Biology subjects

Furuse, M.

Publications and source records attributed to Furuse, M..

12 recordsLinked to original sources

bioRxiv2026

Low-salt diet induces claudin-3 expression and drives adaptive changes in collecting duct of claudin-3-deficient mice

BackgroundRenal sodium reabsorption occurs via both transcellular and paracellular pathways. Tight junction proteins play a key role in mediating paracellular transport. The collecting duct (CD) is critical for the fine-tuning of Na+ balance and is sensitive to changes in dietary salt intake. A low-sodium diet, which increases endogenous aldosterone secretion, stimulates transcellular sodium transport via epithelial Na+ channels (ENaC) and Na,K-ATPase. We hypothesized that a low-sodium diet also modulates paracellular Na+ permeability by regulating the expression or function of claudin-3, a major tight junction protein in the CD, in order to limit the back-leak of reabsorbed sodium and preserve sodium balance. MethodsWe used in vivo mouse models and cultured mouse CD principal cells (mCCDcl1) to assess aldosterones effects on tight junction proteins. In mCCDcl1 cells, aldosterone-induced changes in claudin-3 expression and localization were evaluated via Western blotting and immunofluorescence, and Ussing chamber assays were used to assess paracellular Na+ and Cl- permeability after modulating claudin-3 expression. Wild-type and claudin-3 knockout mice were fed low (0.01%) or normal (0.18%) sodium diets for seven days. In subsets of low sodium diet mice, spironolactone (a mineralocorticoid receptor antagonist) was administered. ResultsIn mice, a low-sodium diet upregulates renal claudin-3 expression. Concordantly, in vitro studies using mCCDcl1 cells showed that aldosterone treatment increased claudin-3 protein levels and promoted its localization to the lateral membrane. Functional analyses demonstrated that claudin-3 overexpression reduced paracellular permeability to both Na+ and Cl-, while claudin-3 silencing increased it. Claudin-3 knockout mice subjected to a low-sodium diet exhibited compensatory upregulation of the - and {gamma}-subunits of ENaC, alongside increased expression of claudin-4, claudin-8, and claudin-10. This highlights an adaptive response that maintains sodium homeostasis in the absence of claudin-3. Importantly, this compensatory mechanism persists even under spironolactone treatment, suggesting that the adaptation of claudin-3-deficient mice occurs independently of mineralocorticoid receptor activation. ConclusionsOur findings demonstrate that aldosterone enhances claudin-3 expression, reinforcing the paracellular barrier to Na+ and complementing its classical role in transcellular Na+ transport. Under low-sodium conditions, claudin-3-deficient mice adapt through complementary mechanisms aimed at increasing sodium reabsorption via ENaC activation and upregulation of claudin-4 and claudin-8, both barrier-forming claudins that restrict paracellular sodium leakage in the CD. This is associated with increased claudin-10 abundance in the thick ascending limb of Henle, a pore-forming claudin that facilitates paracellular sodium permeability. This study advances our understanding of the complex control of renal sodium handling, revealing adaptive mechanisms in response to low-salt diet and claudin-3 deficiency.

physiology↗

bioRxiv2025

Internal desynchrony of the circadian clock system in middle-aged mice under social jet lag-like conditions

Social jet lag (SJL) refers to the discrepancy in sleep patterns between weekdays and weekends, leading to a misalignment between the internal clock and social time. In this study, we investigated the effects of weekly shifts in light-dark (LD) conditions: two days per week with 6-h delayed LD cycles (simulating Saturday and Sunday), followed by a 6-h advance on Monday. Core body temperature rhythms rapidly entrained to the delayed LD cycles on weekends, and these delayed rhythms persisted even after the LD cycle was advanced on Monday. In contrast, plasma corticosterone rhythms on Mondays were aligned with the LD cycle but exhibited reduced amplitude. In the livers of SJL mice on Monday, the expression rhythms of Per1, Per2, and Hsp70 were delayed by 3-5 h compared to that in the controls, whereas Rev-erb expression rhythms remained comparable to those of the controls. The expression of lipid and glucose metabolism-related genes in the liver showed either delayed rhythms or no significant changes. To determine whether the dissociation of gene expression rhythms resulted from gene-specific responses to circadian body temperature and hormonal signals, we conducted ex vivo culture experiments using mouse liver slices. High-temperature stimulation induced Per2 and Hsp70 expression, while dexamethasone induced Per1 expression. High temperature and dexamethasone affected distinct sets of metabolic genes, whereas insulin induced only minor changes. Moreover, these responses were strongly influenced by the age and light exposure of the mice. We also examined the effect of weekly housing by providing environmental enrichment (EE), which had minimal impact on circadian parameters but promoted anti-aging effects on bone density and behavior. Overall, our findings indicate that weekly shifts in LD cycles induce internal desynchronization within the hepatic molecular clock and metabolic pathways by uncoupling core body temperature rhythms, hormonal rhythms, and gene-specific responses to stimuli.

physiology↗

bioRxiv2025

Competitive elimination of ZO-1/ZO-2-deficient cells regulates epithelial barrier homeostasis

Epithelia cover the body and form a barrier to segregate the internal body from the external environment. Epithelial tissues contact the external environment and are exposed to various stresses that are potentially deleterious for the epithelial barrier1. However, how focal epithelial barrier defects are detected and repaired to maintain epithelial barrier homeostasis remains poorly understood. We co-cultured ZO-1/ZO-2 double-knockout (DKO) MDCK II cells, which lack tight junctions, with wild-type MDCK II cells, to understand how epithelial cells respond to focal epithelial barrier defects. When co-cultured with wild-type cells, ZO-1/ZO-2 DKO cells were selectively eliminated by induction of apoptosis. The elimination depended on a purse-string-like contraction of supracellular actomyosin cables formed at the clone boundary, regulated by ROCK. Furthermore, Hippo signaling and adherens junctions in the surrounding wild-type cells were required to eliminate ZO-1/ZO-2 DKO cells. These results demonstrate that selective elimination of ZO-1/ZO-2-deficient cells by cell competition regulates epithelial barrier homeostasis. In briefOtani et al. reveal that epithelial barrier homeostasis is regulated by cell competition-mediated elimination of ZO-1/ZO-2-deficient cells. Supracellular actomyosin cables form at the clone boundary and constrict in a purse-string-like manner to eliminate the loser cells. Mechanosensing in the winning cells is required to eliminate the loser cells. HighlightsO_LIZO-1/ZO-2-deficient cells are eliminated by cell competition C_LIO_LIActomyosin cables form in the winning cells at the clone boundary C_LIO_LIA purse-string-like contraction of actomyosin cables compresses the losing cells C_LIO_LIMechanosensing in the winning cells is important to eliminate the loser cells C_LI

cell biology↗

bioRxiv2024

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo, we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

cell biology↗

bioRxiv2024

ZO-1 shuttles between tight junctions and podosomes by riding ERK activation waves during collective cell migration

Collective cell migration is essential in various physiological processes, including embryonic development, wound healing, and cancer metastasis. However, the mechanisms by which individual cells achieve coordinated movement remain elusive. Here, we demonstrate that zonula occludens-1 (ZO-1), a scaffolding protein of tight junctions (TJs), dynamically translocates to form cell-to- extracellular matrix (ECM) adhesion complexes, podosomes, at the basal cell surface during migration. The translocation of ZO-1 is regulated by extracellular signal-regulated kinase (ERK) activity and its interaction with F-actin. ZO-1 at podosomes contributes to traction force generation and cell invasion, while ZO-1 at TJs mediates intercellular propagation of ERK activation, facilitating collective cell migration. These findings elucidate the versatile roles of ZO-1 in cell adhesion and movement, providing insights into the mechanisms coordinating the discrete movements of individual cells into a cohesive and directed collective cell migration.

cell biology↗

bioRxiv2024

Claudin-11 regulates immunological barrier formation and spermatogonial proliferation through stem cell factor

Tight junctions (TJs) between adjacent Sertoli cells are believed to form immunological barriers that protect spermatogenic cells expressing autoantigens from autoimmune responses. However, there is no direct evidence that Sertoli cell TJs (SCTJs) do indeed form immunological barriers. Here, we analyzed male mice lacking claudin-11 (Cldn11), which encodes a SCTJ component, and found autoantibodies against antigens of spermatocytes/spermatids in their sera. Defective spermatogenesis was not restored in Cldn11-deficient mice on a genetic background mimicking a severely impaired adaptive immune system. This suggests that defective spermatogenesis is not caused by autoimmune responses against spermatogenic cells. Further analyses showed that Cldn11 knockout impaired Sertoli cell polarization, localization of stem cell factor (SCF) (a key molecule for maintaining differentiating spermatogonia) to the basal compartment of seminiferous tubules, and also proliferation of differentiating spermatogonia. We propose that CLDN11 creates a microenvironment for SCF-mediated spermatogonial proliferation at the basal compartment via Sertoli cell polarization.

developmental biology↗

bioRxiv2023

Mechanical transapical coupling of endometrial epithelial cells during implantation

Epithelial cells lining the lumen of organs have an apical-basal polarity, and mechanical cell contacts are located in the lateral and basal regions. During early pregnancy in rodents, the luminal space of the uterus is closed, and opposing epithelial cells attach to each other at apical-apical surfaces. Here, we show that cells are mechanically coupled at the apical cell-cell contacts. The apical plasma membrane was intricately intertwined between opposing cells. Extracellular matrix and tight junction molecules were localized at apical contacts. Claudin inhibition resulted in impaired luminal closure, suggesting a functional requirement of claudins for the establishment of transapical coupling. The present results demonstrate a mechanical cell-cell interaction that occurs between apical-apical surfaces in addition to lateral and basal cell junctions. One Sentence SummariesTransapical coupling of epithelial cells mediated by extracellular matrix and tight junction molecules

cell biology↗

bioRxiv2023

Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions undergo spontaneous fracture when claudin/JAM-A-deficient cells are exposed to mechanical stress. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells. SummaryTight junction membrane proteins claudins, JAM, and CAR coordinately regulate the nanometer-scale organization of ZO-1 molecules, and are required for the mechanical resistance of apical junctions in epithelial cells.

cell biology↗

bioRxiv2022

Reciprocal regulation between cell mechanics and ZO-1 guides tight junction assembly and epithelial morphogenesis

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 regulates cytoskeletal tension at cell-matrix and cell-cell interfaces in an ECM stiffness-regulated manner, possibly via differential organisation of the actin cytoskeleton. ZO-1 depletion inhibited junction assembly and disrupted morphogenesis in an ECM stiffness-dependent manner. Both processes were rescued by inhibition of cell contractility. Although ZO-1-deficient cells could assemble functional barriers at low tension, their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that tension-independent roles of ZO-1 control proper junction organisation.

cell biology↗

bioRxiv2020

A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal and renal system in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. Here, we report a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region having 13-amino acids, and a cytoplasmic region with three repeats of the Tyr-Thr-Pro-Ala motif. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut. Summary statementDepletion of hoka, a gene encoding a novel septate junction protein, from the Drosophila midgut results in the disruption of septate junctions, intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors.

cell biology↗

bioRxiv2020

Angulin-1 seals tricellular contacts independently of tricellulin and claudins

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The COOH-terminus of angulin-1 bound to the TJ scaffold protein ZO-1 and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

cell biology↗

bioRxiv2019

Septate junctions regulate gut homeostasis through regulation of stem cell proliferation and enterocyte behavior in Drosophila

Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route of epithelial cells in the Drosophila midgut. We previously identified three sSJ-associated membrane proteins, Ssk, Mesh, and Tsp2A, and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. Here, we investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ-proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAP kinase and Jak-Stat pathways in stem cells. Loss of cytokines Unpaired2 and Unpaired3, which are involved in Jak-Stat pathway activation, suppressed the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut.\n\nSummary statementDepletion of smooth septate junction-associated proteins from enterocytes in the Drosophila adult midgut results in intestinal hypertrophy accompanied by accumulation of morphologically aberrant enterocytes and increased stem cell proliferation.

cell biology↗