GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis.

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Deep Semantic Segmentation of Angiogenesis Images.

Angiogenesis is the development of new blood vessels from pre-existing ones. It is a complex multifaceted process that is essential for the adequate functioning of human organisms. The investigation of angiogenesis is conducted using various methods. One of the most popular and most serviceable of these methods in vitro is the short-term culture of endothelial cells on Matrigel. However, a significant disadvantage of this method is the manual analysis of a large number of microphotographs. In this regard, it is necessary to develop a technique for automating the annotation of images of capillary-like structures. Despite the increasing use of deep learning in biomedical image analysis, as far as we know, there still has not been a study on the application of this method to angiogenesis images. To the best of our knowledge, this article demonstrates the first tool based on a convolutional Unet++ encoder-decoder architecture for the semantic segmentation of in vitro angiogenesis simulation images followed by the resulting mask postprocessing for data analysis by experts. The first annotated dataset in this field, AngioCells, is also being made publicly available. To create this dataset, participants were recruited into a markup group, an annotation protocol was developed, and an interparticipant agreement study was carried out.

Novel Functions of the Septin Cytoskeleton: Shaping Up Tissue Inflammation and Fibrosis.

Chronic inflammatory diseases cause profound alterations in tissue homeostasis, including unchecked activation of immune and nonimmune cells leading to disease complications such as aberrant tissue repair and fibrosis. Current anti-inflammatory therapies are often insufficient in preventing or reversing these complications. Remodeling of the intracellular cytoskeleton is critical for cell activation in inflamed and fibrotic tissues; however, the cytoskeleton has not been adequately explored as a therapeutic target in inflammation. Septins are GTP-binding proteins that self-assemble into higher order cytoskeletal structures. The septin cytoskeleton exhibits a number of critical cellular functions, including regulation of cell shape and polarity, cytokinesis, cell migration, vesicle trafficking, and receptor signaling. Surprisingly, little is known about the role of the septin cytoskeleton in inflammation. This article reviews emerging evidence implicating different septins in the regulation of host-pathogen interactions, immune cell functions, and tissue fibrosis. Targeting of the septin cytoskeleton as a potential future therapeutic intervention in human inflammatory and fibrotic diseases is also discussed.

Anillin is an emerging regulator of tumorigenesis, acting as a cortical cytoskeletal scaffold and a nuclear modulator of cancer cell differentiation.

Remodeling of the intracellular cytoskeleton plays a key role in accelerating tumor growth and metastasis. Targeting different cytoskeletal elements is important for existing and future anticancer therapies. Anillin is a unique scaffolding protein that interacts with major cytoskeletal structures, e.g., actin filaments, microtubules and septin polymers. A well-studied function of this scaffolding protein is the regulation of cytokinesis at the completion of cell division. Emerging evidence suggest that anillin has other important activities in non-dividing cells, including control of intercellular adhesions and cell motility. Anillin is markedly overexpressed in different solid cancers and its high expression is commonly associated with poor prognosis of patient survival. This review article summarizes rapidly accumulating evidence that implicates anillin in the regulation of tumor growth and metastasis. We focus on molecular and cellular mechanisms of anillin-dependent tumorigenesis that include both canonical control of cytokinesis and novel poorly understood functions as a nuclear regulator of the transcriptional reprogramming and phenotypic plasticity of cancer cells.

Long-term monitoring of the development and extinction of IgA and IgG responses to SARS-CoV-2 infection.

Despite the great interest of the scientific community in the behavior of the human body after contact with the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), long-term (more than 6 months) monitoring of the immunological status of patients with coronavirus disease 2019 (COVID-19) having varying severity degrees and of the people with a low SARS-CoV-2 viral load is practically absent. The aim of this study is a 9-month monitoring of SARS-CoV-2 infection immune response development and extinction using quantitative assessment of IgA and IgG levels in the blood of healthy donors living in the context of the coronavirus pandemic and of the patients who have undergone COVID-19. The project involved 180 volunteers, of whom 51 persons (28.33%) fell ill with COVID-19 during the observation period. All people who underwent COVID-19 developed a stable humoral immune response but their individual immune status had a number of features. Approximately 39.22% (20 of 51 people) of project participants diagnosed with COVID-19 showed an unusual change in plasma anti-SARS-CoV-2 IgA levels. Relatively high levels of IgA (ratio ~ 3) after recovery persisted for a long time (more than 6 months). In one-third (17 of 51 people) of patients with COVID-19, the IgA level exceeded the IgG level. IgA antibodies appeared earlier and showed a stronger and more robust response to the SARS-CoV-2 virus than IgG. Increased levels of anti-SARS-CoV-2 IgA (ratio from 0.8 to 2.36) throughout the observation period were recorded in 28 of 180 project participants (15.56%) of whom only one person fell ill with COVID-19.

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments.

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.

Anillin regulates breast cancer cell migration, growth, and metastasis by non-canonical mechanisms involving control of cell stemness and differentiation.

BACKGROUND: Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis. METHODS: CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice. RESULTS: Loss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells. CONCLUSION: Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

Thermally stabilized operating mode of an erbium-ytterbium laser.

We propose a theoretical method of pumping optimization for the Er-Yb laser based on the concept of a self-cooling laser. The pumping optimization realizes the anti-Stokes fluorescence cooling and excitation transfer by the Yb ions simultaneously. In this case, the Yb ions become the sources of cooling while the Er ions remain the heating sources. With a certain ratio between the cooling and heating sources, the operating temperature of the laser medium can be stabilized. We simulate the pumping process for the parameters of the Er, Yb:YAG system to demonstrate the possibility of getting a thermally stabilized operating mode of the laser for the ion ratios in the range of 40 to 60 Yb ions to one Er ion. The simulations show that the self-cooled laser medium can be implemented for the laser intensities of kW/cm2 in the cavity.

Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. RSV is known to infect epithelial cells and increase the permeability of model airway epithelial monolayers in vitro. We hypothesized that RSV infection also induces airway barrier dysfunction in vivo. C57BL/6 mice were intranasally inoculated with RSV, and on day 4 post-inoculation were examined for viral replication, lung inflammation, and barrier integrity as well as the structure and molecular composition of epithelial junctions. In parallel, primary mouse tracheal epithelial cells (mTEC) were cultured for in vitro studies. RSV-infected mice lost weight and showed significant peribronchial inflammation compared with noninfected controls and UV-inactivated RSV-inoculated animals. RSV infection increased the permeability of the airway epithelial barrier and altered the molecular composition of epithelial tight junctions. The observed RSV-induced barrier disruption was accompanied by decreased expression of several tight-junction proteins and accumulation of cleaved extracellular fragments of E-cadherin in bronchoalveolar lavage and mTEC supernatants. Similarly, in vitro RSV infection of mTEC monolayers resulted in enhanced permeability and disruption of tight-junction structure. Furthermore, incubation of mTEC monolayers with a recombinant fragment of E-cadherin caused tight-junction disassembly. Taken together, these data indicate that RSV infection leads to airway barrier dysfunction in vivo, mediated by either decreased expression or cleavage of junctional proteins. Our observations provide further insights into the pathophysiology of RSV infection and provide a rationale for development of barrier-protecting agents to alleviate the pathogenic effects of RSV infection.

The OncoPPi Portal: an integrative resource to explore and prioritize protein-protein interactions for cancer target discovery.

Motivation: As cancer genomics initiatives move toward comprehensive identification of genetic alterations in cancer, attention is now turning to understanding how interactions among these genes lead to the acquisition of tumor hallmarks. Emerging pharmacological and clinical data suggest a highly promising role of cancer-specific protein-protein interactions (PPIs) as druggable cancer targets. However, large-scale experimental identification of cancer-related PPIs remains challenging, and currently available resources to explore oncogenic PPI networks are limited. Results: Recently, we have developed a PPI high-throughput screening platform to detect PPIs between cancer-associated proteins in the context of cancer cells. Here, we present the OncoPPi Portal, an interactive web resource that allows investigators to access, manipulate and interpret a high-quality cancer-focused network of PPIs experimentally detected in cancer cell lines. To facilitate prioritization of PPIs for further biological studies, this resource combines network connectivity analysis, mutual exclusivity analysis of genomic alterations, cellular co-localization of interacting proteins and domain-domain interactions. Estimates of PPI essentiality allow users to evaluate the functional impact of PPI disruption on cancer cell proliferation. Furthermore, connecting the OncoPPi network with the approved drugs and compounds in clinical trials enables discovery of new tumor dependencies to inform strategies to interrogate undruggable targets like tumor suppressors. The OncoPPi Portal serves as a resource for the cancer research community to facilitate discovery of cancer targets and therapeutic development. Availability and implementation: The OncoPPi Portal is available at http://oncoppi.emory.edu. Contact: andrey.ivanov@emory.edu or hfu@emory.edu.

Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms.

The intestinal epithelium forms a key protective barrier that separates internal organs from the harmful environment of the gut lumen. Increased permeability of the gut barrier is a common manifestation of different inflammatory disorders contributing to the severity of disease. Barrier permeability is controlled by epithelial adherens junctions and tight junctions. Junctional assembly and integrity depend on fundamental homeostatic processes such as cell differentiation, rearrangements of the cytoskeleton, and vesicle trafficking. Alterations of intestinal epithelial homeostasis during mucosal inflammation may impair structure and remodeling of apical junctions, resulting in increased permeability of the gut barrier. In this review, we summarize recent advances in our understanding of how altered epithelial homeostasis affects the structure and function of adherens junctions and tight junctions in the inflamed gut. Specifically, we focus on the transcription reprogramming of the cell, alterations in the actin cytoskeleton, and junctional endocytosis and exocytosis. We pay special attention to knockout mouse model studies and discuss the relevance of these mechanisms to human gastrointestinal disorders.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo.

Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation.

The actin cytoskeleton is a crucial regulator of the intestinal mucosal barrier, controlling the assembly and function of epithelial adherens and tight junctions (AJs and TJs). Junction-associated actin filaments are dynamic structures that undergo constant turnover. Members of the actin-depolymerizing factor (ADF) and cofilin protein family play key roles in actin dynamics by mediating filament severing and polymerization. We examined the roles of ADF and cofilin-1 in regulating the structure and functions of AJs and TJs in the intestinal epithelium. Knockdown of either ADF or cofilin-1 by RNA interference increased the paracellular permeability of human colonic epithelial cell monolayers to small ions. Additionally, cofilin-1, but not ADF, depletion increased epithelial permeability to large molecules. Loss of either ADF or cofilin-1 did not affect the steady-state morphology of AJs and TJs but attenuated de novo junctional assembly. The observed defects in AJ and TJ formation were accompanied by delayed assembly of the perijunctional filamentous actin belt. A total loss of ADF expression in mice did not result in a defective mucosal barrier or in spontaneous gut inflammation. However, ADF-null mice demonstrated increased intestinal permeability and exaggerated inflammation during dextran sodium sulfate-induced colitis. Our findings demonstrate novel roles for ADF and cofilin-1 in regulating the remodeling and permeability of epithelial junctions, as well as the role of ADF in limiting the severity of intestinal inflammation.

Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells.

AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner.

F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells.

Tight junctions (TJ) and adherens junctions (AJ) are key morphological features of differentiated epithelial cells that regulate the integrity and permeability of tissue barriers. Structure and remodeling of epithelial junctions depends on their association with the underlying actomyosin cytoskeleton. Anillin is a unique scaffolding protein interacting with different cytoskeletal components, including actin filaments and myosin motors. Its role in the regulation of mammalian epithelial junctions remains unexplored. Downregulation of anillin expression in human prostate, colonic, and lung epithelial cells triggered AJ and TJ disassembly without altering the expression of junctional proteins. This junctional disassembly was accompanied by dramatic disorganization of the perijunctional actomyosin belt; while the general architecture of the actin cytoskeleton, and activation status of non-muscle myosin II, remained unchanged. Furthermore, loss of anillin disrupted the adducin-spectrin membrane skeleton at the areas of cell-cell contact, selectively decreased γ-adducin expression, and induced cytoplasmic aggregation of αII-spectrin. Anillin knockdown activated c-Jun N-terminal kinase (JNK), and JNK inhibition restored AJ and TJ integrity and cytoskeletal organization in anillin-depleted cells. These findings suggest a novel role for anillin in regulating intercellular adhesion in model human epithelia by mechanisms involving the suppression of JNK activity and controlling the assembly of the perijunctional cytoskeleton.