RESUMO
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 ß-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 ß-cardiac myosin. We are using a recombinantly expressed human ß-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.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Mutação/genética , Miosinas Ventriculares/genética , Fenômenos Biomecânicos/genética , Humanos , Modelos BiológicosRESUMO
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.
Assuntos
Técnicas Biossensoriais , Adesões Focais/química , Integrinas/química , Estresse Mecânico , Citoesqueleto/química , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Microscopia/métodos , NanotecnologiaRESUMO
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.
Assuntos
Colágeno Tipo I/química , Colagenases/química , Humanos , Conformação Molecular , Processamento de Proteína Pós-Traducional , ProteóliseRESUMO
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 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 and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials.
Assuntos
Coagulação Sanguínea , Fibrina/química , Fibrinolisina/química , Animais , Transporte Biológico , Difusão , Fibrinolisina/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Microscopia de Vídeo , Proteólise , Estresse MecânicoRESUMO
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 â¼10 pN in extensional force causes an â¼100-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.
Assuntos
Colágeno/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Fenômenos Mecânicos , Proteólise , Sequência de Aminoácidos , Colágeno/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Hypertrophic cardiomyopathy (HCM) mutations in ß-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 ß-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.
Assuntos
Actinas/metabolismo , Alelos , Substituição de Aminoácidos , Cardiomiopatia Hipertrófica/genética , Mutação , Miosinas/genética , Miosinas/metabolismo , Actinas/química , Sítios de Ligação , Cardiomiopatia Hipertrófica/fisiopatologia , Predisposição Genética para Doença , Humanos , Modelos Moleculares , Contração Miocárdica/genética , Miosinas/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Miosinas Ventriculares/genéticaRESUMO
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 ß-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human ß-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.
Assuntos
Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Contração Miocárdica/genética , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Animais , Miosinas Cardíacas/genética , Linhagem Celular , Movimento Celular/genética , Coração/fisiopatologia , Humanos , Camundongos , Mutação , Mioblastos , Cadeias Pesadas de Miosina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Adesões Focais/metabolismo , Adesões Focais/fisiologia , Fibras de Estresse/fisiologia , Citoesqueleto de Actina/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Fenômenos Biomecânicos/fisiologia , Adesão Celular , Movimento Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/metabolismo , Humanos , Integrinas/metabolismo , Paxilina/metabolismo , Fibras de Estresse/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in ß-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 ß-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.
Assuntos
Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mutação , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Humanos , Modelos Biológicos , Contração MiocárdicaRESUMO
Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in ß-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 ß-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 ß-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.
Assuntos
Actinas/genética , Cardiomiopatia Hipertrófica/genética , Proteínas Motores Moleculares/genética , Miosinas Ventriculares/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Genótipo , Humanos , Mutação , Contração Miocárdica/genética , Miosinas Ventriculares/química , Miosinas Ventriculares/metabolismoRESUMO
The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis(1), atherogenesis(2) and wound healing(3). 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 importance(4-7). Single molecule techniques such as optical trapping(8), atomic force microscopy(9), and magnetic tweezers(10,11) 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 level(12). 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.