Hydrostatic pressure drives sprouting angiogenesis via adherens junction remodelling and YAP signalling.

Endothelial cell physiology is governed by its unique microenvironment at the interface between blood and tissue. A major contributor to the endothelial biophysical environment is blood hydrostatic pressure, which in mechanical terms applies isotropic compressive stress on the cells. While other mechanical factors, such as shear stress and circumferential stretch, have been extensively studied, little is known about the role of hydrostatic pressure in the regulation of endothelial cell behavior. Here we show that hydrostatic pressure triggers partial and transient endothelial-to-mesenchymal transition in endothelial monolayers of different vascular beds. Values mimicking microvascular pressure environments promote proliferative and migratory behavior and impair barrier properties that are characteristic of a mesenchymal transition, resulting in increased sprouting angiogenesis in 3D organotypic model systems ex vivo and in vitro. Mechanistically, this response is linked to differential cadherin expression at the adherens junctions, and to an increased YAP expression, nuclear localization, and transcriptional activity. Inhibition of YAP transcriptional activity prevents pressure-induced sprouting angiogenesis. Together, this work establishes hydrostatic pressure as a key modulator of endothelial homeostasis and as a crucial component of the endothelial mechanical niche.

Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Mechanical factors influence ß-catenin localization and barrier properties.

Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. ß-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that ß-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of ß-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, ß-catenin's nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate ß-catenin localization, and influence endothelial barrier properties. Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of ß-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.

Patterning in stratified epithelia depends on cell-cell adhesion.

Epithelia consist of proliferating and differentiating cells that often display patterned arrangements. However, the mechanism regulating these spatial arrangements remains unclear. Here, we show that cell-cell adhesion dictates multicellular patterning in stratified epithelia. When cultured keratinocytes, a type of epithelial cell in the skin, are subjected to starvation, they spontaneously develop a pattern characterized by areas of high and low cell density. Pharmacological and knockout experiments show that adherens junctions are essential for patterning, whereas the mathematical model that only considers local cell-cell adhesion as a source of attractive interactions can form regions with high/low cell density. This phenomenon, called cell-cell adhesion-induced patterning (CAIP), influences cell differentiation and proliferation through Yes-associated protein modulation. Starvation, which induces CAIP, enhances the stratification of the epithelia. These findings highlight the intrinsic self-organizing property of epithelial cells.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension.

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

Unusual cell-cell cooperative mechanical activity elucidates the process of tissue regeneration.

We have studied wound contraction in three model wounds in animals: excised skin (guinea pig), transected peripheral nerve (rat) and the excised conjunctiva (rabbit). Wound contraction is driven by myofibroblasts bound together by adherens junctions (AJ) that confer cooperative activity to myofibroblasts during wound contraction and synthesis of scar. Grafting with the dermis regeneration template (DRT) cancels cell cooperativity by abolishing AJ connections in myofibroblasts, while also cancelling wound contraction, preventing synthesis of scar and inducing regeneration of excised tissues. The observed definitive prevention of scar synthesis suggests the exploration of DRT scaffolds to regenerate tissues in several other organs and to prevent fibrosis in humans.

A ß-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific ß-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of ß-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of ß-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis.

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Contractile myosin and cell adhesion work together to induce tissue shape changes, but how they are patterned to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While it has been shown that a multicellular gradient of myosin contractility determines folding shape, the impact of multicellular patterning of adherens junction levels on tissue folding is unknown. We identified a novel Drosophila gene moat essential for differential apical constriction and folding behaviors across the ventral epithelium which contains both folding ventral furrow and nonfolding ectodermal anterior midgut (ectoAMG). We show that Moat functions to downregulate polarity-dependent adherens junctions through inhibiting cortical clustering of Bazooka/Par3 proteins. Such downregulation of polarity-dependent junctions is critical for establishing a myosin-dependent pattern of adherens junctions, which in turn mediates differential apical constriction in the ventral epithelium. In moat mutants, abnormally high levels of polarity-dependent junctions promote ectopic apical constriction in cells with low-level contractile myosin, resulting in expansion of infolding from ventral furrow to ectoAMG, and flattening of ventral furrow constriction gradient. Our results demonstrate that tissue-scale distribution of adhesion levels patterns apical constriction and establishes morphogenetic boundaries.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of ß-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-ß-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

HIF2α-dependent Dock4/Rac1-signaling regulates formation of adherens junctions and cell polarity in normoxia.

Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia response accumulating evidence suggests that under certain conditions HIFs, particularly HIF2, may function also under normoxic conditions. Here we report that HIF2α functions under normoxic conditions in kidney epithelial cells to regulate formation of adherens junctions. HIF2α expression was required to induce Dock4/Rac1/Pak1-signaling mediating stability and compaction of E-cadherin at nascent adherens junctions. Impaired adherens junction formation in HIF2α- or Dock4-deficient cells led to aberrant cyst morphogenesis in 3D kidney epithelial cell cultures. Taken together, we show that HIF2α functions in normoxia to regulate epithelial morphogenesis.

Altering Cell Junctional Tension in Spheroids through E-Cadherin Engagement Modulation.

Cadherin-mediated tension at adherens junctions (AJs) is fundamental for cell-cell adhesion and maintaining epithelial integrity. Despite the importance of manipulating AJs to dissect cell-cell interactions, existing three-dimensional (3D) multicellular models have not adequately addressed the precise manipulation of these junctions. To fill this gap, we introduce E-cadherin-modified tension gauge tethers (TGTs) at the junctions within spheroids. The system enables both quantification and modulation of junctional tension with specific DNA triggers. Using rupture-induced fluorescence, we successfully measure mechanical forces in 3D spheroids. Furthermore, mechanically strong TGTs can maintain normal E-cadherin-mediated adhesion. Employing toehold-mediated strand displacement allowed us to disrupt E-cadherin-specific cell-cell adhesion, consequently altering intracellular tension within the spheroids. Our methodology offers a robust and precise way to manipulate cell-cell adhesion and intracellular mechanics in spheroid models.

MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins.

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

Neurotensin modulates ovarian vascular permeability via adherens junctions.

Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes in vitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied in vitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA-Seq) of mOMECs revealed high mRNA expression of the permeability-regulating adherens junction proteins N-cadherin (CDH2) and K-cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell-cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE-cadherin (CDH5) as determined by RNA-Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation-critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.

Pten, PI3K, and PtdIns(3,4,5)P3 dynamics control pulsatile actin branching in Drosophila retina morphogenesis.

Epithelial remodeling of the Drosophila retina depends on the pulsatile contraction and expansion of apical contacts between the cells that form its hexagonal lattice. Phosphoinositide PI(3,4,5)P3 (PIP3) accumulates around tricellular adherens junctions (tAJs) during contact expansion and dissipates during contraction, but with unknown function. Here, we found that manipulations of Pten or PI3-kinase (PI3K) that either decreased or increased PIP3 resulted in shortened contacts and a disordered lattice, indicating a requirement for PIP3 dynamics and turnover. These phenotypes are caused by a loss of branched actin, resulting from impaired activity of the Rac1 Rho GTPase and the WAVE regulatory complex (WRC). We additionally found that during contact expansion, PI3K moves into tAJs to promote the cyclical increase of PIP3 in a spatially and temporally precise manner. Thus, dynamic control of PIP3 by Pten and PI3K governs the protrusive phase of junctional remodeling, which is essential for planar epithelial morphogenesis.

Repeat DNA methylation is modulated by adherens junction signaling.

Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.