Adherens junctions as molecular regulators of emergent tissue mechanics.

Tissue and organ development during embryogenesis relies on the collective and coordinated action of many cells. Recent studies have revealed that tissue material properties, including transitions between fluid and solid tissue states, are controlled in space and time to shape embryonic structures and regulate cell behaviours. Although the collective cellular flows that sculpt tissues are guided by tissue-level physical changes, these ultimately emerge from cellular-level and subcellular-level molecular mechanisms. Adherens junctions are key subcellular structures, built from clusters of classical cadherin receptors. They mediate physical interactions between cells and connect biochemical signalling to the physical characteristics of cell contacts, hence playing a fundamental role in tissue morphogenesis. In this Review, we take advantage of the results of recent, quantitative measurements of tissue mechanics to relate the molecular and cellular characteristics of adherens junctions, including adhesion strength, tension and dynamics, to the emergent physical state of embryonic tissues. We focus on systems in which cell-cell interactions are the primary contributor to morphogenesis, without significant contribution from cell-matrix interactions. We suggest that emergent tissue mechanics is an important direction for future research, bridging cell biology, developmental biology and mechanobiology to provide a holistic understanding of morphogenesis in health and disease.

c-Src induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Organization and dynamics of the cortical complexes controlling insulin secretion in ß-cells.

Insulin secretion in pancreatic ß-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 the non-neuronal proteins LL5ß (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5ß or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have 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.

Enhanced RhoA signalling stabilizes E-cadherin in migrating epithelial monolayers.

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.

TMEM59 potentiates Wnt signaling by promoting signalosome formation.

Wnt/ß-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 multimeric 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.

Control of apico-basal epithelial polarity by the microtubule minus-end-binding protein CAMSAP3 and spectraplakin ACF7.

The microtubule cytoskeleton regulates cell polarity by spatially organizing membrane trafficking and signaling processes. In epithelial cells, microtubules form parallel arrays aligned along the apico-basal axis, and recent work has demonstrated that the members of CAMSAP/Patronin family control apical tethering of microtubule minus ends. Here, we show that in mammalian intestinal epithelial cells, the spectraplakin ACF7 (also known as MACF1) specifically binds to CAMSAP3 and is required for the apical localization of CAMSAP3-decorated microtubule minus ends. Loss of ACF7 but not of CAMSAP3 or its homolog CAMSAP2 affected the formation of polarized epithelial cysts in three-dimensional cultures. In short-term epithelial polarization assays, knockout of CAMSAP3, but not of CAMSAP2, caused microtubule re-organization into a more radial centrosomal array, redistribution of Rab11-positive (also known as Rab11A) endosomes from the apical cell surface to the pericentrosomal region and inhibition of actin brush border formation at the apical side of the cell. We conclude that ACF7 is an important regulator of apico-basal polarity in mammalian intestinal cells and that a radial centrosome-centered microtubule organization can act as an inhibitor of epithelial polarity.

A Lifeact-EGFP quail for studying actin dynamics in vivo.

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.

Epithelial cell spreading assay on E-cadherin-coated glass or PDMS substrates for microscopy-based analysis of cadherin adhesions.

Adherens junctions (AJs) are multi-protein adhesion structures that couple contractile actomyosin networks of epithelial cells within a tissue. Here, we present an epithelial cell spreading assay on E-cadherin-coated glass or polydimethylsiloxane (PDMS) substrates for detailed microscopy-based analysis of cadherin adhesions. We describe steps for preparation of glass coverslips and PDMS gels, E-cadherin coating, and epithelial cell spreading. Epithelial cells can be seeded on E-cadherin-coated surfaces, thereby mimicking AJ formation in X-Y dimension, making it suitable for microscopy analysis. For complete details on the use and execution of this protocol, please refer to Noordstra et al. (2023).1.

Cadherins and the cortex: A matter of time?

Cell adhesion systems commonly operate in close partnership with the cytoskeleton. Adhesion receptors bind to the cortex and regulate its dynamics, organization and mechanics; conversely, the cytoskeleton influences aspects of adhesion, including strength, stability and ductility. In this review we consider recent advances in elucidating such cooperation, focusing on interactions between classical cadherins and actomyosin. The evidence presents an apparent paradox. Molecular mechanisms of mechanosensation by the cadherin-actin apparatus imply that adhesion strengthens under tension. However, this does not always translate to the broader setting of confluent tissues, where increases in fluctuations of tension can promote intercalation due to the shrinkage of adherens junctions. Emerging evidence suggests that understanding of timescales may be important in resolving this issue, but that further work is needed to understand the role of adhesive strengthening across scales.

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis.

Epithelia 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.

An E-cadherin-actin clutch translates the mechanical force of cortical flow for cell-cell contact to inhibit epithelial cell locomotion.

Adherens junctions (AJs) allow cell contact to inhibit epithelial migration yet also permit epithelia to move as coherent sheets. How, then, do cells identify which contacts will inhibit locomotion? Here, we show that in human epithelial cells this arises from the orientation of cortical flows at AJs. When the leader cells from different migrating sheets make head-on contact with one another, they assemble AJs that couple together oppositely directed cortical flows. This applies a tensile signal to the actin-binding domain (ABD) of α-catenin, which provides a clutch to promote lateral adhesion growth and inhibit the lamellipodial activity necessary for migration. In contrast, AJs found between leader cells in the same migrating sheet have cortical flows aligned in the same direction, 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 signal for contact to inhibit locomotion.

Rapid lamellipodial responses by neighbor cells drive epithelial sealing in response to pyroptotic cell death.

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.

For whom the cell tolls.

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.

Live Imaging of Apoptotic Extrusion and Quantification of Apical Extrusion in Epithelial Cells.

Apoptotic cell death eliminates unhealthy cells and maintains homeostatic cell numbers within tissues. Epithelia, which serve as fundamental tissue barriers for the body, depend on a physical expulsion of dying cells (apoptotic cell extrusion) to remain sealed and intact. Apoptotic cell extrusion has been widely studied over recent years, with researchers using various approaches to induce apoptotic cell death. Unfortunately, the majority of chemical-based approaches for cell death induction rely on sporadically occurring apoptosis and extrusion, making imagining lengthy, often unsuccessful, and difficult to capture in high-quality images because of the frequent frame sampling needed to visualise the key molecular processes that drive extrusion. Here, we present a protocol that describes steps needed for laser-mediated induction of apoptosis in a cell of choice, followed by imaging of apoptotic extrusion in confluent monolayers of epithelial cells. Moreover, we provide the description of a new approach involving the mixing of labelled and unlabelled cells. In particular, this protocol characterises how cells surrounding apoptotic cells behave, with high spatial and temporal resolution. This can be achieved without the optical interference that apoptotic cells cause as they are physically expelled from the monolayer and move out of focus for imaging. Finally, the protocol is accompanied by detailed procedures describing cell preparation for apoptotic extrusion experiments, as well as post-acquisition analysis required to evaluate rates of successful extrusion.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

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.

Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor 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.

Linking cortical microtubule attachment and exocytosis.

Exocytosis is a fundamental cellular process whereby secreted molecules are packaged into vesicles that move along cytoskeletal filaments and fuse with the plasma membrane. To function optimally, cells are strongly dependent on precisely controlled delivery of exocytotic cargo. In mammalian cells, microtubules serve as major tracks for vesicle transport by motor proteins, and thus microtubule organization is important for targeted delivery of secretory carriers. Over the years, multiple microtubule-associated and cortical proteins have been discovered that facilitate the interaction between the microtubule plus ends and the cell cortex. In this review, we focus on mammalian protein complexes that have been shown to participate in both cortical microtubule capture and exocytosis, thereby regulating the spatial organization of secretion. These complexes include microtubule plus-end tracking proteins, scaffolding factors, actin-binding proteins, and components of vesicle docking machinery, which together allow efficient coordination of cargo transport and release.

Mesenchymal Cell Invasion Requires Cooperative Regulation of Persistent Microtubule Growth by SLAIN2 and CLASP1.

Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function.

Molecular Pathway of Microtubule Organization at the Golgi Apparatus.

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes.