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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.

Summary Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

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.

The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

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.

Quantitative analysis of the pairwise dynamics of the actin cytoskeleton, focal adhesions, and ECM fibrils reveals how cytoskeletal dynamics drive matrix deformation and cell motility for primary human fibroblasts embedded in a 3D fibrin matrix., The ability of cells to impart forces and deformations on their surroundings underlies cell migration and extracellular matrix (ECM) remodeling and is thus an essential aspect of complex, metazoan life. Previous work has resulted in a refined understanding, commonly termed the molecular clutch model, of how cells adhering to flat surfaces such as a microscope coverslip transmit cytoskeletally generated forces to their surroundings. Comparatively less is known about how cells adhere to and exert forces in soft, three-dimensional (3D), and structurally heterogeneous ECM environments such as occur in vivo. We used time-lapse 3D imaging and quantitative image analysis to determine how the actin cytoskeleton is mechanically coupled to the surrounding matrix for primary dermal fibroblasts embedded in a 3D fibrin matrix. Under these circumstances, the cytoskeletal architecture is dominated by contractile actin bundles attached at their ends to large, stable, integrin-based adhesions. Time-lapse imaging reveals that α-actinin-1 puncta within actomyosin bundles move more quickly than the paxillin-rich adhesion plaques, which in turn move more quickly than the local matrix, an observation reminiscent of the molecular clutch model. However, closer examination did not reveal a continuous rearward flow of the actin cytoskeleton over slower moving adhesions. Instead, we found that a subset of stress fibers continuously elongated at their attachment points to integrin adhesions, providing stable, yet structurally dynamic coupling to the ECM. Analytical modeling and numerical simulation provide a plausible physical explanation for this result and support a picture in which cells respond to the effective stiffness of local matrix attachment points. The resulting dynamic equilibrium can explain how cells maintain stable, contractile connections to discrete points within ECM during cell migration, and provides a plausible means by which fibroblasts contract provisional matrices during wound healing.

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.

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human β-cardiac myosin at the molecular level. We observe extremely large increases (25% – 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant myosin motors compared to wild type myosin. In contrast to previous studies of HCM-causing mutations in human β-cardiac myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human β-cardiac myosin.

Hypertrophic cardiomyopathy (HCM) is a genetic disease that affects 1 in 500 people. In infants it is particularly severe and is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in beta cardiac myosin, the motor protein responsible for ventricular contraction. This study explores how pediatric-specific HCM mutations in beta cardiac myosin (D239N, H251N, and P710R) alter the biochemical and biomechanical properties of beta cardiac myosin at the molecular and cellular levels. Because these mutations manifest early in life, we hypothesize that they will be more severe than mutations that are not pediatric-specific. We investigated the biomechanical properties of these mutations at the molecular level using a purified S1 domain of human beta cardiac myosin. Specifically, we looked at the effects of these mutations on the ATPase activity and the unloaded velocity (in vitro motility) of beta cardiac S1. Upon comparison with WT beta cardiac S1, we find significant differences between the biomechanical properties of the WT and the pediatric-specific myosin mutations, which are more drastic as compared to mutations that are not pediatric-specific. To fully characterize the system, we are currently performing single molecule optical trap force measurements to determine the intrinsic force of these mutations. Future studies will involve creating iPSC-derived cardiomyocytes to study the mechanics of these mutations at the cellular level. The results from this study will increase the understanding of how genetic mutations that cause HCM lead to the presentation of the disease, and will uncover potential targets for therapeutic design to treat the cause of the disease instead of the symptoms.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

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.

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

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.

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7. Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

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.

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and cardiac hypercontractility. Mutations in β cardiac myosin heavy chain (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single β-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 or more years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in early onset disease may show more severe molecular changes in function compared to later onset mutations. In this work, we performed steady-state and transient kinetic analyses of myosins carrying 1 of 7 missense mutations in the motor domain. Of these 7, 4 have been identified in early onset cardiomyopathy screens. The derived parameters were used to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related Duty Ratio (DR; fraction of myosin in strongly attached force-holding states) and the measured ATPases all change in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the 7 HCM mutations are clearly distinct from a set of DCM mutations previously characterized.

Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are important causes of heart failure, arrhythmia, and sudden cardiac death. HCM is the most common heritable cardiovascular disorder, affecting 1 in 500 individuals, and is caused primarily by mutations that alter proteins of the cardiac sarcomere. Sarcomeric protein mutations are an increasingly recognized cause of familial DCM as well. HCM is associated with severe thickening of the left ventricular wall, preserved/increased systolic (contractile) and impaired diastolic (relaxation) function of the heart. DCM hearts have dilated left ventricular chambers and suffer from inadequate systolic activity. More than 300 point mutations in beta-cardiac myosin are associated with HCM or DCM. It is believed that HCM and DCM mutations increase and decrease respectively the power output of cardiac myosin which leads to a cascade of downstream signaling that gradually leads to the disease phenotype. To test this hypothesis it is essential to measure the power output of all HCM/DCM mutants in a high throughput manner. To quantitatively measure the load dependent myosin power output, we first improved the loaded in vitro motility assay (LIMA) by replacing load generating molecule alpha-actinin with alpha-catenin, which was generously provided by James Nelson at Stanford University. For accurate-and-fast data analysis, we developed a software called FAST that runs over parallel CPUs. Our initial results indicate that two HCM mutants (R719W, R403Q) have higher power output than the wildtype cardiac myosin. FAST-LIMA will be an essential tool for screening drugs that revert the effects of HCM/DCM mutations on the power output of beta-cardiac myosin.

The mechanical forces exerted and detected by living cells play integral roles in diverse biological phenomena, including growth and development, wound healing, and cancer metastasis. In the past decade, techniques such as traction force microscopy and micropost arrays have proven to be powerful tools for measuring the forces generated by cells. In particular, traction force microscopy has recently been extended to three-dimensional cell culture environments by embedding tracer beads in either a synthetic polyethylene glycol hydrogel (PEG; Legant et al., Nat. Meth. 2010) or in collagen gels (Koch et al., PLoS ONE 2012). The embedded beads move in response to cell-generated distortions of the matrix, allowing cell-generated forces to be calculated. We sought to develop an experimental system that would exhibit the excellent mechanical properties of the PEG hydrogel while using a naturally occurring biological matrix. Fibrin gels fulfill both of these requirements: fibrin is elastic up to ∼50% strain (Brown et al., Science 2009) and is also widely used for 3D cell culture. Here we describe the use of fluorescently labeled fibrin gels to measure the forces generated by cells in 3D culture. We observe dramatic but elastic deformations of the fibrin matrix surrounding cells as they grow, divide, and migrate. Further, we find that the dynamic forces generated by the cell can be measured using the deformations of the matrix itself, providing a direct observation of how the cell modifies its surroundings. We discuss the use of this new technique in studying matrix remodeling and cell migration.

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.