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Hypertrophic cardiomyopathy–causing mutations disrupt a key regulatory off state of myosin in thick filaments., Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin and myosin binding protein-C (MyBP-C) lead to hypercontractility of the heart, an early hallmark of HCM. We show that hypercontractility caused by the HCM-causing mutation R663H cannot be explained by changes in fundamental myosin contractile parameters, much like the HCM-causing mutation R403Q. Using enzymatic assays with purified human β-cardiac myosin, we provide evidence that both mutations cause hypercontractility by increasing the number of functionally accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of MyBP-C. Furthermore, addition of C0-C7 decreases the wild-type myosin basal ATPase single turnover rate, while the mutants do not show a similar reduction. These data suggest that a primary mechanism of action for these mutations is to increase the number of myosin heads functionally available for interaction with actin, which could contribute to hypercontractility.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif (IHM) lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, which could lead to hypercontractility at the molecular level., Hypertrophic cardiomyopathy (HCM) leads to hyper-contractility of the heart and is often caused by mutations in human β-cardiac myosin. Here authors show that four separate β-cardiac myosin mutations can modulate myosin activity by disrupting intramolecular interactions.

Hypertrophic cardiomyopathy (HCM) mutations in ß-cardiac myosin and myosin binding protein-C (MyBP-C) cause hypercontractility of the heart. We show that hypercontractility caused by the HCM myosin mutation R663H cannot be explained by changes in the fundamental parameters such as actin-activated ATPase, intrinsic force, velocity of pure actin or regulated thin filaments, or the pCa50 of the velocity of regulated thin filaments. The same conclusion was made earlier for the HCM myosin mutation R403Q (Nag et al. 2015). Using enzymatic assays for the number of functionally-available heads in purified human ß-cardiac myosin preparations, we provide evidence that both R403Q and R663H HCM myosin mutations cause hypercontractility by increasing the number of functionally-accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of myosin binding protein-C.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

A working model for β-cardiac myosin in the sequestered state and binding assays reveal interactions between the myosin head and tail that are disrupted by mutations associated with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

The sarcomere is an exquisitely designed apparatus that is capable of generating force, which in the case of the heart results in the pumping of blood throughout the body. At the molecular level, an ATP-dependent interaction of myosin with actin drives the contraction and force generation of the sarcomere. Over the past six decades, work on muscle has yielded tremendous insights into the workings of the sarcomeric system. We now stand on the cusp where the acquired knowledge of how the sarcomere contracts and how that contraction is regulated can be extended to an understanding of the molecular mechanisms of sarcomeric diseases, such as hypertrophic cardiomyopathy (HCM). In this review we present a picture that combines current knowledge of the myosin mesa, the sequestered state of myosin heads on the thick filament, known as the interacting-heads motif (IHM), their possible interaction with myosin binding protein C (MyBP-C) and how these interactions can be abrogated leading to hyper-contractility, a key clinical manifestation of HCM. We discuss the structural and functional basis of the IHM state of the myosin heads and identify HCM-causing mutations that can directly impact the equilibrium between the 'on state' of the myosin heads (the open state) and the IHM 'off state'. We also hypothesize a role of MyBP-C in helping to maintain myosin heads in the IHM state on the thick filament, allowing release in a graded manner upon adrenergic stimulation. By viewing clinical hyper-contractility as the result of the destabilization of the IHM state, our aim is to view an old disease in a new light.

Living cells are exquisitely responsive to mechanical cues, yet how cells produce and detect mechanical force remains poorly understood due to a lack of methods that visualize cell-generated forces at the molecular scale. Here we describe Förster resonance energy transfer (FRET)-based molecular tension sensors that allow us to directly visualize cell-generated forces with single-molecule sensitivity. We apply these sensors to determine the distribution of forces generated by individual integrins, a class of cell adhesion molecules with prominent roles throughout cell and developmental biology. We observe strikingly complex distributions of tensions within individual focal adhesions. FRET values measured for single probe molecules suggest that relatively modest tensions at the molecular level are sufficient to drive robust cellular adhesion.

Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum. We observe that the application of 16 pN of force causes an 8-fold increase in collagen proteolysis rates by MMP-1 but does not affect cleavage rates by Clostridium collagenase. Quantitative analysis of these data allows us to infer the structural changes in collagen associated with proteolytic cleavage by both enzymes. Our data support a model in which MMP-1 cuts a transient, stretched conformation of its recognition site. In contrast, our findings suggest that Clostridium collagenase is able to cleave the fully wound collagen triple helix, accounting for its lack of force sensitivity and low sequence specificity. We observe that the cleavage of heterotrimeric collagen is less force sensitive than the proteolysis of a homotrimeric collagen model peptide, consistent with studies suggesting that the MMP-1 recognition site in heterotrimeric collagen I is partially unwound at equilibrium.

Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ∼10 pN in extensional force causes an ∼100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.

During direct cell-to-cell communication, lipids on the extracellular side of plasma membranes reorganize, and membrane-associated communication-related molecules colocalize. At colocalization sites, sometimes identified as rafts, the local cell surface topography and reactivity are altered. The processes regulating these changes are largely unknown. On model lipid membranes, study of simplified processes that control surface topography and reactivity may potentially contribute to the understanding and control of related cell functions and associated diseases. Integration of these processes on nanometer-sized lipid vesicles used as drug delivery carriers would precisely control their interactions with diseased cells minimizing toxicities. Here we design such basic pH-dependent processes on model functionalized lipid bilayers, and we demonstrate reversible sharp changes in binding reactivity within a narrow pH window. Cholesterol enables tuning of the membrane reorganization to occur at pH values not necessarily close to the reported pK(a)'s of the constituent titratable lipids, and bilayer reorganization over repeated cycles of induced pH changes exhibits hysteresis.

Mechanical interactions between cells and the extracellular matrix (ECM) exert a profound influence on cell migration, proliferation, and stem cell differentiation. However, fundamental aspects of how cells detect and generate mechanical forces at the cell-ECM interface remain poorly understood. Here we describe a new technique, termed Molecular Force Microscopy (MFM) that visualizes the forces experienced by single cellular adhesion molecules with nanometer, piconewton, and sub-second resolutions. MFM uses a new class of FRET-based molecular tension sensors that bind to an avidin-coated glass coverslip at one end and present an integrin binding site at the other. Cellular integrins transmit force to the FRET pair, resulting in decreased FRET with increasing load. Unlike previously reported force sensors, MFM sensor molecules allow quantitative FRET imaging at the single molecule level. We found that human foreskin fibroblasts (HFFs) adhered to and spread on surfaces functionalized with the MFM probes, and developed mature focal adhesions as evidenced by paxillin localization and actin stress fiber formation. We observed a bimodal distribution of FRET efficiency values for MFM sensor molecules beneath HFFs, with one peak corresponding to zero load and the other indicating a distribution of forces between 1 and 4 pN. Despite evidence of robust adhesion, the forces we measured were ∼10-fold lower than the force necessary to break individual integrin-ECM bonds. Our data provide the first direct measurement of the tension per integrin molecule necessary to form stable contacts with the ECM. The relatively narrow range of forces that we observed suggests that mechanical tension at individual adhesion molecules is subject to exquisite feedback and control. Ongoing work uses the unique capabilities of MFM to elucidate the mechanical signal transduction mechanisms that underlie cell migration and adhesion.

The generation and coordination of cellular traction forces play important roles in cell migration and extracellular matrix (ECM) remodeling, and hence in the development and repair of biological tissues. Historically, models for how cells generate and sense mechanical force have been derived from observations of cells adhering to two-dimensional (2D) surfaces. Here we sought to understand how the insights garnered from studies of cellular motility in 2D might apply to cell migration and traction force generation in more biologically realistic, 3D environments. We embedded primary human fibroblasts in a 3D fibrin matrix, and used multicolor, time-lapse confocal microscopy to simultaneously image matrix deformation induced by cellular traction and the dynamics of the actomyosin cytoskeleton and cell-matrix adhesions. We observed that traction forces were transduced to the matrix through contractile actomyosin fibers coupled to paxillin-rich adhesions. Spatial decomposition of the matrix deformation tensors, as quantified using digital volume correlation (DVC), revealed that cell-generated matrix deformations were largely tangential to the cell surface. Within protrusions, matrix deformations occurred in both the retrograde and anterograde directions relative to the protrusion tip. Automated tracking of paxillin-rich adhesions revealed persistent movements in both the retrograde and anterograde directions, with apparent slippage between adhesions and the underlying matrix. We tracked actin motion using both photoactivatable mCherry-actin and via automated particle tracking of alpha-actinin-EGFP puncta. These data revealed simultaneous anterograde and retrograde cytoskeletal motion within individual protrusions. In addition, the actin cytoskeleton moved at velocities higher than those of proximal paxillin plaques or proximal ECM. Together, these data suggest that a modified version of the molecular clutch model of cytoskeletal force transmission, which was originally developed to describe cell migration on flat surfaces, can be applied to understand cell migration in some 3D contexts.

Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases exponentially with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity within fibrin networks offers a means of tuning the transport of proteins and other soluble factors within fibrin-based biomaterials, potentially addressing a key challenge in engineering complex tissues in vitro.

The generation and coordination of cellular traction forces plays important roles in cell adhesion, cell migration, and extracellular matrix (ECM) remodeling, and hence in the development and repair of biological tissues. Historically, models for how cells generate and sense mechanical force have been derived from observations of cells grown on hard, two-dimensional (2D) surfaces. However, the cytoskeletal geometry of cells embedded in porous, 3D environments is inherently different than those found in 2D. Here we seek to understand the spatio-temporal regulation of cellular traction forces in a 3D environment. We embed primary human fibroblasts in a fluorescently labeled fibrin matrix, and use multicolor, time-lapse confocal microscopy to simultaneously image matrix deformation induced by cellular traction and the dynamics of the acto-myosin cytoskeleton and paxillin-rich cell-matrix adhesions. We observe significant traction forces transduced to the matrix through dynamic actomyosin-rich protrusions. Flux analysis of myosin concentrations in the protrusions reveals recruitment of myosin towards the protrusion tip during extension, and localization of myosin farther from the tip during retraction. Moreover, we observe both retrograde and anterograde movement of F-actin and myosin during cell traction generation, as well as similar bi-directional dynamics for paxillin-rich adhesions. Our data suggests a model of force generation and mechanotransduction different from the canonical continuous lamellipodial retrograde flow implicated in 2D cell migration. Ongoing work examines the role of signal transduction pathways in regulating cellular force generation, with the end goal of understanding how cells couple force generation and mechanotransduction in fully 3D environments.