Publications by authors named "Michael J Greenberg"

40 Publications

A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding.

Mol Biol Cell 2021 Jun 23:mbcE21020082. Epub 2021 Jun 23.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.
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http://dx.doi.org/10.1091/mbc.E21-02-0082DOI Listing
June 2021

Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation.

J Gen Physiol 2021 May;153(5)

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO.

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.
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http://dx.doi.org/10.1085/jgp.202012787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054178PMC
May 2021

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

JACC Basic Transl Sci 2021 Apr 26;6(4):331-345. Epub 2021 Feb 26.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

There is ongoing debate as to whether cardiac complications of coronavirus disease-2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.
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http://dx.doi.org/10.1016/j.jacbts.2021.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909907PMC
April 2021

Complexity in genetic cardiomyopathies and new approaches for mechanism-based precision medicine.

J Gen Physiol 2021 Mar;153(3)

Department of Biomedical Engineering, University of Arizona, Tucson, AZ.

Genetic cardiomyopathies have been studied for decades, and it has become increasingly clear that these progressive diseases are more complex than originally thought. These complexities can be seen both in the molecular etiologies of these disorders and in the clinical phenotypes observed in patients. While these disorders can be caused by mutations in cardiac genes, including ones encoding sarcomeric proteins, the disease presentation varies depending on the patient mutation, where mutations even within the same gene can cause divergent phenotypes. Moreover, it is challenging to connect the mutation-induced molecular insult that drives the disease pathogenesis with the various compensatory and maladaptive pathways that are activated during the course of the subsequent progressive, pathogenic cardiac remodeling. These inherent complexities have frustrated our ability to understand and develop broadly effective treatments for these disorders. It has been proposed that it might be possible to improve patient outcomes by adopting a precision medicine approach. Here, we lay out a practical framework for such an approach, where patient subpopulations are binned based on common underlying biophysical mechanisms that drive the molecular disease pathogenesis, and we propose that this function-based approach will enable the development of targeted therapeutics that ameliorate these effects. We highlight several mutations to illustrate the need for mechanistic molecular experiments that span organizational and temporal scales, and we describe recent advances in the development of novel therapeutics based on functional targets. Finally, we describe many of the outstanding questions for the field and how fundamental mechanistic studies, informed by our more nuanced understanding of the clinical disorders, will play a central role in realizing the potential of precision medicine for genetic cardiomyopathies.
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http://dx.doi.org/10.1085/jgp.202012662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852459PMC
March 2021

Computational Tool for Ensemble Averaging of Single-Molecule Data.

Biophys J 2021 01 26;120(1):10-20. Epub 2020 Nov 26.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Molecular motors couple chemical transitions to conformational changes that perform mechanical work in a wide variety of biological processes. Disruption of this coupling can lead to diseases, and therefore there is a need to accurately measure mechanochemical coupling in motors in both health and disease. Optical tweezers with nanometer spatial and millisecond temporal resolution have provided valuable insights into these processes. However, fluctuations due to Brownian motion can make it difficult to precisely resolve these conformational changes. One powerful analysis technique that has improved our ability to accurately measure mechanochemical coupling in motor proteins is ensemble averaging of individual trajectories. Here, we present a user-friendly computational tool, Software for Precise Analysis of Single Molecules (SPASM), for generating ensemble averages of single-molecule data. This tool utilizes several conceptual advances, including optimized procedures for identifying single-molecule interactions and the implementation of a change-point algorithm, to more precisely resolve molecular transitions. Using both simulated and experimental data, we demonstrate that these advances allow for accurate determination of the mechanics and kinetics of the myosin working stroke with a smaller set of data. Importantly, we provide our open-source MATLAB-based program with a graphical user interface that enables others to readily apply these advances to the analysis of their own data.
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http://dx.doi.org/10.1016/j.bpj.2020.10.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820714PMC
January 2021

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

bioRxiv 2020 Nov 5. Epub 2020 Nov 5.

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.
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http://dx.doi.org/10.1101/2020.11.04.364315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654892PMC
November 2020

Beyond genomics-technological advances improving the molecular characterization and precision treatment of heart failure.

Heart Fail Rev 2021 Mar 3;26(2):405-415. Epub 2020 Sep 3.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8231, St. Louis, MO, 63110, USA.

Dilated cardiomyopathy (DCM) is a major cause of heart failure and cardiovascular mortality. In the past 20 years, there has been an overwhelming focus on developing therapeutics that target common downstream disease pathways thought to be involved in all forms of heart failure independent of the initial etiology. While this strategy is effective at the population level, individual responses vary tremendously and only approximately one third of patients receive benefit from modern heart failure treatments. In this perspective, we propose that DCM should be considered as a collection of diseases with a common phenotype of left ventricular dilation and systolic dysfunction rather than a single disease entity, and that mechanism-based classification of disease subtypes will revolutionize our understanding and clinical approach towards DCM. We discuss how these efforts are central to realizing the potential of precision medicine and how they are empowered by the development of new tools that allow investigators to strategically employ genomic and transcriptomic information. Finally, we outline an investigational strategy to (1) define DCM at the patient level, (2) develop new tools to model and mechanistically dissect subtypes of human heart failure, and (3) harness these insights for the development of precision therapeutics.
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http://dx.doi.org/10.1007/s10741-020-10021-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897225PMC
March 2021

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties.

Elife 2020 05 29;9. Epub 2020 May 29.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine in St. Louis, St. Louis, United States.

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.
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http://dx.doi.org/10.7554/eLife.55132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259954PMC
May 2020

Variant R94C in -Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

J Am Heart Assoc 2020 03 26;9(5):e015111. Epub 2020 Feb 26.

Division of Paediatric Cardiology Department of Pediatrics Duke University School of Medicine Durham NC.

Background Pediatric-onset restrictive cardiomyopathy (RCM) is associated with high mortality, but underlying mechanisms of disease are under investigated. RCM-associated diastolic dysfunction secondary to variants in -encoded cardiac troponin T (TNNT2) is poorly described. Methods and Results Genetic analysis of a proband and kindred with RCM identified TNNT2-R94C, which cosegregated in a family with 2 generations of RCM, ventricular arrhythmias, and sudden death. TNNT2-R94C was absent among large, population-based cohorts Genome Aggregation Database (gnomAD) and predicted to be pathologic by in silico modeling. Biophysical experiments using recombinant human TNNT2-R94C demonstrated impaired cardiac regulation at the molecular level attributed to reduced calcium-dependent blocking of myosin's interaction with the thin filament. Computational modeling predicted a shift in the force-calcium curve for the R94C mutant toward submaximal calcium activation compared within the wild type, suggesting low levels of muscle activation even at resting calcium concentrations and hypercontractility following activation by calcium. Conclusions The pathogenic TNNT2-R94C variant activates thin-filament-mediated sarcomeric contraction at submaximal calcium concentrations, likely resulting in increased muscle tension during diastole and hypercontractility during systole. This describes the proximal biophysical mechanism for development of RCM in this family.
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http://dx.doi.org/10.1161/JAHA.119.015111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335540PMC
March 2020

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy.

Proc Natl Acad Sci U S A 2019 09 19;116(36):17831-17840. Epub 2019 Aug 19.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, ΔK210. We determined the molecular mechanism of ΔK210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that ΔK210 not only reduces contractility but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results help link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.
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http://dx.doi.org/10.1073/pnas.1910962116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731759PMC
September 2019

Computational Tool to Study Perturbations in Muscle Regulation and Its Application to Heart Disease.

Biophys J 2019 06 7;116(12):2246-2252. Epub 2019 May 7.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in the sarcomere and generate pulling forces. This process is regulated by calcium, and it can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations (e.g., β-adrenergic stimulation), and pharmacological interventions. Therefore, it is important to have a methodology to robustly determine the impact of these perturbations and statistically evaluate their effects. Here, we present an approach to measure the equilibrium constants that govern muscle activation, estimate uncertainty in these parameters, and statistically test the effects of perturbations. We provide a MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials that make this approach accessible. The hypothesis testing and error estimation approaches described here are broadly applicable, and the provided tools work with other types of data, including cellular measurements. To demonstrate the utility of the approach, we apply it to elucidate the biophysical mechanism of a mutation that causes familial hypertrophic cardiomyopathy. This approach is generally useful for studying muscle diseases and therapeutic interventions that target muscle contraction.
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http://dx.doi.org/10.1016/j.bpj.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588827PMC
June 2019

Cooperative Changes in Solvent Exposure Identify Cryptic Pockets, Switches, and Allosteric Coupling.

Biophys J 2019 03 25;116(5):818-830. Epub 2019 Jan 25.

Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri; Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, Missouri. Electronic address:

Proteins are dynamic molecules that undergo conformational changes to a broad spectrum of different excited states. Unfortunately, the small populations of these states make it difficult to determine their structures or functional implications. Computer simulations are an increasingly powerful means to identify and characterize functionally relevant excited states. However, this advance has uncovered a further challenge: it can be extremely difficult to identify the most salient features of large simulation data sets. We reasoned that many functionally relevant conformational changes are likely to involve large, cooperative changes to the surfaces that are available to interact with potential binding partners. To examine this hypothesis, we introduce a method that returns a prioritized list of potentially functional conformational changes by segmenting protein structures into clusters of residues that undergo cooperative changes in their solvent exposure, along with the hierarchy of interactions between these groups. We term these groups exposons to distinguish them from other types of clusters that arise in this analysis and others. We demonstrate, using three different model systems, that this method identifies experimentally validated and functionally relevant conformational changes, including conformational switches, allosteric coupling, and cryptic pockets. Our results suggest that key functional sites are hubs in the network of exposons. As a further test of the predictive power of this approach, we apply it to discover cryptic allosteric sites in two different β-lactamase enzymes that are widespread sources of antibiotic resistance. Experimental tests confirm our predictions for both systems. Importantly, we provide the first evidence, to our knowledge, for a cryptic allosteric site in CTX-M-9 β-lactamase. Experimentally testing this prediction did not require any mutations and revealed that this site exerts the most potent allosteric control over activity of any pockets found in β-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 β-lactamase demonstrates the utility of exposons.
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http://dx.doi.org/10.1016/j.bpj.2018.11.3144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400826PMC
March 2019

Genetic and Tissue Engineering Approaches to Modeling the Mechanics of Human Heart Failure for Drug Discovery.

Front Cardiovasc Med 2018 19;5:120. Epub 2018 Sep 19.

InvivoSciences Inc., Madison, WI, United States.

Heart failure is the leading cause of death in the western world and as such, there is a great need for new therapies. Heart failure has a variable presentation in patients and a complex etiology; however, it is fundamentally a condition that affects the mechanics of cardiac contraction, preventing the heart from generating sufficient cardiac output under normal operating pressures. One of the major issues hindering the development of new therapies has been difficulties in developing appropriate model systems of human heart failure that recapitulate the essential changes in cardiac mechanics seen in the disease. Recent advances in stem cell technologies, genetic engineering, and tissue engineering have the potential to revolutionize our ability to model and study heart failure . Here, we review how these technologies are being applied to develop personalized models of heart failure and discover novel therapeutics.
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http://dx.doi.org/10.3389/fcvm.2018.00120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156537PMC
September 2018

Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke.

Nat Commun 2018 09 21;9(1):3838. Epub 2018 Sep 21.

Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, 700A Clinical Research Building, Philadelphia, PA, 19104-6085, USA.

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin's working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.
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http://dx.doi.org/10.1038/s41467-018-06193-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155018PMC
September 2018

Measuring the Kinetic and Mechanical Properties of Non-processive Myosins Using Optical Tweezers.

Methods Mol Biol 2017 ;1486:483-509

Department of Physiology, The Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

The myosin superfamily of molecular motors utilizes energy from ATP hydrolysis to generate force and motility along actin filaments in a diverse array of cellular processes. These motors are structurally, kinetically, and mechanically tuned to their specific molecular roles in the cell. Optical trapping techniques have played a central role in elucidating the mechanisms by which myosins generate force and in exposing the remarkable diversity of myosin functions. Here, we present thorough methods for measuring and analyzing interactions between actin and non-processive myosins using optical trapping techniques.
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http://dx.doi.org/10.1007/978-1-4939-6421-5_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506543PMC
January 2018

MEMLET: An Easy-to-Use Tool for Data Fitting and Model Comparison Using Maximum-Likelihood Estimation.

Biophys J 2016 Jul;111(2):273-282

Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

We present MEMLET (MATLAB-enabled maximum-likelihood estimation tool), a simple-to-use and powerful program for utilizing maximum-likelihood estimation (MLE) for parameter estimation from data produced by single-molecule and other biophysical experiments. The program is written in MATLAB and includes a graphical user interface, making it simple to integrate into the existing workflows of many users without requiring programming knowledge. We give a comparison of MLE and other fitting techniques (e.g., histograms and cumulative frequency distributions), showing how MLE often outperforms other fitting methods. The program includes a variety of features. 1) MEMLET fits probability density functions (PDFs) for many common distributions (exponential, multiexponential, Gaussian, etc.), as well as user-specified PDFs without the need for binning. 2) It can take into account experimental limits on the size of the shortest or longest detectable event (i.e., instrument "dead time") when fitting to PDFs. The proper modification of the PDFs occurs automatically in the program and greatly increases the accuracy of fitting the rates and relative amplitudes in multicomponent exponential fits. 3) MEMLET offers model testing (i.e., single-exponential versus double-exponential) using the log-likelihood ratio technique, which shows whether additional fitting parameters are statistically justifiable. 4) Global fitting can be used to fit data sets from multiple experiments to a common model. 5) Confidence intervals can be determined via bootstrapping utilizing parallel computation to increase performance. Easy-to-follow tutorials show how these features can be used. This program packages all of these techniques into a simple-to-use and well-documented interface to increase the accessibility of MLE fitting.
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http://dx.doi.org/10.1016/j.bpj.2016.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968482PMC
July 2016

A Perspective on the Role of Myosins as Mechanosensors.

Biophys J 2016 Jun;110(12):2568-2576

Pennsylvania Muscle Institute and Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms.
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http://dx.doi.org/10.1016/j.bpj.2016.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919425PMC
June 2016

Mechanochemical tuning of myosin-I by the N-terminal region.

Proc Natl Acad Sci U S A 2015 Jun 8;112(26):E3337-44. Epub 2015 Jun 8.

Pennsylvania Muscle Institute and the Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104

Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post-power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms.
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http://dx.doi.org/10.1073/pnas.1506633112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491760PMC
June 2015

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Traffic 2015 Jul 29;16(7):691-711. Epub 2015 Apr 29.

Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.
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http://dx.doi.org/10.1111/tra.12282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945106PMC
July 2015

Inherent force-dependent properties of β-cardiac myosin contribute to the force-velocity relationship of cardiac muscle.

Biophys J 2014 Dec;107(12):L41-L44

Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

The heart adjusts its power output to meet specific physiological needs through the coordination of several mechanisms, including force-induced changes in contractility of the molecular motor, the β-cardiac myosin (βCM). Despite its importance in driving and regulating cardiac power output, the effect of force on the contractility of a single βCM has not been measured. Using single molecule optical-trapping techniques, we found that βCM has a two-step working stroke. Forces that resist the power stroke slow the myosin-driven contraction by slowing the rate of ADP release, which is the kinetic step that limits fiber shortening. The kinetic properties of βCM are affected by load, suggesting that the properties of myosin contribute to the force-velocity relationship in intact muscle and play an important role in the regulation of cardiac power output.
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http://dx.doi.org/10.1016/j.bpj.2014.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269798PMC
December 2014

A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning.

Proc Natl Acad Sci U S A 2014 Feb 27;111(6):2116-21. Epub 2014 Jan 27.

Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104.

Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor's lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
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http://dx.doi.org/10.1073/pnas.1321022111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926069PMC
February 2014

Regulation and control of myosin-I by the motor and light chain-binding domains.

Trends Cell Biol 2013 Feb 29;23(2):81-9. Epub 2012 Nov 29.

The Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085, USA.

Members of the myosin-I family of molecular motors are expressed in many eukaryotes, where they are involved in a multitude of critical processes. Humans express eight distinct members of the myosin-I family, making it the second largest family of myosins expressed in humans. Despite the high degree of sequence conservation in the motor and light chain-binding domains (LCBDs) of these myosins, recent studies have revealed surprising diversity of function and regulation arising from isoform-specific differences in these domains. Here we review the regulation of myosin-I function and localization by the motor and LCBDs.
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http://dx.doi.org/10.1016/j.tcb.2012.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558615PMC
February 2013

Myosin IC generates power over a range of loads via a new tension-sensing mechanism.

Proc Natl Acad Sci U S A 2012 Sep 20;109(37):E2433-40. Epub 2012 Aug 20.

The Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104-6085, USA.

Myosin IC (myo1c), a widely expressed motor protein that links the actin cytoskeleton to cell membranes, has been associated with numerous cellular processes, including insulin-stimulated transport of GLUT4, mechanosensation in sensory hair cells, endocytosis, transcription of DNA in the nucleus, exocytosis, and membrane trafficking. The molecular role of myo1c in these processes has not been defined, so to better understand myo1c function, we utilized ensemble kinetic and single-molecule techniques to probe myo1c's biochemical and mechanical properties. Utilizing a myo1c construct containing the motor and regulatory domains, we found the force dependence of the actin-attachment lifetime to have two distinct regimes: a force-independent regime at forces < 1 pN, and a highly force-dependent regime at higher loads. In this force-dependent regime, forces that resist the working stroke increase the actin-attachment lifetime. Unexpectedly, the primary force-sensitive transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. This force-sensing behavior is unique amongst characterized myosins and clearly demonstrates mechanochemical diversity within the myosin family. Based on these results, we propose that myo1c functions as a slow transporter rather than a tension-sensitive anchor.
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http://dx.doi.org/10.1073/pnas.1207811109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443183PMC
September 2012

Calcium regulation of myosin-I tension sensing.

Biophys J 2012 Jun 19;102(12):2799-807. Epub 2012 Jun 19.

Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Myo1b is a myosin that is exquisitely sensitive to tension. Its actin-attachment lifetime increases > 50-fold when its working stroke is opposed by 1 pN of force. The long attachment lifetime of myo1b under load raises the question: how are actin attachments that last >50 s in the presence of force regulated? Like most myosins, forces are transmitted to the myo1b motor through a light-chain binding domain that is structurally stabilized by calmodulin, a calcium-binding protein. Thus, we examined the effect of calcium on myo1b motility using ensemble and single-molecule techniques. Calcium accelerates key biochemical transitions on the ATPase pathway, decreases the working-stroke displacement, and greatly reduces the ability of myo1b to sense tension. Thus, calcium provides an effective mechanism for inhibiting motility and terminating long-duration attachments.
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http://dx.doi.org/10.1016/j.bpj.2012.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379621PMC
June 2012

Kinetic schemes for post-synchronized single molecule dynamics.

Biophys J 2012 Mar 20;102(6):L23-5. Epub 2012 Mar 20.

Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Recordings from single molecule experiments can be aggregated to determine average kinetic properties of the system under observation. The kinetics after a synchronized reaction step can be interpreted using all of the standard tools developed for ensemble perturbation experiments. The kinetics leading up to a synchronized event, determined by the lifetimes of the preceding states; however, are not as obvious if the reaction has reversible steps or branches. Here we describe a general procedure for dealing with these situations.
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http://dx.doi.org/10.1016/j.bpj.2012.01.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309285PMC
March 2012

Kinetic schemes for post-synchronized single molecule dynamics.

Biophys J 2012 Mar 20;102(6):L23-5. Epub 2012 Mar 20.

Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Recordings from single molecule experiments can be aggregated to determine average kinetic properties of the system under observation. The kinetics after a synchronized reaction step can be interpreted using all of the standard tools developed for ensemble perturbation experiments. The kinetics leading up to a synchronized event, determined by the lifetimes of the preceding states; however, are not as obvious if the reaction has reversible steps or branches. Here we describe a general procedure for dealing with these situations.
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http://dx.doi.org/10.1016/j.bpj.2012.01.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309285PMC
March 2012

A hearing loss-associated myo1c mutation (R156W) decreases the myosin duty ratio and force sensitivity.

Biochemistry 2011 Mar 15;50(11):1831-8. Epub 2011 Feb 15.

Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, United States.

myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch 1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c(3IQ)) and mutant myo1c (R156W-myo1c(3IQ)) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited >4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but the rate of actin gliding is substantially reduced at low surface densities of R156W-myo1c(3IQ). We used a frictional loading assay to measure the affect of resisting forces on the rate of actin gliding and found that R156W-myo1c(3IQ) is less force-sensitive than myo1c(3IQ). Taken together, these results indicate that myo1c with the R156W mutation has a lower duty ratio than the wild-type protein and motile properties that are less sensitive to resisting forces.
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http://dx.doi.org/10.1021/bi1016777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059334PMC
March 2011

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry.

Proc Natl Acad Sci U S A 2010 Oct 20;107(40):17403-8. Epub 2010 Sep 20.

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q- and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes.
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http://dx.doi.org/10.1073/pnas.1009619107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951453PMC
October 2010

The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors.

Cytoskeleton (Hoboken) 2010 May;67(5):273-85

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Molecular motors convert chemical energy into mechanical movement, generating forces necessary to accomplish an array of cellular functions. Since molecular motors generate force, they typically work under loaded conditions where the motor mechanochemistry is altered by the presence of a load. Several biophysical techniques have been developed to study the loaded behavior and force generating capabilities of molecular motors yet most of these techniques require specialized equipment. The frictional loading assay is a modification to the in vitro motility assay that can be performed on a standard epifluorescence microscope, permitting the high-throughput measurement of the loaded mechanochemistry of molecular motors. Here, we describe a model for the molecular basis of the frictional loading assay by modeling the load as a series of either elastic or viscoelastic elements. The model, which calculates the frictional loads imposed by different binding proteins, permits the measurement of isotonic kinetics, force-velocity relationships, and power curves in the motility assay. We show computationally and experimentally that the frictional load imposed by alpha-actinin, the most widely employed actin binding protein in frictional loading experiments, behaves as a viscoelastic rather than purely elastic load. As a test of the model, we examined the frictional loading behavior of rabbit skeletal muscle myosin under normal and fatigue-like conditions using alpha-actinin as a load. We found that, consistent with fiber studies, fatigue-like conditions cause reductions in myosin isometric force, unloaded sliding velocity, maximal power output, and shift the load at which peak power output occurs.
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http://dx.doi.org/10.1002/cm.20441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861725PMC
May 2010
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