Coupling during collective cell migration is controlled by a vinculin mechanochemical switch.

The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors.

Rho activation drives luminal collapse and eversion in epithelial acini.

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. Although some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternative pathway for altered polarity. Because mechanical alterations are common in human cancer, including an upregulation of RhoA-mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of ß1-integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model that balances tension at cell-cell interfaces revealed a fivefold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts.

Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-ß-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell's contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through ß-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.

E-cadherin adhesion dynamics as revealed by an accelerated force ramp are dependent upon the presence of α-catenin.

Tissue remodeling and shape changes often rely on force-induced cell rearrangements occurring via cell-cell contact dynamics. Epithelial cell-cell contact shape changes are particularly dependent upon E-cadherin adhesion dynamics which are directly influenced by cell-generated and external forces. While both the mobility of E-cadherin adhesions and their adhesion strength have been reported before, it is not clear how these two aspects of E-cadherin adhesion dynamics are related. Here, using magnetic pulling cytometry, we applied an accelerated force ramp on the E-cadherin adhesion between an E-cadherin-coated magnetic microbead and an epithelial cell to ascertain this relationship. Our approach enables the determination of the adhesion strength and force-dependent mobility of individual adhesions, which revealed a direct correlation between these key characteristics. Since α-catenin has previously been reported to play a role in both E-cadherin mobility and adhesion strength when studied independently, we also probed epithelial cells in which α-catenin has been knocked out. We found that, in the absence of α-catenin, E-cadherin adhesions not only had lower adhesion strength, as expected, but were also more mobile. We observed that α-catenin was required for the recovery of strained cell-cell contacts and propose that the adhesion strength and force-dependent mobility of E-cadherin adhesions act in tandem to regulate cell-cell contact homeostasis. Our approach introduces a method which relates the force-dependent adhesion mobility to adhesion strength and highlights the morphological role played by α-catenin in E-cadherin adhesion dynamics.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Enhancing European Management of Analgesia, Sedation, and Delirium: A Multinational, Prospective, Interventional Before-After Trial.

BACKGROUND: The objective of this study was to analyze the impact of a structured educational intervention on the implementation of guideline-recommended pain, agitation, and delirium (PAD) assessment. METHODS: This was a prospective, multinational, interventional before-after trial conducted at 12 intensive care units from 10 centers in Germany, Austria, Switzerland, and the UK. Intensive care units underwent a 6-week structured educational program, comprising online lectures, instructional videos, educational handouts, and bedside teaching. Patient-level PAD assessment data were collected in three 1-day point-prevalence assessments before (T1), 6 weeks after (T2), and 1 year after (T3) the educational program. RESULTS: A total of 430 patients were included. The rate of patients who received all three PAD assessments changed from 55% (107/195) at T1 to 53% (68/129) at T2, but increased to 73% (77/106) at T3 (p = 0.003). The delirium screening rate increased from 64% (124/195) at T1 to 65% (84/129) at T2 and 77% (82/106) at T3 (p = 0.041). The pain assessment rate increased from 87% (170/195) at T1 to 92% (119/129) at T2 and 98% (104/106) at T3 (p = 0.005). The rate of sedation assessment showed no signficiant change. The proportion of patients who received nonpharmacological delirium prevention measures increased from 58% (114/195) at T1 to 80% (103/129) at T2 and 91% (96/106) at T3 (p < 0.001). Multivariable regression revealed that at T3, patients were more likely to receive a delirium assessment (odds ratio [OR] 2.138, 95% confidence interval [CI] 1.206-3.790; p = 0.009), sedation assessment (OR 4.131, 95% CI 1.372-12.438; p = 0.012), or all three PAD assessments (OR 2.295, 95% CI 1.349-3.903; p = 0.002) compared with T1. CONCLUSIONS: In routine care, many patients were not assessed for PAD. Assessment rates increased significantly 1 year after the intervention. Clinical trial registration ClinicalTrials.gov: NCT03553719.

Progerin-expressing endothelial cells are unable to adapt to shear stress.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. Patients with HGPS experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study, we examined how progerin-expressing endothelial cells adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control endothelial cells to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in endothelial cells, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that endothelial cells either overexpressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). Endothelial cells overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing endothelial cells had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, the prealignment of cells to shear stress before progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of endothelial cells to properly adapt to shear stress.

Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network.

Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca2+-dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases.

The Elephant in the Cell: Nuclear Mechanics and Mechanobiology.

The 2021 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C) featured a workshop titled "The Elephant in the Room: Nuclear Mechanics and Mechanobiology." The goal of this workshop was to provide a perspective from experts in the field on the current understanding of nuclear mechanics and its role in mechanobiology. This paper reviews the major themes and questions discussed during the workshop, including historical context on the initial methods of measuring the mechanical properties of the nucleus and classifying the primary structures dictating nuclear mechanics, physical plasticity of the nucleus, the emerging role of the linker of nucleoskeleton and cytoskeleton (LINC) complex in coupling the nucleus to the cytoplasm and driving the behavior of individual cells and multicellular assemblies, and the computational models currently in use to investigate the mechanisms of gene expression and cell signaling. Ongoing questions and controversies, along with promising future directions, are also discussed.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration.

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.

Spatial Proliferation of Epithelial Cells Is Regulated by E-Cadherin Force.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. Prior experiments have shown that externally applied forces, such as stretch, result in increased proliferation in an E-cadherin force-dependent manner. In this study, the spatial regulation of cell proliferation in large epithelial colonies was examined. Surprisingly, cells at the center of the colony still had increased proliferation as compared to cells in confluent monolayers. E-cadherin forces were found to be elevated for both cells at the edge and center of these larger colonies when compared to confluent monolayers. To determine if high levels of E-cadherin force were necessary to induce proliferation at the center of the colony, a lower-force mutant of E-cadherin was developed. Cells with lower E-cadherin force had significantly reduced proliferation for cells at the center of the colony but minimal differences for cells at the edges of the colony. Similarly, increasing substrate stiffness was found to increase E-cadherin force and increase the proliferation rate across the colony. Taken together, these results show that forces through cell-cell junctions regulate proliferation across large groups of epithelial cells. In addition, an important finding of this study is that junction forces are dynamic and modulate cellular function even in the absence of externally applied loads.

Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.

Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 amino acid repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.

The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.

Flow-dependent cellular mechanotransduction in atherosclerosis.

Atherosclerosis depends on risk factors such as hyperlipidemia, smoking, hypertension and diabetes. Although these risk factors are relatively constant throughout the arterial circulation, atherosclerotic plaques occur at specific sites where flow patterns are disturbed, with lower overall magnitude and complex changes in speed and direction. Research over the past few decades has provided new insights into the cellular mechanisms of force transduction and how mechanical effects act in concert with conventional risk factors to mediate plaque formation and progression. This Commentary summarizes our current understanding of how mechanotransduction pathways synergize with conventional risk factors in atherosclerosis. We attempt to integrate cellular studies with animal and clinical data, and highlight major questions that need to be answered to develop more effective therapies.

A homeostatic role of nucleus-actin filament coupling in the regulation of cellular traction forces in fibroblasts.

Cellular traction forces are contractile forces that depend on the material/substrate stiffness and play essential roles in sensing mechanical environments and regulating cell morphology and function. Traction forces are primarily generated by the actin cytoskeleton and transmitted to the substrate through focal adhesions. The cell nucleus is also believed to be involved in the regulation of this type of force; however, the role of the nucleus in cellular traction forces remains unclear. In this study, we explored the effects of nucleus-actin filament coupling on cellular traction forces in human dermal fibroblasts cultured on substrates with varying stiffness (5, 15, and 48 kPa). To investigate these effects, we transfected the cells with a dominant-negative Klarsicht/ANC-1/Syne homology (DN-KASH) protein that was designed to displace endogenous linker proteins and disrupt nucleus-actin cytoskeleton connections. The force that exists between the cytoskeleton and the nucleus (nuclear tension) was also evaluated with a fluorescence resonance energy transfer (FRET)-based tension sensor. We observed a biphasic change in cellular traction forces with a peak at 15 kPa, regardless of DN-KASH expression, that was inversely correlated with the nuclear tension. In addition, the relative magnitude and distribution of traction forces in nontreated wild-type cells were similar across different stiffness conditions, while DN-KASH-transfected cells exhibited a different distribution pattern that was impacted by the substrate stiffness. These results suggest that the nucleus-actin filament coupling play a homeostatic role by maintaining the relative magnitude of cellular traction forces in fibroblasts under different stiffness conditions.

Desmosomal Cadherin Tension Loss in Pemphigus Vulgaris Mediated by the Inhibition of Active RhoA at Cell-Cell Adhesions.

Binding of autoantibodies to keratinocyte surface antigens, primarily desmoglein 3 (Dsg3) of the desmosomal complex, leads to the dissociation of cell-cell adhesion in the blistering disorder pemphigus vulgaris (PV). After the initial disassembly of desmosomes, cell-cell adhesions actively remodel in association with the cytoskeleton and focal adhesions. Growing evidence highlights the role of adhesion mechanics and mechanotransduction at cell-cell adhesions in this remodeling process, as their active participation may direct autoimmune pathogenicity. However, a large part of the biophysical transformations after antibody binding remains underexplored. Specifically, it is unclear how tension in desmosomes and cell-cell adhesions changes in response to antibodies, and how the altered tensional states translate to cellular responses. Here, we showed a tension loss at Dsg3 using fluorescence resonance energy transfer (FRET)-based tension sensors, a tension loss at the entire cell-cell adhesion, and a potentially compensatory increase in junctional traction force at cell-extracellular matrix adhesions after PV antibody binding. Further, our data indicate that this tension loss is mediated by the inhibition of RhoA at cell-cell contacts, and the extent of RhoA inhibition may be crucial in determining the severity of pathogenicity among different PV antibodies. More importantly, this tension loss can be partially restored by altering actomyosin based cell contractility. Collectively, these findings provide previously unattainable details in our understanding of the mechanisms that govern cell-cell interactions under physiological and autoimmune conditions, which may open the window to entirely new therapeutics aimed at restoring physiological balance to tension dynamics that regulates the maintenance of cell-cell adhesion.

A comparison of noninvasive bioreactance with oesophageal Doppler estimation of stroke volume during open abdominal surgery: an observational study.

CONTEXT: The anaesthetist must maintain tissue perfusion by ensuring optimal perioperative fluid balance. This can be achieved using less invasive cardiac output monitors such as oesophageal Doppler monitoring (ODM). Other less invasive cardiac output monitors using bio-impedence technology (noninvasive cardiac output monitoring, NICOM) may have a role in monitoring the circulation and informing fluid management decisions. OBJECTIVE: To compare estimates of stroke volume from ODM with those from NICOM, a noninvasive monitor using bioreactance, a modification of transthoracic bio-impedence. DESIGN: An observational study. SETTING: Manchester Royal Infirmary, UK. Data collected in 2011 and 2012. PARTICIPANTS: Twenty-two patients scheduled for major, open abdominal surgery. Reasons for noninclusion: atrial fibrillation; heart failure; oesophageal disease; lack of capacity; and known sensitivity to colloid. INTERVENTION: All patients had oesophageal Doppler cardiac output monitoring as a standard element of anaesthesia care. We placed NICOM Bioreactance electrodes and recorded stroke volume estimates from both devices. Fluid challenges were given by the anaesthetist and the haemodynamic responses were recorded. MAIN OUTCOME MEASURE: Stroke volume during surgery. The Bland-Altman method was used to compare bias and limits of agreement for stroke volume and cardiac output. Fluid responders were defined as patients who increased stroke volume by at least 10% after fluid loading. The precision of each device was calculated during periods of haemodynamic stability. RESULTS: We made 788 acceptable measurements of cardiac output. The bias was -6.9 ml and the limits of agreement were -22.9 to 36.8 ml. The percentage error was 57%. Average precision for both the ODM and NICOM were similar, 8.5% (SD 5.4%) and 8.7% (SD 3.2%). The concordance for the stroke volume change following fluid challenge was 90.5%. Both devices produced unacceptable readings with electrical diathermy. CONCLUSION: Simultaneous stroke volume estimations made by noninvasive Bioreactance (NICOM) and oesophageal Doppler showed bias and limits of agreement that are not clinically acceptable. The measurements made by these two devices cannot be regarded as interchangeable.

α-Catenin Dependent E-cadherin Adhesion Dynamics as Revealed by an Accelerated Force Ramp.

Tissue remodeling and shape changes often rely on force-induced cell rearrangements occurring via cell-cell contact dynamics. Epithelial cell-cell contact shape changes are particularly dependent upon E-cadherin adhesion dynamics which are directly influenced by cell-generated and external forces. While both the mobility of E-cadherin adhesions and their adhesion strength have been reported before, it is not clear how these two aspects of E-cadherin adhesion dynamics are related. Here, using magnetic pulling cytometry, we applied an accelerated force ramp on the E-cadherin adhesion between an E-cadherin-coated magnetic microbead and an epithelial cell to ascertain this relationship. Our approach enables the determination of the adhesion strength and force-dependent mobility of individual adhesions, which revealed a direct correlation between these key characteristics. Since α-catenin has previously been reported to play a role in both E-cadherin mobility and adhesion strength when studied independently, we also probed epithelial cells in which α-catenin has been knocked out. We found that, in the absence of α-catenin, E-cadherin adhesions not only had lower adhesion strength, as expected, but were also more mobile. We observed that α-catenin was required for the recovery of strained cell-cell contacts and propose that the adhesion strength and force-dependent mobility of E-cadherin adhesions act in tandem to regulate cell-cell contact homeostasis. Our approach introduces a method which relates the force-dependent adhesion mobility to adhesion strength and highlights the morphological role played by α-catenin in E-cadherin adhesion dynamics.

Coupling During Collective Cell Migration is Controlled by a Vinculin Mechanochemical Switch.

Collective cell migration (CCM) plays important roles in development, physiological, and pathological processes. A key feature of CCM is the dynamic mechanical coupling between cells, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to adapt to forces. Recent efforts have identified key proteins and implicated cellular-scale mechanical properties, but how key proteins give rise to these larger-scale mechanical processes is unclear. Using force-sensitive biosensors, cell migration assays, and molecular clutch models, we sought a molecular understanding of adhesion strengthening that could bridge this gap. We found that the mechanical linker protein vinculin bears substantial loads at AJs, FAs, and in the cytoplasm during epithelial sheet migration, and we identified a switch-like residue on vinculin that regulates its conformation and loading at the AJs during CCM. In vinculin KO-rescue, this switch jointly controlled the speed and coupling length-scale of CCM, which suggested changes in adhesion-based friction. To test this, we developed molecularly detailed friction clutch models of the FA and AJ. They show that open, loaded vinculin increases friction in adhesive structures, with larger affects observed in AJs. Thus, this work elucidates how load-bearing linker proteins can be regulated to alter mechanical properties of cells and enable rapid tuning of mechanical coupling in CCM.

Nuclear lamina strain states revealed by intermolecular force biosensor.

Nuclear lamins have been considered an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that the nanobody-based approach allows construction of biosensors for complex protein structures for mechanobiology studies.