Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores.

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

Dividing cells regulate their lipid composition and localization.

Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology.

A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges.

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.

Molecular Fluctuations as a Ruler of Force-Induced Protein Conformations.

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis examines protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically stable states scales with the protein's contour length increment, in agreement with the sequence-independent freely jointed chain model of polymer physics. Our findings will help elucidate the conformational dynamics of proteins exposed to mechanical force at high resolution which are of central importance in mechanosensing and mechanotransduction.

The nanomechanics of individual proteins.

Mechanical forces regulate a large variety of cellular functionalities, encompassing e.g. motility, differentiation and muscle contractility. To adapt to the dynamic change in mechanical stress, the constitutive individual proteins need to reversibly stretch and recoil over long periods of time. Yet, the molecular mechanisms controlling the mechanical unfolding and refolding of proteins cannot be accessed by protein folding biochemistry experiments conducted in the bulk, because they cannot typically apply forces to individual proteins. The advent of single-molecule nanomechanical techniques, often combined with bespoke protein engineering strategies, has enabled monitoring the conformational dynamics of proteins under force with unprecedented length-, time- and force-resolution. This review focuses on the fundamental operational principles of the main single-molecule nanomechanical techniques, placing particular emphasis on the most common analytical approaches used to extract information directly from the experiments. The breadth of enabling applications highlights the most exciting and promising outputs from the nanomechanics field to date.

Controlling Anomalous Diffusion in Lipid Membranes.

Diffusion in cell membranes is not just simple two-dimensional Brownian motion but typically depends on the timescale of the observation. The physical origins of this anomalous subdiffusion are unresolved, and model systems capable of quantitative and reproducible control of membrane diffusion have been recognized as a key experimental bottleneck. Here, we control anomalous diffusion using supported lipid bilayers containing lipids derivatized with polyethylene glycol (PEG) headgroups. Bilayers with specific excluded area fractions are formed by control of PEG lipid mole fraction. These bilayers exhibit a switch in diffusive behavior, becoming anomalous as bilayer continuity is disrupted. Using a combination of single-molecule fluorescence and interferometric imaging, we measure the anomalous behavior in this model over four orders of magnitude in time. Diffusion in these bilayers is well described by a power-law dependence of the mean-square displacement with observation time. Anomaleity in this system can be tailored by simply controlling the mole fraction of PEG lipid, producing bilayers with diffusion parameters similar to those observed for anomalous diffusion in biological membranes.

Single-molecule Force Spectroscopy Predicts a Misfolded, Domain-swapped Conformation in human γD-Crystallin Protein.

Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the ß1 and ß2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.

The Nanomechanics of Lipid Multibilayer Stacks Exhibits Complex Dynamics.

The nanomechanics of lipid membranes regulates a large number of cellular functions. However, the molecular mechanisms underlying the plastic rupture of individual bilayers remain elusive. This study uses force clamp spectroscopy to capture the force-dependent dynamics of membrane failure on a model diphytanoylphosphatidylcholine multilayer stack, which is devoid of surface effects. The obtained kinetic measurements demonstrate that the rupture of an individual lipid bilayer, occurring in the bilayer parallel plane, is a stochastic process that follows a log-normal distribution, compatible with a pore formation mechanism. Furthermore, the vertical individual force-clamp trajectories, occurring in the bilayer orthogonal bilayer plane, reveal that rupturing process occurs through distinct intermediate mechanical transition states that can be ascribed to the fine chemical composition of the hydrated phospholipid moiety. Altogether, these results provide a first description of unanticipated complexity in the energy landscape governing the mechanically induced bilayer rupture process.

Direct Observation of the Dynamics of Self-Assembly of Individual Solvation Layers in Molecularly Confined Liquids.

Confined liquids organize in solidlike layers at the liquid-substrate interface. Here we use force-clamp spectroscopy AFM to capture the equilibrium dynamics between the broken and reformed states of an individual solvation layer in real time. Kinetic measurements demonstrate that the rupture of each individual solvation layer in structured liquids is driven by the rupture of a single interaction for 1-undecanol and by two interactions in the case of the ionic liquid ethylammonium nitrate. Our results provide a first description of the energy landscape governing the molecular motions that drive the packing and self-assembly of each individual liquid layer.

Rate limit of protein elastic response is tether dependent.

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D(eff) ~ 1,200 nm(2)/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D(eff) ~ 10(8) nm(2)/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with D(eff) ~ 10(4)-10(6) nm(2)/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.

The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.

Acinar-ductal cell rearrangement drives branching morphogenesis of the murine pancreas in an IGF/PI3K-dependent manner.

During organ formation, progenitor cells need to acquire different cell identities and organize themselves into distinct structural units. How these processes are coordinated and how tissue architecture(s) is preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which leads to acini splitting. Newly formed acini remain connected to the bifurcated branches generated by ductal cell rearrangement. Insulin growth factor (IGF)/phosphatidylinositol 3-kinase (PI3K) pathway finely regulates this process by controlling pancreatic ductal tissue fluidity, with a simultaneous impact on branching and cell fate acquisition. Together, our results explain how acinar structure multiplication and branch bifurcation are synchronized during pancreas organogenesis.

Structural anisotropy results in mechano-directional transport of proteins across nuclear pores.

The nuclear pore complex regulates nucleocytoplasmic transport by means of a tightly synchronized suite of biochemical reactions. The physicochemical properties of the translocating cargos are emerging as master regulators of their shuttling dynamics. As well as being affected by molecular weight and surface-exposed amino acids, the kinetics of the nuclear translocation of protein cargos also depend on their nanomechanical properties, yet the mechanisms underpinning the mechanoselectivity of the nuclear pore complex are unclear. Here we show that proteins with locally soft regions in the vicinity of the nuclear-localization sequence exhibit higher nuclear-import rates, and that such mechanoselectivity is specifically impaired upon knocking down nucleoporin 153, a key protein in the nuclear pore complex. This allows us to design a short, easy-to-express and chemically inert unstructured peptide tag that accelerates the nuclear-import rate of stiff protein cargos. We also show that U2OS osteosarcoma cells expressing the peptide-tagged myocardin-related transcription factor import this mechanosensitive protein to the nucleus at higher rates and display faster motility. Locally unstructured regions lower the free-energy barrier of protein translocation and might offer a control mechanism for nuclear mechanotransduction.

Spontaneous dimerization of titin protein Z1Z2 domains induces strong nanomechanical anchoring.

Muscle elasticity strongly relies on the mechanical anchoring of the giant protein titin to both the sarcomere M-band and the Z-disk. Such strong attachment ensures the reversible dynamics of the stretching-relaxing cycles determining the muscle passive elasticity. Similarly, the design of biomaterials with enhanced elastic function requires experimental strategies able to secure the constituent molecules to avoid mechanical failure. Here we show that an engineered titin-mimicking protein is able to spontaneously dimerize in solution. Our observations reveal that the titin Z1Z2 domains are key to induce dimerization over a long-range distance in proteins that would otherwise remain in their monomeric form. Using single molecule force spectroscopy, we measure the threshold force that triggers the noncovalent transition from protein dimer to monomer, occurring at ∼700 piconewtons. Such extremely high mechanical stability is likely to be a natural protective mechanism that guarantees muscle integrity. We propose a simple molecular model to understand the force-induced dimer-to-monomer transition based on the geometric distribution of forces occurring within a dimeric protein under mechanical tension.

Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.

The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.

The role of single protein elasticity in mechanobiology.

In addition to biochemical signals and genetic considerations, mechanical forces are rapidly emerging as a master regulator of human physiology. Yet the molecular mechanisms that regulate force-induced functionalities across a wide range of scales, encompassing the cell, tissue or organ levels, are comparatively not so well understood. With the advent, development and refining of single molecule nanomechanical techniques, enabling to exquisitely probe the conformational dynamics of individual proteins under the effect of a calibrated force, we have begun to acquire a comprehensive knowledge on the rich plethora of physicochemical principles that regulate the elasticity of single proteins. Here we review the major advances underpinning our current understanding of how the elasticity of single proteins regulates mechanosensing and mechanotransduction. We discuss the present limitations and future challenges of such a prolific and burgeoning field.

Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape.

Statistical mechanics can describe the major conformational ensembles determining the equilibrium free-energy landscape of a folding protein. The challenge is to capture the full repertoire of low-occurrence conformations separated by high kinetic barriers that define complex landscapes. Computationally, enhanced sampling methods accelerate the exploration of molecular rare events. However, accessing the entire protein's conformational space in equilibrium experiments requires technological developments to enable extended observation times. We developed single-molecule magnetic tweezers to capture over a million individual transitions as a single talin protein unfolds and refolds under force in equilibrium. When observed at classically-probed timescales, talin folds in an apparently uncomplicated two-state manner. As the sampling time extends from minutes to days, the underlying energy landscape exhibits gradually larger signatures of complexity, involving a finite number of well-defined rare conformations. A fluctuation analysis allows us to propose plausible structures of each low-probability conformational state. The physiological relevance of each distinct conformation can be connected to the binding of the cytoskeletal protein vinculin, suggesting an extra layer of complexity in talin-mediated mechanotransduction. More generally, our experiments directly test the fundamental notion that equilibrium dynamics depend on the observation timescale.

Enzyme-less nanopore detection of post-translational modifications within long polypeptides.

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through the nanopore, with directional co-translocational unfolding occurring unit by unit from either the C or N terminus. Chaotropic reagents at non-denaturing concentrations accelerate the analysis. By monitoring the ionic current flowing through the nanopore, we locate post-translational modifications deep within the polypeptide chains, laying the groundwork for compiling inventories of the proteoforms in cells and tissues.

Myc-dependent dedifferentiation of Gata6+ epidermal cells resembles reversal of terminal differentiation.

Dedifferentiation is the process by which terminally differentiated cells acquire the properties of stem cells. During mouse skin wound healing, the differentiated Gata6-lineage positive cells of the sebaceous duct are able to dedifferentiate. Here we have integrated lineage tracing and single-cell mRNA sequencing to uncover the underlying mechanism. Gata6-lineage positive and negative epidermal stem cells in wounds are transcriptionally indistinguishable. Furthermore, in contrast to reprogramming of induced pluripotent stem cells, the same genes are expressed in the epidermal dedifferentiation and differentiation trajectories, indicating that dedifferentiation does not involve adoption of a new cell state. We demonstrate that dedifferentiation is not only induced by wounding, but also by retinoic acid treatment or mechanical expansion of the epidermis. In all three cases, dedifferentiation is dependent on the master transcription factor c-Myc. Mechanotransduction and actin-cytoskeleton remodelling are key features of dedifferentiation. Our study elucidates the molecular basis of epidermal dedifferentiation, which may be generally applicable to adult tissues.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.