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Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance., Li et al. investigates the role of N-linked glycosylation for the function of mechanosensitive ion channel Piezo1. They show that disease-linked loss of function mutations in Piezo1 that are trafficking defective lack N-linked glycosylation.

Talin and vinculin are part of a multi-component system involved in mechanosensing in cell-matrix adhesions. Both exist in auto-inhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin’s interaction with vinculin has not been investigated. Here by quantifying the force-dependent talin-vinculin interactions and kinetics using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an auto-inhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for higher-order glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

Filopodia are ubiquitous membrane projections that play crucial role in guiding cell migration on rigid substrates and through extracellular matrix by utilizing yet unknown mechanosensing molecular pathways. As recent studies show that Ca2+channels localized to filopodia play an important role in regulation of their formation and since some Ca2+channels are known to possess mechanosensing properties, activity of filopodial Ca2+channels might be tightly interlinked with the filopodia mechanosensing function. We tested this hypothesis by monitoring changes in the intra-filopodial Ca2+level in response to application of stretching force to individual filopodia of several cell types. It has been found that stretching forces of tens of pN strongly promote Ca2+influx into filopodia, causing persistent Ca2+oscillations that last for minutes even after the force is released. Most of the known mechanosensitive Ca2+channels, such as Piezo 1, Piezo 2 and TRPV4, were found to be dispensable for the observed force-dependent Ca2+influx. In contrast, L-type Ca2+channels appear to be a key component in the discovered phenomenon. Since previous studies have shown that intra-filopodial transient Ca2+signals play an important role in guidance of cell migration, our results suggest that the force-dependent activation of L-type Ca2+channels may contribute to this process. Overall, our study reveals an intricate interplay between mechanical forces and Ca2+signaling in filopodia, providing novel mechanistic insights for the force-dependent filopodia functions in guidance of cell migration.Significance statementWe found that tensile forces of tens of pN applied to individual filopodia trigger Ca2+influx through L-type Ca2+channels, producing persistent Ca2+oscillations inside mechanically stretched filopodia. Resulting elevation of the intra-filopodial Ca2+level in turn leads to downstream activation of calpain protease, which is known to play a crucial role in regulation of the cell adhesion dynamics. Thus, our work suggests that L-type channel-dependent Ca2+signaling and the mechanosensing function of filopodia are coupled to each other, synergistically governing cell adhesion and motion in a force-dependent manner. Since L-type Ca2+channels have been previously found in many different cell types, such as neural or cancer cells, the above mechanism is likely to be widespread among various cell lines.

Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems – focal adhesions and mechanosensitive ion channels, can convert mechanical features of the microenvironment into biochemical signals. We report here surprisingly that the mechanosensitive Ca2+-channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting adhesion maturation and cell spreading in normal but not in tumor cells. In the absence of Piezo1, matrix adhesions are smaller in normal cells mimicking transformed cells where adhesions do not change with or without Piezo1. A novel adhesion-targeted calcium sensor shows robust Piezo1-dependent, calcium influx at adhesions in normal cells; but not in transformed cells. A linker domain in Piezo1 is needed for binding to adhesions and overexpression of the domain blocks Piezo1 binding to adhesions decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a novel component of focal adhesions in non-transformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

The force-dependent unfolding/refolding of protein domains and ligand-receptor association/dissociation are crucial for mechanosensitive functions, while many aspects of how force affects the transition rate still remain poorly understood. Here, we report a new analytical expression of the force-dependent rate of molecules for transitions overcoming a single barrier. Unlike previous models derived in the framework of Kramers theory that requires a presumed one-dimensional free energy landscape, our model is derived based on the structural-elastic properties of molecules which are not restricted by the shape and dimensionality of the underlying free energy landscape. Importantly, the parameters of this model provide direct information on the structural-elastic features of the molecules between their transition and initial states. We demonstrate the applications of this model by applying it to explain force-dependent transition kinetics for several molecules and predict the structural-elastic properties of the transition states of these molecules.

For individual cells in tissues to create the diverse forms of biological organisms, it is necessary that they must reliably sense and generate the correct forces over the correct distances and directions. There is considerable evidence that the mechanical aspects of the cellular microenvironment provide critical physical parameters to be sensed. How proteins sense forces and cellular geometry to create the correct morphology is not understood in detail but protein unfolding appears to be a major component in force and displacement sensing. Thus, the crystallographic structure of a protein domain provides only a starting point to then analyze what will be the effects of physiological forces through domain unfolding or catch-bond formation. In this review, we will discuss the recent studies of cytoskeletal and adhesion proteins that describe protein domain dynamics. Forces applied to proteins can activate or inhibit enzymes, increase or decrease protein-protein interactions, activate or inhibit protein substrates, induce catch bonds and regulate interactions with membranes or nucleic acids. Further, the dynamics of stretch-relaxation can average forces or movements to reliably regulate morphogenic movements. In the few cases where single molecule mechanics are studied under physiological conditions such as titin and talin, there are rapid cycles of stretch-relaxation that produce mechanosensing signals. Fortunately, the development of new single molecule and super-resolution imaging methods enable the analysis of single molecule mechanics in physiologically relevant conditions. Thus, we feel that stereotypical changes in cell and tissue shape involve mechanosensing that can be analyzed at the nanometer level to determine the molecular mechanisms involved.

Cancer cells differ from normal cells in several important features like anchorage independence, Warburg effect and mechanosensing. Further, in recent studies, they respond aberrantly to external mechanical distortion. Consistent with altered mechano-responsiveness, we find that cyclic stretching of tumor cells from many different tissues reduces growth rate and causes apoptosis on soft surfaces. Surprisingly, normal cells behave similarly when transformed by depletion of the rigidity sensor protein (Tropomyosin 2.1). Restoration of rigidity sensing in tumor cells promotes rigidity dependent mechanical behavior, i.e. cyclic stretching enhances growth and reduces apoptosis on soft surfaces. The mechanism of mechanical apoptosis (mechanoptosis) of transformed cells involves calcium influx through the mechanosensitive channel, Piezo1 that activates calpain 2 dependent apoptosis through the BAX molecule and subsequent mitochondrial activation of caspase 3 on both fibronetin and collagen matrices. Thus, it is possible to selectively kill tumor cells by mechanical perturbations, while stimulating the growth of normal cells.

Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell–extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of talin are utilized in the diverse talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of talin rod domains make talin a very effective force buffer that sets a physiological force range of only a few pNs in the talin-mediated force transmission pathway., Talin is a mechanosensing cytoplasmic adaptor that links integrin cell adhesion receptors to the actin cytoskeleton. Here the authors measure the force-dependent folding and refolding kinetics of all talin rod domains to propose that talin can function as a force buffer under physiologically relevant conditions.

Transformed cancer cells differ from normal cells in several important features like anchorage independence, Warburg effect and mechanosensing. Consequently, transformed cancer cells develop an anaplastic morphology and respond aberrantly to external mechanical forces. Consistent with altered mechano-responsiveness, here we show that transformed cancer cells from many different tissues have reduced growth and become apoptotic upon cyclic stretch as do normal cells after the transformation. When matrix rigidity sensing is restored in transformed cancer cells, they survive and grow faster on soft surface upon cyclic stretch like normal cells but undergo anoikis without stretch by activation of death associated protein kinase1 (DAPK1). In contrast, stretch-dependent apoptosis (mechanoptosis) of transformed cells is driven by stretch-mediated calcium influx and calcium-dependent calpain 2 protease activation on both collagen and fibronectin matrices. Further, mechanosensitive calcium channel, Piezo1 is needed for mechanoptosis. Thus, cyclic stretching of transformed cells from different tissues activates apoptosis, whereas similar stretching of normal cells stimulates growth.

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~386 kDa surface protein, comprised of 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium- binding and the folded domains have differential mechanical stabilities. Importantly, we identify that >100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (?M) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion.

α-actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. In this work, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1 as well as the mechanical strength of the α-actinin 1 dimerization interaction. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Based on the mechanical stability and kinetics of α-actinin 1, a novel role of the α-actinin 1 as molecular suspensions for the cytoskeleton network is revealed. Our results provide the first comprehensive analysis of the force dependent stability and interactions of α-actinin 1, which sheds new light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions.

Summary: α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Further, a role of the α-actinin 1 as molecular shock absorber for the cytoskeleton network is revealed. Our results provide a comprehensive analysis of the force-dependent stability and interactions of α-actinin 1, which sheds important light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions. : α-Actinins are critical actin crosslinking proteins that organize actin cytoskeletal networks. Le et al. determine the mechanical stability and dynamics of human α-actinin 1 and the force-dependence of vinculin binding to α-actinin 1, which sheds light on the molecular mechanisms of mechanotransmission and mechanosensing. Keywords: α-actinin 1, cytoskeleton, mechanosensing, mechanostransmission, vinculin binding, magnetic tweezers, single molecule manipulation, molecular shock absorber

A fundamental question in mechanobiology is how mechanical stimuli are sensed by mechanosensing proteins and converted into signals that direct cells to adapt to the external environment. A key function of cell adhesion to the extracellular matrix (ECM) is to transduce mechanical forces between cells and their extracellular environment. Talin, a cytoplasmic adapter essential for integrin-mediated adhesion to the ECM, links the actin cytoskeleton to integrin at the plasma membrane. Here, we review recent progress in the understanding of talin-dependent mechanosensing revealed by stretching single talin molecules. Rapid progress in single-molecule force manipulation technologies has made it possible to directly study the impact of mechanical force on talin’s conformations and its interactions with other signaling proteins. We also provide our views on how findings from such studies may bring new insights into understanding the principles of mechanobiology on a broader scale, and how such fundamental knowledge may be harnessed for mechanopharmacology.

Unfolded protein, a disordered structure found before folding of newly synthesized protein or after protein denaturation, is a substrate for binding by many cellular factors such as heat-stable proteins, chaperones, and many small molecules. However, it is challenging to directly probe such interactions in physiological solution conditions because proteins are largely in their folded state. In this work we probed small molecule binding to mechanically unfolded polyprotein using sodium dodecyl sulfate (SDS) as an example. The effect of binding is quantified based on changes in the elasticity and refolding of the unfolded polyprotein in the presence of SDS. We show that this single-molecule mechanical detection of binding to unfolded polyprotein can serve, to our knowledge, as a novel label-free assay with a great potential to study many factors that interact with unfolded protein domains, which underlie many important biological processes.

Nature Communications 5: Article number: 4525 (2014); Published: 31 July 2014; Updated: 4 March 2015 The affiliation details for Jie Yan are incorrect in this Article. The correct affiliation details for this author are given below: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.

The mechanical stability of force-bearing proteins is crucial for their functions. However, slow transition rates of complex protein domains have made it challenging to investigate their equilibrium force-dependent structural transitions. Using ultra stable magnetic tweezers, we report the first equilibrium single-molecule force manipulation study of the classic titin I27 immunoglobulin domain. We found that individual I27 in a tandem repeat unfold/fold independently. We obtained the force-dependent free energy difference between unfolded and folded I27 and determined the critical force (∼5.4 pN) at which unfolding and folding have equal probability. We also determined the force-dependent free energy landscape of unfolding/folding transitions based on measurement of the free energy cost of unfolding. In addition to providing insights into the force-dependent structural transitions of titin I27, our results suggest that the conformations of titin immunoglobulin domains can be significantly altered during low force, long duration muscle stretching.

Focal adhesion protein talin has emerged as a key protein the mechanotransduction pathway linking integrin based cell adhesion to actin cytoskeleton. The force-dependent structural transitions of the c-terminal rod domain, consisting of 13 alpha-helical bundles, are critical for talin's mechanosensing function during cell adhesion and spreading but remain poorly understood. We systematically determined the force-dependent unfolding/refolding kinetics of all talin rod domains and obtained the dynamics of force fluctuation of talin rod under experimentally measured extension fluctuation time trajectories. The results revealed talin rod's role as a viscoelastic spring and tension buffer that maintains at a level < 10 pN along talin mediated force-transmission pathway. These results provide the first quantitative understanding of how talin serves as a mechanical clutch during cell spreading.

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 MI vinculin-binding domain (VBD) to MIII 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 MI domain in a 1:1 ratio with nanomolar affinity, preventing MI 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.

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein.

Interaction between talin and vinculin plays a critical role in stabilizing the focal adhesions (FAs) and regulates cell motility. In previous study, we showed that force applied to talin rod domains R1-R3 is necessary to expose the vinculin binding site, which drastically promotes the binding of the vinculin head domain. In this work, using magnetic tweezers single-molecule manipulation, we show that full-length vinculin also binds to mechanically unfolded talin R1-R3. It indicates that binding to mechanically unfolded talin R1-R3 results in dissociation of the vinculin tail domain from the head, implying release of the auto-inhibition conformation of full-length vinculin. Together, these results suggest that the force-dependent interaction between talin and vinculin likely plays a crucial role in vinculin activation in vivo.

Shaping, maintenance and repair of adult tissues require fine-tuning of cell-cell adhesion. α-catenin, a cytoplasmic adapter links the actin cytoskeleton to cell-cell junctions, plays a central role in regulation of the cell-cell adherence. This regulation requires binding of α-catenin to vinculin in a force dependent manner. By stretching single α-catenin construct using magnetic tweezers, we find that force in physiological range can expose the vinculin-binding sites buried in α-catenin, drastically promoting subsequent binding of the head domain of vinculin with a nanoMolar affinity. The bound vinculin head then irreversibly locks α-catenin in its unfolded conformations after force is released. The bound vinculin head can however be displaced at high forces > 30 pN, resulting in a biphasic force dependent binding of α-catenin to vinculin head. Further, we find that full-length vinculin also binds to mechanically unfolded α-catenin, implying release of the auto-inhibition conformation of full-length vinculin. Together, these results provide important insights into mechanosensing at cell-cell adherence.