Biology subjects

Otani, T.

Publications and source records attributed to Otani, T..

13 recordsLinked to original sources

bioRxiv2026

Wnt signaling and tissue tension reconstitute planar cell polarity in vitro

Planar cell polarity (PCP) aligns cells within the plane of a tissue through asymmetric localization of core PCP proteins. In vivo, PCP arises from integration of biochemical signaling and mechanical inputs, making it challendging to reconstruct in vitro. Here we reconstitute PCP in cultured MDCK epithelial cells using Wnt signaling, collective migration, and Prickle3 overexpression. Loss of Vangl1 disrupts reconstituted PCP, directional collective migration, and propagation of ERK traveling waves. Orthogonal manipulation of signaling and mechanics show that tissue tension contributes to establishing a polarity axis, but not its direction, whereas local Wnt input specifies polarity direction. Together, these inputs generate tissue-wide vectorial polarity. This system provides a tractable framework for dissecting how signaling and mechanics are integrated to organize tissues. TeaserWnt signaling and tissue tension reconstitute planar cell polarity in cultured epithelial cells through core-component interdependence and ERK dynamics.

synthetic biology↗

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

Cell position-based evaluation of mechanical features of cells in multicellular systems

Measurement of mechanical forces of cell-cell interactions is important for studying the emergence of diverse three-dimensional morphologies of multicellular organisms. We previously reported an image-based statistical method for inferring effective pairwise forces of cell-cell interactions (i.e., attractive/repulsive forces), where a cell particle model was fitted to cell tracking data acquired by live imaging. However, because the particle model is a coarse-grained model, it remains unclear how the pairwise forces relates to sub-cellular mechanical components including cell-cell adhesive forces. Here we applied our inference method to cell tracking data generated by vertex models that assumed sub-cellular components. Through this approach, we investigated the relationship between the effective pairwise forces and various sub-cellular components: cell-cell adhesion forces, cell surface tensions, cell-extracellular matrix (ECM) adhesion, traction forces between cells and ECM, cell growth, etc. We found that the cell-cell adhesion forces were attractive, and both the cell surface tensions and cell-ECM adhesive forces were repulsive, etc. These results indicate that sub-cellular mechanical components can contribute to the effective attractive/repulsive forces of cell-cell interactions. This comprehensive analysis provides theoretical bases for linking the pairwise forces to the sub-cellular mechanical components: this showcase is useful for speculating the sub-cellular mechanical components from the information of cell positions, and for interpreting simulation results based on particle models.

biophysics↗

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

Multivalent Afadin Interaction Promotes IDR-Mediated Condensate Formation and Junctional Separation of Epithelial Cells

In epithelial cells, cell-cell adhesion is mediated by the apical junctional complex (AJC), which consists of tight junctions (TJs) and adherens junctions (AJs) aligned from the apical to the basal axis. However, the mechanism of AJC formation on the apical side and the separation of these junctions within AJCs are poorly understood. We found that multivalent interactions of afadin with adhesion molecules and the cytoskeleton lead to condensate formation in an intrinsically disordered region (IDR)-dependent manner, which promotes efficient accumulation in the linear AJ during initial junction formation. Furthermore, we found that afadin and ZO-1 were able to induce different condensate formations in the cell and that these molecules were differentially distributed from each other. These properties of afadin explain how it strictly localizes to AJs in epithelial cells and is involved in regulating the segregation of AJ and TJ within the AJC.

cell 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

Effective mechanical potential of cell-cell interaction in tissues harboring cavity and in cell sheet toward morphogenesis

Measuring mechanical forces of cell-cell interactions is important for studying morphogenesis in multicellular organisms. We previously reported an image-based statistical method for inferring effective mechanical potentials of pairwise cell-cell interactions by fitting cell tracking data with a theoretical model. However, whether this method is applicable to tissues with non-cellular components such as cavities remains elusive. Here we evaluated the applicability of the method to cavity-harboring tissues. Using synthetic data generated by simulations, we found that the effect of expanding cavities was added to the pregiven potentials used in the simulations, resulting in the inferred effective potentials having an additional repulsive component derived from the expanding cavities. Interestingly, simulations by using the effective potentials reproduced the cavity-harboring structures. Then, we applied our method to the mouse blastocysts, and found that the inferred effective potentials can reproduce the cavity-harboring structures. Pairwise potentials with additional repulsive components were also detected in two-dimensional cell sheets, by which curved sheets including tubes and cups were simulated. We conclude that our inference method is applicable to tissues harboring cavities and cell sheets, and the resultant effective potentials are useful to simulate the morphologies.

biophysics↗

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↗

bioRxiv2021

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane (ONM) via its transmembrane domain and also associated with the inner nuclear membrane (INM) via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that overaccumulation of Atg39 causes the NE to protrude towards the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 assembly in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

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

Effective physical properties of cell-cell interaction explain basic structural units of three-dimensional morphogenesis

Mechanical forces are critical for the emergence of diverse three-dimensional morphologies of multicellular systems. However, it remains unclear what kind of mechanical parameters at cellular level substantially contribute to tissue morphologies. This is largely due to technical limitations of live measurements of cellular forces. Here we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, we approximated cells as particles, where mean forces (i.e. effective forces) of pairwise cell-cell interactions are considered. Then, the forces were statistically inferred by fitting the mathematical model to cell tracking data. This method was validated by using synthetic cell tracking data resembling various in vivo situations. Application of our method to the cells in the early embryos of mice and the nematode Caenorhabditis elegans revealed that cell-cell interaction forces can be written as a pairwise potential energy in a manner dependent on cell-cell distances. Importantly, the profiles of the pairwise potentials were quantitatively different among species and embryonic stages, and the quantitative differences correctly described the differences of their morphological features such as spherical vs. distorted cell aggregates, and tightly vs. non-tightly assembled aggregates. We conclude that the effective pairwise potential of cell-cell interactions is a live measurable parameter whose quantitative differences can be a parameter describing three-dimensional tissue morphologies. Author summaryEmergence of diverse three-dimensional morphologies of multicellular organisms is one of the most intriguing phenomena in nature. Due to the complex situations in living systems (e.g. a lot of genes are involved in morphogenesis.), a model for describing the emergent properties of multicellular systems has not been established. To approach this issue, approximation of the complex situations to limited numbers of parameters is required. Here, we searched for mechanical parameters for describing morphologies. We developed a statistical method for inferring mechanical potential energy of cell-cell interactions in three-dimensional tissues; the mechanical potential is an approximation of various mechanical components such as cell-cell adhesive forces, cell surface tensions, etc. Then, we showed that the quantitative differences in the potential is sufficient to reproduce basic three-dimensional morphologies observed during the mouse and C. elegans early embryogenesis, revealing a direct link between cellular level mechanical parameters and three-dimensional morphologies. Our framework provides a noninvasive tool for measuring spatiotemporal cellular forces, which would be useful for studying morphogenesis of larger tissues including organs and their regenerative therapy.

biophysics↗