Ezrin/radixin/moesin Research Articles

Interaction of SARS-CoV-2 Spike protein with ACE2 induces cortical actin modulation, including dephosphorylation of ERM proteins and reduction of cortical stiffness.

Cell surface cortical actin is a regulatory target for viral infection. We aimed to investigate the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on host cell cortical stiffness, an indicator of cortical actin structure. The receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein induced a reduction in cortical stiffness in ACE2-expressing cells. The interaction of RBD with ACE2 caused the inactivation of Ezrin/Radixin/Moesin (ERM) proteins. We further investigated the effects of the RBD of SARS-CoV-2 Omicron variants, BA.1 and BA.5. These RBDs influenced cortical stiffness depending on their affinity for ACE2. Our study provides the first evidence that the interaction of the SARS-CoV-2 S protein with ACE2 induces mechanobiological signals and attenuates the cortical actin.

Manipulation of radixin phosphorylation in the nucleus accumbens core modulates risky choice behavior

Ezrin-Radixin-Moesin (ERM) proteins are actin-binding proteins that contribute to morphological changes in dendritic spines. Despite their significant role in regulating spine structure, the role of ERM proteins in the nucleus accumbnes (NAcc) is not well known, especially in in the context of risk-reward decision-making. Here, we measured the relationship between synaptic excitation and inhibition (E/I ratio) from medium spiny neurons in the NAcc core obtained in the rat after a rat gambling task (rGT). Then, after surgery of a phosphomimetic pseudo-active mutant form of radixin (Rdx-T564D) in the NAcc core, we examined its role in synaptic plasticity and the accompanying risk-choice behavior in rGT. We found that basal E/I ratio in the NAcc core was higher in risk-averse rats than risk-seeking rats. However, it was significantly reduced in risk-averse rats similar to that in risk-seeking rats in the presence of Rdx-T564D in the NAcc core. Furthermore, the head sizes of spines were shifted in risk-averse rats expressing Rdx-T564D in the NAcc core, similar to those observed in risk-seeking rats. The effects of Rdx-T564D in risk-averse rats were again manifested as behavioral changes, with reduced selection of optimal choices and increased selection of disadvantageous ones. In this study, we demonstrated that manipulation of radixin phosphorylation status in the NAcc core can alter glutamatergic synaptic transmission and spine structure at this site, as well as risk choice behaviors in the rGT. These novel findings illustrate that radixin in the NAcc core plays a significant role in determining risk preference during the rGT.

Moesin controls cell-cell fusion and osteoclast function.

Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including fertilisation and the formation of placenta, muscle and osteoclasts, multinucleated cells that are unique in their ability to resorb bone. The mechanisms of osteoclast multinucleation involve dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. Here, we found that moesin, a cytoskeletal linker protein member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role in both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin inhibition favors their ability to fuse into multinucleated osteoclasts. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes (TNTs), F-actin-based intercellular bridges that we reveal here to trigger cell-cell fusion. Moesin also controls HIV-1- and inflammation-induced cell fusion. In addition, moesin regulates the formation of the sealing zone, the adhesive structure determining osteoclast bone resorption area, and thus controls bone degradation, via a β3-integrin/RhoA/SLK pathway. Supporting our results, moesin - deficient mice present a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclast activity in bone disease therapy.

Hippo pathway inactivation through subcellular localization of NF2/merlin in outer cells of mouse embryos.

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.

Structural Basis for the Interaction between the Ezrin FERM-Domain and Human Aquaporins.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

Characterization of thymic architecture and lymphocyte populations in X-MAID due to an underlying pathogenic moesin mutation

Background: X-linked Moesin Associated Immunodeficiency (X-MAID) is a combined immunodeficiency caused by deficiency in the moesin protein. Moesin, which is encoded by the MSN gene, is part of the ezrin-radixin-moesin (ERM) family of transmembrane proteins that interact with the actin cytoskeleton and regulate the shape and migration of cells. Deficiency of moesin is associated with aberrant T cell migration and inadequate immune synapse formation, leading to significant immunodeficiency and recurrent infections. While the clinical presentation of X-MAID is diverse, to date, no thymus histopathology findings have been reported. Aim: Describe the thymus histopathology of a patient with X-MAID. Results: Our patient is a 10-year-old male who presented early in life with recurrent infections, dysmorphic features, and severe pulmonary venous stenosis which required a double lung transplant at the age of 4 years. Prior to transplant, he was referred to Immunology for assessment and was subsequently found to harbour a hemizygous variant in the MSN gene (c.278dupT; p.L93FfsX21). Thymus histopathology findings showed significant cortical atrophy and dysplasia and was accompanied by reduction in CD3+ cells in the cortex. Abnormally low numbers of suppressor T cells and T helper cells in the thymic cortex and medulla were noted. Conclusion: Thymic findings in X-MAID can include cortical atrophy, dysplasia, and decreased cellularity. This provides further evidence for the importance of moesin on T cell development and migration in the thymus. Statement of novelty: Description of thymus histopathology in a patient with X-MAID.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Microscopic clusters feature the composition of biochemical tetraspanin-assemblies and constitute building-blocks of tetraspanin enriched domains

Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic criterion differentiating between web and TEMs is lacking. Using super-resolution microcopy, we identify co-assemblies between the tetraspanins CD9 and CD81 and CD151 and CD81. CD9 assemblies contain as well the CD9/CD81-interaction partner EWI-2. Moreover, CD9 clusters are proximal to clusters of the CD81-interaction partner CD44 and CD81-/EWI-2-interacting ezrin–radixin–moesin proteins. Assemblies scatter unorganized across the cell membrane; yet, upon EWI-2 elevation, they agglomerate into densely packed arranged-crowds in a process independent from actin dynamics. In conclusion, microscopic clusters are equivalent to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration enriches tetraspanins, which makes agglomerations to a microscopic complement of TEMs. The microscopic classification of tetraspanin assemblies advances our understanding of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections.

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

Exploring the relationship between novel MSN exon mutations and altered expression of other cytoskeleton genes in archival FFPE meningioma tissues

Meningioma, a prevalent brain tumor, can severely impact patients' health and quality of life. However, there is a scarcity of data on Ezrin/Radixin/Moesin (ERM) and the quantification of mutations in the Moesin gene. In an effort to bridge this knowledge gap, we undertook an extensive study to measure the expression of ERM genes in meningioma patients, with a concurrent assessment of mutations in the moesin gene. We analyzed the expression patterns of these genes in 50 formalin-fixed paraffin-embedded meningioma tissues, using the xylene method to deparaffinize the tissues and the TRIzol technique to isolate total RNA for qPCR expression analysis. Our results indicated that Ezrin and Radixin expression levels were significantly upregulated (p < 0.05), while Moesin expression was downregulated (p < 0.05). In addition, we analyzed Moesin exons 2–6 for mutations using sanger sequencing and identified 10 novel variants, including 5 missense, 3 synonymous, and 2 frameshift mutations. Our findings suggests that a potential role of ERM genes in meningioma pathogenesis, and our identification of MSN gene mutations may contribute to understanding their harmful or gain-of-function effects. These results offer new insights into the mechanisms underlying meningioma carcinogenesis, which may facilitate the development of targeted therapies in the future.

Contribution of radixin and ezrin to the maintenance of hepatocytes' excretory function in health and disease

Background & aimsExcretory liver failure is frequently associated with poor prognosis in critically ill patients. It is characterized by the loss of canalicular membrane export pumps at the hepatocyte membrane. The membrane export pump Multidrug resistant-associated protein (MRP) 2 is pivotal in hepatocytes for brushed membrane morphology and transport of various metabolites. In addition, MRP2 anchoring proteins of the Ezrin/Radixin/Moesin (ERM) family are crucial for the correct MRP2 location, integration, and function in different tissues. In hepatocytes, altered ERM signaling is elementary for developing excretory liver failure. MethodsPolarized human HepaRG cells, primary human hepatocytes, and hepatocyte-specific Ezrin knockout mice are employed to investigate ERM expression and function in health and the bile duct ligation model of obstructive cholestasis. ResultsERM-scaffolding protein Ezrin has no relevant function in maintaining the canalicular structure in hepatocytes during health and disease. ConclusionsHomeostasis of the canalicular pole in hepatocytes is maintained exclusively by Radixin but not Ezrin, and Radixin dysfunction promotes cholestasis.

High levels of PD-L1 on platelets of NSCLC patients contributes to the pharmacological activity of Atezolizumab

Several studies have associated platelets (PLTs) to NSCLC prognosis. To understand the role of PLTs in immunotherapy-treated patients, we used blood samples of NSCLC patients at different TNM stage. We found that PLTs count and the expression of PD-L1 (pPD-L1) were significantly higher in NSCLC patients at Stage IV than Stage I-III and healthy subjects. The presence of high pPD-L1 was associated to upregulated genes for the extracellular matrix organization and tumor immunosuppression. When patients’ survival was correlated to the levels of pPD-L1, longer survival rate was observed, but not when progression disease occurred. The in vitro stimulation of pPD-L1 with Atezolizumab induced CXCL4 release, accompanied by higher levels of TGFβ at the time of drug resistance when the levels of CD16, CD32 and CD64 significantly increased. Leiden-clustering method defined the phenotype of PLTs which showed that the ezrin-radixin-moesin (ERM) family proteins, underlying the PD-L1 signalosome, were involved in high pPD-L1 and higher survival rate. These data imply that Stage IV NSCLC patients characterized by high pPD-L1 are associated with longer progression-free survival rate because the blockade of pPD-L1 by Atezolizumab avoids the exacerbation of a T cell-mediated immune-suppressive environment. pPD-L1 could be an easy-to-use clinical approach to predict ICI responsiveness.

A novel role for the chloride intracellular channel protein Clic5 in ciliary function

CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left–right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation.

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Potential Role of Moesin in Regulating Mast Cell Secretion.

Mast cells have existed for millions of years in species that never suffer from allergic reactions. Hence, in addition to allergies, mast cells can play a critical role in homeostasis and inflammation via secretion of numerous vasoactive, pro-inflammatory and neuro-sensitizing mediators. Secretion may utilize different modes that involve the cytoskeleton, but our understanding of the molecular mechanisms regulating secretion is still not well understood. The Ezrin/Radixin/Moesin (ERM) family of proteins is involved in linking cell surface-initiated signaling to the actin cytoskeleton. However, how ERMs may regulate secretion from mast cells is still poorly understood. ERMs contain two functional domains connected through a long α-helix region, the N-terminal FERM (band 4.1 protein-ERM) domain and the C-terminal ERM association domain (C-ERMAD). The FERM domain and the C-ERMAD can bind to each other in a head-to-tail manner, leading to a closed/inactive conformation. Typically, phosphorylation on the C-terminus Thr has been associated with the activation of ERMs, including secretion from macrophages and platelets. It has previously been shown that the ability of the so-called mast cell "stabilizer" disodium cromoglycate (cromolyn) to inhibit secretion from rat mast cells closely paralleled the phosphorylation of a 78 kDa protein, which was subsequently shown to be moesin, a member of ERMs. Interestingly, the phosphorylation of moesin during the inhibition of mast cell secretion was on the N-terminal Ser56/74 and Thr66 residues. This phosphorylation pattern could lock moesin in its inactive state and render it inaccessible to binding to the Soluble NSF attachment protein receptors (SNAREs) and synaptosomal-associated proteins (SNAPs) critical for exocytosis. Using confocal microscopic imaging, we showed moesin was found to colocalize with actin and cluster around secretory granules during inhibition of secretion. In conclusion, the phosphorylation pattern and localization of moesin may be important in the regulation of mast cell secretion and could be targeted for the development of effective inhibitors of secretion of allergic and inflammatory mediators from mast cells.

Identification of Moesin (MSN) as a Potential Therapeutic Target for Colorectal Cancer via the β-Catenin-RUNX2 Axis.

CRC is the second leading cause of cancer-related death. The complex mechanisms of metastatic CRC limit available therapeutic choice. Thus, identifying new CRC therapeutic targets is essential. Moesin (MSN), a member of the ezrin-radixin-moesin family, connects the cell membrane to the actin-based cytoskeleton and regulates cell morphology. We investigated the role of MSN in the progression of CRC. GENT2 and oncomine were used to study MSN expression and CRC patient outcomes. MSN-specific shRNAs or MSN-overexpressed plasmid were used to establish MSN-KD and MSN overexpressed cell lines, respectively. SRB, migration, wound healing, and flow cytometry were used to test cell survival and migration. Propidium iodide and annexin V stain were used to analyze the cell cycle and apoptosis. MSN expression was found to be higher in CRC tissues than in normal tissues. Higher MSN expression is associated with poor overall survival, disease-free survival, and relapse-free survival rates in CRC patients. MSN silencing inhibits cell proliferation, adhesion, migration, and invasion in vitro, whereas MSN overexpression accelerates cell proliferation, adhesion, migration, and invasion. RNA sequencing was used to investigate differentially expressed genes, and RUNX2 was discovered as a possible downstream target for MSN. In CRC patients, RUNX2 expression was significantly correlated with MSN expression. We also found that MSN silencing decreased cytoplasmic and nuclear β-catenin levels. Additionally, pharmacological inhibition of β-catenin in MSN-overexpressed cells led to a reduction of RUNX2, and activating β-catenin signaling by inhibiting GSK3β rescued the RUNX2 downregulation in MSN-KD cells. This confirms that MSN regulates RUNX2 expression via activation of β-catenin signaling. Finally, our result further determined that RUNX2 silencing reduced the ability of MSN overexpression cells to proliferate and migrate. MSN accelerated CRC progression via the β-catenin-RUNX2 axis. As a result, MSN holds the potential to become a new target for CRC treatment.

Radixin modulates the plasma membrane localization of CD47 in human uterine cervical adenocarcinoma cells

Despite the dramatic success of immune checkpoint blockers in treating numerous cancer cell types, current therapeutic modalities provide clinical benefits to a subset of patients with cervical cancers. CD47 is commonly overexpressed in a broad variety of cancer cells, correlates with poor clinical prognosis, and acts as a dominant macrophage checkpoint by interacting with receptors expressed on macrophages. It allows cancer cells to escape from the innate immune system and hence is a potential therapeutic target for developing novel macrophage checkpoint blockade immunotherapies. As the intracellular scaffold proteins, ezrin/radixin/moesin (ERM) family proteins post-translationally regulate the cellular membrane localization of numerous transmembrane proteins, by crosslinking them with the actin cytoskeleton. We demonstrated that radixin modulates the plasma membrane localization and functionality of CD47 in HeLa cells. Immunofluorescence analysis and co-immunoprecipitation assay using anti-CD47 antibody showed the colocalization of CD47 and all three ERM families in the plasma membrane, and the molecular interactions between CD47 and all three ERM. Interestingly, gene silencing of only radixin, reduced the CD47 plasma membrane localization and functionality by means of flow cytometry and phagocytosis assay but had little influence on its mRNA expression. Together, in HeLa cells radixin may function as a principal scaffold protein responsible for the CD47 plasma membrane localization.

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.

CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

Cellular Membrane Localization of Innate Immune Checkpoint Molecule CD47 Is Regulated by Radixin in Human Pancreatic Ductal Adenocarcinoma Cells.

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent "do not eat me" signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells.