Biology subjects

Ivanov, A. I.

Publications and source records attributed to Ivanov, A. I..

8 recordsLinked to original sources

bioRxiv2026

SETD3 ACTIN HISTIDINE METHYLTRANSFERASE REGULATES INTESTINAL EPITHELIAL BARRIER INTEGRITY AND MUCOSAL INFLAMMATION

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

cell biology↗

bioRxiv2026

Cdc42 small GTPase is a novel regulator of the fibrogenic activation of human intestinal myofibroblasts

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts. In briefZafar et al. unravel a novel role of Cdc42 small GTPase in fibrogenic activation of human intestinal myofibroblasts. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta} induced expression of contractile cytoskeletal proteins and extracellular matrix proteins in activated myofibroblasts. The mechanisms underlying such profibrotic activity of Cdc42 involve regulation of de novo protein translation via activation of the AKT-mTOR signaling module. HighlightsO_LICdc42 regulates TGF-{beta} dependent activation of human intestinal myofibroblasts C_LIO_LIProfibrotic activity of Cdc42 involves regulation of de novo protein translation C_LIO_LICdc42 regulates myofibroblast activation via AKT-mTOR signaling pathway C_LIO_LITargeting Cdc42-Akt-mTOR signaling may facilitate the development of novel antifibrotic therapies C_LI

cell biology↗

bioRxiv2025

MYOSIN IIA MOTOR REGULATES ATTACHING-EFFACING BACTERIA INTERACTIONS WITH INTESTINAL EPITHELIUM

Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium, colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton.

cell biology↗

bioRxiv2024

The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

Background and AimsIntestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the cytoskeleton is critical for maintaining gut barrier-integrity, the role of the septins- the newest family of cytoskeletal proteins- is unknown. To address this knowledge gap, we evaluate the role of SEPT9- a critical component of the septin-cytoskeleton- in intestinal epithelial cell (IEC) barrier permeability and inflammation. MethodsWe developed SEPT9-NeonGreen knockin mice, inducible intestinal epithelial cell (IEC)-specific SEPT9 knockout (KO) mice, and SEPT9-KO human IEC lines. SEPT9 localization was analyzed using super-resolution microscopy. Barrier-integrity was assessed via transepithelial electrical resistance, FITC-dextran flux, and visualization of tight junction (TJ) and adherens junction (AJ) proteins. Dextran sodium sulfate-induced experimental colitis was evaluated in control and KO mice through measuring cytokine expression, immune cell infiltration, and IEC death. SEPT9 expression was examined in intestinal tissue of IBD patients. ResultsSEPT9 overlapped with TJs and AJs at IEC apical junctions. SEPT9 loss resulted in a leaky epithelial barrier due to mislocalization of junctional proteins. SEPT9 interacted with non-muscle myosin IIC (NMIIC) at the IEC apical-junctional actomyosin belt, and its ablation displaced NMIIC from IEC junctions. Loss of NMIIC also caused barrier disruption. SEPT9 KO mice exhibited increased susceptibility to experimental-colitis. SEPT9 expression was significantly reduced in intestinal mucosa of IBD patients. ConclusionSEPT9 regulates intestinal barrier integrity, supporting TJ and AJ assembly through NMIIC recruitment to the actomyosin belt. SEPT9 safeguards the intestinal mucosa during acute inflammation, and its reduced expression in IBD suggests a loss of this protective function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/629767v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5141dcorg.highwire.dtl.DTLVardef@bae524org.highwire.dtl.DTLVardef@19c8eaorg.highwire.dtl.DTLVardef@d5b218_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions

BackgroundFibroblasts play a key role in stricture formation in Crohns 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. MethodsWe performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, 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. ResultsOur 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 upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. ConclusionA 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 opens potential therapeutic developments.

immunology↗

bioRxiv2021

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. While biochemical mechanisms that regulate 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 levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 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 epithelial 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.

cell biology↗

bioRxiv2020

Spatio-temporal heterogeneity in hippocampal metabolism in control and epilepsy conditions.

The hippocampuss dorsal and ventral parts are involved in different operative circuits, which functions vary in time during the night and day cycle. These functions are altered in epilepsy. Since energy production is tailored to function, we hypothesized that energy production would be space- and time-dependent in the hippocampus and that such organizing principle would be modified in epilepsy. Using metabolic imaging and metabolite sensing ex vivo, we show that the ventral hippocampus favors aerobic glycolysis over oxidative phosphorylation as compared to the dorsal part in the morning in control mice. In the afternoon, aerobic glycolysis is decreased, and oxidative phosphorylation increased. In the dorsal hippocampus, the metabolic activity varies less between these two times but is weaker than in the ventral. Thus, the energy metabolism is different along the dorsoventral axis and changes as a function of time in control mice. In an experimental model of epilepsy, we find a large alteration of such spatio-temporal organization. In addition to a general hypometabolic state, the dorsoventral difference disappears in the morning, when seizure probability is low. In the afternoon, when seizure probability is high, the aerobic glycolysis is enhanced in both parts, the increase being stronger in the ventral area. We suggest that energy metabolism is tailored to the functions performed by brain networks, which vary over time. In pathological conditions, the alterations of these general rules may contribute to network dysfunctions. SIGNIFICANCE STATEMENTThe dorsal part of the hippocampus is involved in spatial learning and memory processes, while the ventral is implicated in motivational and emotional behavior. These functions change during the night and day cycle, and they are altered in epilepsy. Here we show that energy production (glycolysis versus oxidative phosphorylation) varies along the dorsoventral axis in a circadian manner ex vivo in control mice. These rules are altered in experimental epilepsy. Thus, energy production may be tailored to the function performed by hippocampal subdivisions and to the time of the day. Alterations in epilepsy may contribute to seizure generation and cognitive deficits.

neuroscience↗

bioRxiv2019

The GABA-Glutamate supramammillary-dorsal Dentate Gyrus pathway controls theta and gamma oscillations in the DG during paradoxical sleep

Several studies suggest that neurons from the lateral region of the SuM (SuML) innervating the dorsal dentate gyrus (DG) display a dual GABAergic and glutamatergic transmission and are specifically activated during paradoxical (REM) sleep (PS). The objective of the present study is to fully characterize the anatomical, neurochemical and electrophysiological properties of the SuML-DG projection neurons and to determine how they control DG oscillations and neuronal activation during PS and other vigilance states. For this purpose, we combine structural connectivity techniques using neurotropic viral vectors (rabies virus, AAV), neurochemical anatomy (immunohistochemistry, in situ hybridization) and imaging (light, electron and confocal microscopy) with in vitro (patch clamp) and in vivo (LFP, EEG) optogenetic and electrophysiological recordings performed in transgenic VGLUT2-cre male mice. At the cellular level, we show that the SuML-DG neurons co-release GABA and glutamate on dentate granule cells and increase the activity of a subset of DG granule cells. At the network level, we show that activation of the SuML-DG pathway increases theta power and frequency during PS as well as gamma power during PS and waking in the DG. At the behavioral level, we show that the activation of this pathway does not change animal behavior during PS, induces awakening during slow wave sleep and increases motor activity during waking. These results suggest that the SuML-DG pathway is capable of supporting the increase of theta and gamma power in the DG observed during PS and plays an important modulatory role of DG network activity during this state.\n\nSignificant statementAn increase of theta and gamma power in the dentate gyrus (DG) is an hallmark of paradoxical (REM) sleep (PS) and is suggested to promote learning and memory consolidation by synchronizing hippocampal networks and increasing its outputs to cortical targets. However the neuronal networks involved in such control of DG activity during PS are poorly understood. The present study identifies a population of GABA/Glutamate neurons in the lateral supramammllary nucleus (SuML) innervating the DG that could support such control during PS. Indeed, we show that activation of these SuML-DG projections increase theta power and frequency as well as gamma power in the DG specifically during PS and modulate activity of a subset of DG granule cells.

neuroscience↗