential therapeutic niche for celastrol-based intervention of cancer progression in hepatocellular carcinoma.

Ezrin protects bovine spermatozoa from spontaneous acrosome reaction

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR.

Nuclear factor IX promotes glioblastoma development through transcriptional activation of Ezrin

Enhanced migration is pivotal for the malignant development of glioblastoma (GBM), but the underlying molecular mechanism that modulates the migration of the GBM cells remains obscure. Here we show that nuclear factor IX (NFIX) is significantly upregulated in human GBM lesions compared with normal or low-grade gliomas. NFIX deficiency impairs the migration of GBM cells and inhibits the tumor growth in the hippocampus of immunodeficient nude mice. Mechanistically, NFIX silencing suppresses the expression of Ezrin, a protein that crosslinks actin cytoskeleton and plasma membrane, which is also positively correlated with GBM malignancy. NFIX depletion induced migration inhibition of GBM cells can be rescued by the replenishment of Ezrin. Furthermore, we identify a NFIX response element (RE) between −840 and −825 bp in the promoter region of the Ezrin gene. Altogether, our findings show, for the first time that NFIX can transcriptionally upregulate the expression of Ezrin and contribute to the enhanced migration of GBM cells, suggesting that NFIX is a potential target for GBM therapy.

RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinoma

ObjectivesIncreased expression of REarranged during Transfection (RET) kinase is reported in 10–20 % of lung adenocarcinomas (LUAD) and is associated with metastasis and reduced survival. Ezrin is a scaffold protein that promotes protein interactions with the actin cytoskeleton to regulate cell migration and is also associated with invasion and metastasis in cancers. RET isoforms interact with unique combinations of scaffold proteins to promote distinct signaling pathways. We hypothesized that RET isoforms associate distinctly with Ezrin for cytoskeletal reorganization and LUAD cell migration processes. MethodsHCC1833 and A549 LUAD, SH-SY5Y neuroblastoma or HEK-293 cells expressing RET and Ezrin were stimulated with the RET ligand glial cell line-derived neurotrophic factor (GDNF) and treated with RET, Ezrin or Src inhibitors. Co-immunoprecipitation or pull-down assays coupled to immunoblotting were used to investigate protein activation and interactions. Immunofluorescence confocal microscopy assessed LUAD cytoskeletal reorganization and colocalization of RET and Ezrin. Live-cell fluorescence imaging was used to measure cell migration and chemotaxis. ResultsGDNF promoted activation, interaction and colocalization of RET51 isoform and Ezrin. Inhibition of RET or Src impaired Ezrin interactions with RET and Src. GDNF stimulation enhanced the formation of actin-rich filopodia, in which both RET and Ezrin were enriched, and promoted chemotaxis in LUAD cells. However, inhibition of RET, Src or Ezrin suppressed filopodia formation, reduced colocalization of Ezrin with RET, and impaired cell migration and/ or chemotaxis. We further showed that GDNF-mediated activation of RET and Ezrin promoted RhoA-GTPase activity and signaling of ROCK1 and ROCK2 in LUAD cells. ConclusionsExpression and activation of RET51 mediates unique protein interactions with Ezrin to promote LUAD cell chemotaxis for cancer cell dissemination, which may have implications in LUAD metastatic progression.

CLIC4 and CLIC1 bridge plasma membrane and cortical actin network for a successful cytokinesis.

CLIC4 and CLIC1 are members of the well-conserved chloride intracellular channel proteins (CLICs) structurally related to glutathione-S-transferases. Here, we report new roles of CLICs in cytokinesis. At the onset of cytokinesis, CLIC4 accumulates at the cleavage furrow and later localizes to the midbody in a RhoA-dependent manner. The cell cycle-dependent localization of CLIC4 is abolished when its glutathione S-transferase activity-related residues (C35A and F37D) are mutated. Ezrin, anillin, and ALIX are identified as interaction partners of CLIC4 at the cleavage furrow and midbody. Strikingly, CLIC4 facilitates the activation of ezrin at the cleavage furrow and reciprocally inhibition of ezrin activation diminishes the translocation of CLIC4 to the cleavage furrow. Furthermore, knockouts of CLIC4 and CLIC1 cause abnormal blebbing at the polar cortex and regression of the cleavage furrow at late cytokinesis leading to multinucleated cells. We conclude that CLIC4 and CLIC1 function together with ezrin where they bridge plasma membrane and actin cytoskeleton at the polar cortex and cleavage furrow to promote cortical stability and successful completion of cytokinesis in mammalian cells.

Perspectives for Ezrin and Radixin in Astrocytes: Kinases, Functions and Pathology.

Astrocytes are increasingly perceived as active partners in physiological brain function and behaviour. The structural correlations of the glia–synaptic interaction are the peripheral astrocyte processes (PAPs), where ezrin and radixin, the two astrocytic members of the ezrin-radixin-moesin (ERM) family of proteins are preferentially localised. While the molecular mechanisms of ERM (in)activation appear universal, at least in mammalian cells, and have been studied in great detail, the actual ezrin and radixin kinases, phosphatases and binding partners appear cell type specific and may be multiplexed within a cell. In astrocytes, ezrin is involved in process motility, which can be stimulated by the neurotransmitter glutamate, through activation of the glial metabotropic glutamate receptors (mGluRs) 3 or 5. However, it has remained open how this mGluR stimulus is transduced to ezrin activation. Knowing upstream signals of ezrin activation, ezrin kinase(s), and membrane-bound binding partners of ezrin in astrocytes might open new approaches to the glial role in brain function. Ezrin has also been implicated in invasive behaviour of astrocytomas, and glial activation. Here, we review data pertaining to potential molecular interaction partners of ezrin in astrocytes, with a focus on PKC and GRK2, and in gliomas and other diseases, to stimulate further research on their potential roles in glia-synaptic physiology and pathology.

Abstract 4594: Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression

Abstract Background: Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family, promotes tumor progression and metastasis. We previously revealed that the tumor-promoting role of LOXL2 is mediated by perturbing the architecture of the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying LOXL2-driven ESCC progression remain elusive, and the cytoskeletal proteins that directly interact with LOXL2 remain uncharacterized. Methods: Knockdown LOXL2 in ESCC cells was used by a lentivirus shRNA targeting LOXL2 and analyzed by RNA-sequencing and bioinformatics analysis. Proteomic analysis following by co-immunoprecipitation and immunofluorescence were applied for identification and validation of interacting partners of LOXL2 and its splicing isoform L2Δ13. Cell migration and invasion were measured by wound healing and transwell assays. By risk score calculations, immunohistochemistry of tissue microarrays were adopted to evaluate the prognosis of ESCC patients. Results: We found that knockdown of LOXL2 in ESCC cells suppresses cell migration and invasion, whereas re-expressions of LOXL2 and its non-enzymatic splicing isoform L2Δ13 rescue these cell behaviors. Silencing of LOXL2 inhibits filopodia formation, and induces changes in the expression of cytoskeleton-associated genes. Our interactome analysis identified four actin-binding proteins, i.e. ezrin (EZR), facsin (FSCN1), heat shock protein beta-1 (HSPB1) and tropomodulin-3 (TMOD3), as novel interactors of LOXL2 and L2Δ13. These novel LOXL2/L2Δ13 interaction networks carry important prognostic values in ESCC, as molecular signatures of combinations of LOXL2/L2Δ13 and their cytoskeletal interactors are associated with clinical outcome in ESCC patients. Mechanistically, the ezrin-LOXL2 interaction promotes PKCα-stimulated phosphorylation of ezrin at Thr 567 (T567), an essential residue for ezrin activation, which enhances cell motility and cell invasion in ESCC. Correlating with this, double-high expression of LOXL2 and phosphorylated ezrin-T567 predicts significantly shorter overall survival in ESCC patients. Conclusion: In conclusion, our findings have uncovered a novel molecular mechanism underlying the tumor-promoting role of cytoplasmic LOXL2, which may open new avenues for the therapeutic targeting of LOXL2 in ESCC. (This study is supported by the Natural Science Foundation of China (No. 81472613) and Li Ka Shing Foundation). Citation Format: Xiuhui Zhan, Jiwei Jiao, Haifeng Zhang, Jianzhong He, Runliu Li, Haiying Zou, Zhiyong Wu, Shaohong Wang, Xiue Xu, Jianyi Wu, Liandi Liao, Yinwei Cheng, Kai Zhang, Gera Neufeld, Liyan Xu, Enmin Li. Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4594.