Search

SummaryEpithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP)1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ)3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening4–10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself11,12, or proximate to the junctions13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

Toll receptors are key determinants of planar polarity during Drosophila gastrulation. Two papers in the current issue of Developmental Cell now identify key features of their downstream signaling that allow cell symmetry to be broken by apparently non-polarized Toll receptors.

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

SUMMARYAdherens junctions allow cell contact to inhibit epithelial migration. But a long-standing puzzle is how locomotion is downregulated when E-cadherin adhesions form at surfaces perpendicular, but not those parallel, to the direction of migration. We now show that this arises from coupling between E-cadherin adhesions and the retrograde cortical flows of leader cells in migrating epithelia. At interfaces perpendicular to the direction of motion, such flows are antiparallel, which generates a tensile signal that induces the actin-binding domain of α-catenin to promote lateral growth of nascent adhesions and inhibit the lamellipodial activity necessary for migration. At interfaces parallel to the direction of motion, by contrast, cortical flows are aligned and no such mechanical inhibition takes place. Therefore, α-catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and trigger the first signal for contact to inhibit locomotion.

Epithelia migrate as physically coherent populations of cells. Earlier studies revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also reflects an increase in RhoA-mediated junctional contractility. We find that active RhoA levels were stimulated by p114 RhoGEF at the junctions between migrating MCF-7 monolayers, and this is accompanied by increased levels of actomyosin and mechanical tension. By applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin during epithelial migration. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion against the mechanical stresses of migration.

Epithelia migrate as physically coherent populations of cells. Previous studies have revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell–cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also increases due to an increase in RhoA-mediated junctional contractility. We found that active RhoA levels were stimulated by p114 RhoGEF (also known as ARHGEF18) at the junctions between migrating MCF-7 monolayers, and this was accompanied by increased levels of actomyosin and mechanical tension. Applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin (CDH1) during epithelial migration and promoted orderly collective movement. We suggest that stabilization of E-cadherin by RhoA serves to increase cell–cell adhesion to protect against the mechanical stresses of migration. This article has an associated First Person interview with the first author of the paper.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

Cell injury poses a substantial challenge for epithelia homeostasis. Several cellular processes preserve epithelial barriers in response to apoptosis, but less is known about other forms of cell death, such as pyroptosis. Here we use an inducible caspase-1 system to analyze how colon epithelial monolayers respond to pyroptosis. We confirm that sporadic pyroptotic cells are physically eliminated from confluent monolayers by apical extrusion. This is accompanied by a transient defect in barrier function at the site of the pyroptotic cells. By visualizing cell shape changes and traction patterns in combination with cytoskeletal inhibitors, we show that rapid lamellipodial responses in the neighbor cells are responsible for correcting the leakage and resealing the barrier. Cell contractility is not required for this resealing response, in contrast to the response to apoptosis. Therefore, pyroptosis elicits a distinct homeostatic response from the epithelium that is driven by the stimulation of lamellipodia in neighbor cells.

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P₂) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P₂, or disabling the interaction between FMNL2 and PtdIns(4,5)P₂. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton. Refereed/Peer-reviewed

Mechanical tension governs epithelial morphogenesis and homeostasis, but its regulation remains poorly understood. Tension is commonly contractile, arising when the actomyosin cortices of cells are mechanically coupled together by cadherin adhesion. Here we report that caveolae control levels of epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) in the surrounding epithelium, but not in the oncogene-expressing cells, blocked extrusion leading to the retention and proliferation of transformed cells within the monolayer. Tensile stress was aberrantly elevated in CAV1-depleted monolayers due to elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) causing increased recruitment of the formin, FMNL2. Oncogenic extrusion was restored to CAV1-deficient monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, by controlling lipid signalling to the actin cytoskeleton, caveolae regulate mechanical tension for epithelial homeostasis.

Wnt/β-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multi-meric Wnt–FZD assemblies via intramembrane interactions. Subsequently, these Wnt–FZD–TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions., This work is part of the Oncode Institute which is partly financed by the Dutch Cancer Society. This work was supported by European Research Council Starting Grant 242958 (to M.M.M.), European Union Grant FP7 Marie Curie ITN 608180 “WntsApp” (to M.M.M.), and the Netherlands Organization for Scientific Research NWO VICI Grant 91815604 and ECHO Grant 711.013.012 (to M.M.M.). T.Y.L. and A.J.R.H. are supported by large-scale proteomics facility Proteins@Work Project 184.032.201 embedded in the Netherlands Proteomics Centre and supported by the Netherlands Organization for Scientific Research. They were additionally supported through European Union Horizon 2020 Program FET-OPEN Project MSmed, Project 686547. F.X.P. was supported by Grants SAF2014-53320R and SAF2017-88390R from the Spanish Government.