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By modelling epithelial cells as active nematic liquid crystals, stresses induced at the sites of topological defects are found to be the primary drivers of extrusion and cell death. A functional role for topological defects in epithelial monolayers Epithelial monolayers remove excess cells by extrusion. Benoit Ladoux and colleagues now report a purely mechanical route to cell extrusion at the site of topological defects within the cell monolayer. By modelling the epithelium as an active nematic liquid crystal, they show that cell extrusion is driven by stresses induced by distortions in cell orientation. Extrusion hotspots were controlled by geometrically inducing defects through microcontact printing of patterned monolayers. The authors also investigated the mechanotransductive effect of stress localization and found that signals related to cell death were induced at these sites of compressive stress. Additionally, tampering with the intercellular adhesion complexes led to a weakening of cell–cell interactions and resulted in an increased number of defects and extrusions. This finding is in line with nematic theory, which predicts that the number of topological defects is inversely related to the orientational elasticity. Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling 1 , oncogenic transformation 2 , 3 and overcrowding of cells 4 , 5 , 6 . Despite the important linkage of cell extrusion to developmental 7 , homeostatic 5 and pathological processes 2 , 8 such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments 9 , 10 in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity 11 , caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell–cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo , with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

α-catulin is identified as a marker to locate functional haematopoiteic stem cells in deep imaging experiments of bone marrow, showing that α-catulin –GFP + c-kit + cells commonly reside in perisinusoidal niches throughout the bone marrow. A marker for bone marrow stem cells The location of haematopoietic stem cells (HSCs) in the bone marrow remains unclear. The absence of a single marker that can identify HSCs, the rarity of HSCs and the limitations in imaging techniques in the bone marrow have prevented the resolution of this question. Sean Morrison and colleagues have now identified α-catulin as marker of functional HSCs that can be used to visualize the cells in optically cleared bone marrow using deep confocal imaging and digital tissue reconstruction. They find that α-catulin GFP + c-kit + cells are more common in central marrow than near bone surfaces, and in the diaphysis region of the bone relative to the metaphysis. The cells identified in this fashion are found in perisinusoidal niches throughout bone marrow and in close proximity to leptin receptor positive and Cxcl12 high cells. Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial 1 . HSCs are rare and few can be found in thin tissue sections 2 , 3 or upon live imaging 4 , making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α - catulin GFP ), we discover that α - catulin GFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α - catulin −GFP + c-kit + cells give long-term multilineage reconstitution of irradiated mice, indicating that α - catulin −GFP + c-kit + cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α - catulin –GFP + c-kit + cells and to digitally reconstruct large segments of bone marrow. The distribution of α - catulin –GFP + c-kit + cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr + ) and Cxcl12 high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67 + ) and non-dividing (Ki-67 − ), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr + Cxcl12 high cells throughout the bone marrow.

Force sensing at cadherin-mediated adhesions is critical for their proper function. α-Catenin, which links cadherins to actomyosin, has a crucial role in this mechanosensing process. It has been hypothesized that force promotes vinculin binding, although this has never been demonstrated. X-ray structure further suggests that α-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. Here, by stretching single α-catenin molecules using magnetic tweezers, we show that the subdomains M I vinculin-binding domain (VBD) to M III unfold in three characteristic steps: a reversible step at ~5 pN and two non-equilibrium steps at 10–15 pN. 5 pN unfolding forces trigger vinculin binding to the M I domain in a 1:1 ratio with nanomolar affinity, preventing M I domain refolding after force is released. Our findings demonstrate that physiologically relevant forces reversibly unfurl α-catenin, activating vinculin binding, which then stabilizes α-catenin in its open conformation, transforming force into a sustainable biochemical signal. At cell–cell adhesions, α-catenin contains a cryptic vinculin-binding site. Here, Yao et al . demonstrate, using magnetic tweezers, that physiologically relevant forces unfurl α-catenin to reveal the vinculin-binding site, and allow the reversible binding of vinculin to mechanically reinforce the adhesion.

The cadherin–catenin adhesion complex is the central component of the cell–cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible “tongue” that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin–catenin adhesion complex in mechanotransduction.

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, β-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that β-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 β-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 β-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin. Adherens junctions mediate force transmission and connect the cytoskeleton of adjacent cells. This study demonstrates that force transmission can be achieved through direct and functional β-catenin/vinculin interaction in the absence of α-catenin.

αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin–β-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1–D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1–D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or \"coincidence\" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.

The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force; however, it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show single piconewton forces applied solely to F-actin enhance binding by the human version of the essential cell-cell adhesion protein αE-catenin but not its homolog vinculin. Cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin’s C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin’s C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through α-catenin. All of the cells in our bodies rely on cues from their surrounding environment to alter their behavior. As well sending each other chemical signals, such as hormones, cells can also detect pressure and physical forces applied by the cells around them. These physical interactions are coordinated by a network of proteins called the cytoskeleton, which provide the internal scaffold that maintains a cell’s shape. However, it is not well understood how forces transmitted through the cytoskeleton are converted into mechanical signals that control cell behavior. The cytoskeleton is primarily made up protein filaments called actin, which are frequently under tension from external and internal forces that push and pull on the cell. Many proteins bind directly to actin, including adhesion proteins that allow the cell to ‘stick’ to its surroundings. One possibility is that when actin filaments feel tension, they convert this into a mechanical signal by altering how they bind to other proteins. To test this theory, Mei et al. isolated and studied an adhesion protein called α-catenin which is known to interact with actin. This revealed that when tiny forces – similar to the amount cells experience in the body – were applied to actin filaments, this caused α-catenin and actin to bind together more strongly. However, applying the same level of physical force did not alter how well actin bound to a similar adhesion protein called vinculin. Further experiments showed that this was due to differences in a small, flexible region found on both proteins. Manipulating this region revealed that it helps α-catenin attach to actin when a force is present, and was thus named a ‘force detector’. Proteins that bind to actin are essential in all animals, making it likely that force detectors are a common mechanism. Scientists can now use this discovery to identify and manipulate force detectors in other proteins across different cells and animals. This may help to develop drugs that target the mechanical signaling process, although this will require further understanding of how force detectors work at the molecular level.

α-catenin is a crucial protein at cell junctions that provides connection between the actin cytoskeleton and the cell membrane. At adherens junctions (AJs), α-catenin forms heterodimers with β-catenin that are believed to resist force on F-actin. Outside AJs, α-catenin forms homodimers that regulates F-actin organization and directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown. By using ultra-fast laser tweezers we found that a single α-β-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 α-β-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in α-catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an α-catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that α-catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface. By using laser tweezers, the authors show that a single α-catenin molecule does not resist force on F-actin. However, clustering of multiple molecules and force applied toward F-actin pointed end engage a molecular switch in α-catenin, which unfolds and strongly binds F-actin.

Annexin-1 (ANXA1) has shown neuroprotective effects and microglia play significant roles during central nervous system injury, yet the underlying mechanisms remain unclear. This study sought to determine whether ANXA1 regulates microglial response to oxygen–glucose deprivation/reperfusion (OGD/R) treatment and to clarify the downstream molecular mechanism. In rat hippocampal slices, OGD/R treatment enhanced the ANXA1 expression in neuron, the formyl peptide receptor (FPRs) expression in microglia, and the microglial activation in the CA1 region (cornu ammonis 1). These effects were reversed by the FPRs antagonist Boc1. The cell membrane currents amplitude of BV-2 microglia (the microglial like cell-line) was increased when treated with Ac2-26, the N-terminal peptide of ANXA1. Ac2-26 treatment enhanced BV-2 microglial migration whereas Boc1 treatment inhibited the migration. In BV-2 microglia, both the expression of the CK2 target phosphorylated α-E-catenin and the binding of casein kinase II (CK2) with α-E-catenin were elevated by Ac2-26, these effects were counteracted by the CK2 inhibitor TBB and small interfering (si) RNA directed against transcripts of CK2 and FPRs. Moreover, both TBB and siRNA-mediated inhibition of CK2 blocked Ac2-26-mediated BV-2 microglia migration. Our findings indicate that ANXA1 promotes microglial activation and migration during OGD/R via FPRs, and CK2 target α-E-catenin phosphorylation is involved in this process.

Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1–specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin–mediated tumor suppression in breast cancer cells.