Single Myosin Molecule Research Articles

Single-Molecule Investigation of Load-Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin.

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native β-cardiac myosin (βM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for βM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins.

New insights into the cellular and molecular mechanisms of skeletal muscle fatigue: the Marion J. Siegman Award Lectureships.

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases.

Mechanisms of myosin II force generation: insights from novel experimental techniques and approaches.

Myosin II is a molecular motor that converts chemical energy derived from ATP hydrolysis into mechanical work. Myosin II isoforms are responsible for muscle contraction and a range of cell functions relying on the development of force and motion. When the motor attaches to actin, ATP is hydrolyzed and inorganic phosphate (Pi) and ADP are released from its active site. These reactions are coordinated with changes in the structure of myosin, promoting the so-called "power stroke" that causes the sliding of actin filaments. The general features of the myosin-actin interactions are well accepted, but there are critical issues that remain poorly understood, mostly due to technological limitations. In recent years, there has been a significant advance in structural, biochemical, and mechanical methods that have advanced the field considerably. New modeling approaches have also allowed researchers to understand actomyosin interactions at different levels of analysis. This paper reviews recent studies looking into the interaction between myosin II and actin filaments, which leads to power stroke and force generation. It reviews studies conducted with single myosin molecules, myosins working in filaments, muscle sarcomeres, myofibrils, and fibers. It also reviews the mathematical models that have been used to understand the mechanics of myosin II in approaches focusing on single molecules to ensembles. Finally, it includes brief sections on translational aspects, how changes in the myosin motor by mutations and/or posttranslational modifications may cause detrimental effects in diseases and aging, among other conditions, and how myosin II has become an emerging drug target.

Variants of the myosin interacting-heads motif.

Under relaxing conditions, the two heads of myosin II interact with each other and with the proximal part (S2) of the myosin tail, establishing the interacting-heads motif (IHM), found in myosin molecules and thick filaments of muscle and nonmuscle cells. The IHM is normally thought of as a single, unique structure, but there are several variants. In the simplest ("canonical") IHM, occurring in most relaxed thick filaments and in heavy meromyosin, the interacting heads bend back and interact with S2, and the motif lies parallel to the filament surface. In one variant, occurring in insect indirect flight muscle, there is no S2-head interaction and the motif is perpendicular to the filament. In a second variant, found in smooth and nonmuscle single myosin molecules in their inhibited (10S) conformation, S2 is shifted ∼20 Å from the canonical form and the tail folds twice and wraps around the interacting heads. These molecule and filament IHM variants have important energetic and pathophysiological consequences. (1) The canonical motif, with S2-head interaction, correlates with the super-relaxed (SRX) state of myosin. The absence of S2-head interaction in insects may account for the lower stability of this IHM and apparent absence of SRX in indirect flight muscle, contributing to the quick initiation of flight in insects. (2) The ∼20 Å shift of S2 in 10S myosin molecules means that S2-head interactions are different from those in the canonical IHM. This variant therefore cannot be used to analyze the impact of myosin mutations on S2-head interactions that occur in filaments, as has been proposed. It can be used, instead, to analyze the structural impact of mutations in smooth and nonmuscle myosin.

Microscale thermophoresis suggests a new model of regulation of cardiac myosin function via interaction with cardiac myosin-binding protein C

The cardiac isoform of myosin-binding protein C (cMyBP-C) is a key regulatory protein found in cardiac myofilaments that can control the activation state of both the actin-containing thin and myosin-containing thick filaments. However, in contrast to thin filament–based mechanisms of regulation, the mechanism of myosin-based regulation by cMyBP-C has yet to be defined in detail. To clarify its function in this process, we used microscale thermophoresis to build an extensive interaction map between cMyBP-C and isolated fragments of β-cardiac myosin. We show here that the regulatory N-terminal domains (C0C2) of cMyBP-C interact with both the myosin head (myosin S1) and tail domains (myosin S2) with micromolar affinity via phosphorylation-independent and phosphorylation-dependent interactions of domain C1 and the cardiac-specific m-motif, respectively. Moreover, we show that the interaction sites with the highest affinity between cMyBP-C and myosin S1 are localized to its central domains, which bind myosin with submicromolar affinity. We identified two separate interaction regions in the central C2C4 and C5C7 segments that compete for the same binding site on myosin S1, suggesting that cMyBP-C can crosslink the two myosin heads of a single myosin molecule and thereby stabilize it in the folded OFF state. Phosphorylation of the cardiac-specific m-motif by protein kinase A had no effect on the binding of either the N-terminal or the central segments to the myosin head domain, suggesting this might therefore represent a constitutively bound state of myosin associated with cMyBP-C. Based on our results, we propose a new model of regulation of cardiac myosin function by cMyBP-C.

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

A reverse stroke characterizes the force generation of cardiac myofilaments, leading to an understanding of heart function

Changes in the molecular properties of cardiac myosin strongly affect the interactions of myosin with actin that result in cardiac contraction and relaxation. However, it remains unclear how myosin molecules work together in cardiac myofilaments and which properties of the individual myosin molecules impact force production to drive cardiac contractility. Here, we measured the force production of cardiac myofilaments using optical tweezers. The measurements revealed that stepwise force generation was associated with a higher frequency of backward steps at lower loads and higher stall forces than those of fast skeletal myofilaments. To understand these unique collective behaviors of cardiac myosin, the dynamic responses of single cardiac and fast skeletal myosin molecules, interacting with actin filaments, were evaluated under load. The cardiac myosin molecules switched among three distinct conformational positions, ranging from pre- to post-power stroke positions, in 1 mM ADP and 0 to 10 mM phosphate solution. In contrast to cardiac myosin, fast skeletal myosin stayed primarily in the post-power stroke position, suggesting that cardiac myosin executes the reverse stroke more frequently than fast skeletal myosin. To elucidate how the reverse stroke affects the force production of myofilaments and possibly heart function, a simulation model was developed that combines the results from the single-molecule and myofilament experiments. The results of this model suggest that the reversal of the cardiac myosin power stroke may be key to characterizing the force output of cardiac myosin ensembles and possibly to facilitating heart contractions.

Saturation of Actin-Myosin Kinetics and Mechanics

Actin-myosin kinetics and mechanics are well-characterized at the level of individual myosin molecules; however, the collective behavior of an ensemble of myosin molecules differs significantly from that of the sum of single myosin molecules. For instance, actin movement generated by one myosin molecule is associated with actin-myosin binding, or attachment, whereas actin movement generated by many myosin molecules, N, is influenced by actin-myosin detachment. Major gaps exist in our understanding of the basic chemical kinetics underlying ensemble myosin mechanics.

Collective Force Generator Model of Muscle Contraction

Muscle contraction results from cyclic interactions of actin-myosin binding coupled to the catalyzed hydrolysis of ATP. Although the mechanics and kinetics of muscle myosins are well established, it is less evident how these single molecule behaviors scale up to the collective behaviors of many myosin molecules working together. We have developed a computer simulation of collective force generation by myosin molecules based on well-established kinetic and mechanical properties of single myosin molecules and use an in-vitro motility assay to test model predictions. A collective force model predicts that even in in vitro motility assays myosin heads collectively generate force in common compliant elements and at sufficiently high numbers, N, reach a stall force Consistent with our collective force model, we show that ATPase activity in a motility assay increases linearly at low myosin density, saturating with actin sliding velocities, V, at high myosin densities. We show that activation of V upon addition of inorganic phosphate, Pi, results from Pi decreasing the auto-inhibiting collective force, which in turn activates the ATPase activity. Collective force generation by myosin results in unexpected emergent mechanical behaviors that offer new interpretations for how muscle works.

The mesa trail and the interacting heads motif of myosin II

Myosin II molecules in the thick filaments of striated muscle form a structure in which the heads interact with each other and fold back onto the tail. This structure, the “interacting heads motif” (IHM), provides a mechanistic basis for the auto-inhibition of myosin in relaxed thick filaments. Similar IHM interactions occur in single myosin molecules of smooth and nonmuscle cells in the switched-off state. In addition to the interaction between the two heads, which inhibits their activity, the IHM also contains an interaction between the motor domain of one head and the initial part (subfragment 2, S2) of the tail. This is thought to be a crucial anchoring interaction that holds the IHM in place on the thick filament. S2 appears to cross the head at a specific location within a broader region of the motor domain known as the myosin mesa. Here, we show that the positive and negative charge distribution in this part of the mesa is complementary to the charge distribution on S2. We have designated this the “mesa trail” owing to its linear path across the mesa. We studied the structural sequence alignment, the location of charged residues on the surface of myosin head atomic models, and the distribution of surface charge potential along the mesa trail in different types of myosin II and in different species. The charge distribution in both the mesa trail and the adjacent S2 is relatively conserved. This suggests a common basis for IHM formation across different myosin IIs, dependent on attraction between complementary charged patches on S2 and the myosin head. Conservation from mammals to insects suggests that the mesa trail/S2 interaction plays a key role in the inhibitory function of the IHM.

Acidosis decreases the Ca2+ sensitivity of thin filaments by preventing the first actomyosin interaction.

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca2+) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca2+ sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly (P < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8-pCa 6) but not at maximal Ca2+ concentration (pCa 5-pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin's powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca2+]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca2+]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca2+ concentrations that might contribute to the loss of force during muscle fatigue.

Stronger than they look

An experimental study of living cells suggests that single myosin molecules are capable of generating unusually large forces. The observation is supported by a theoretical model — and demonstrates the complexity of in vivo force generation.

Controlling load-dependent kinetics of β-cardiac myosin at the single-molecule level.

Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate from actin of myosin, which determines the time that myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which myosin works. Here, we measure the detachment rate of single molecules of human β-cardiac myosin and its load dependence. We find that both can be modulated by both small molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating vs. inhibitory perturbations of cardiac myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and that cardiac contractility can be controlled by tuning the load-dependent kinetics of single myosin molecules.

All play and no work: skits and models in teaching skeletal muscle physiology.

All play and no work: skits and models in teaching skeletal muscle physiology.

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.

Molecular structure of muscle filaments determined by electron microscopy.

Electron microscopy and X-ray diffraction have together played a key role in our understanding of the molecular structure and mechanism of contraction of muscle. This review highlights the role of electron microscopy, from early insights into thick and thin filament structure by negative staining, to studies of single myosin molecule structure, and finally to recent high-resolution structures by cryo-electron microscopy. Muscle filaments are designed for movement. Their labile structures thus present challenges to obtaining near-atomic detail, which are also discussed.

No Difference in Myosin Kinetics and Spatial Distribution of the Lever Arm in the Left and Right Ventricles of Human Hearts.

The systemic circulation offers larger resistance to the blood flow than the pulmonary system. Consequently, the left ventricle (LV) must pump blood with more force than the right ventricle (RV). The question arises whether the stronger pumping action of the LV is due to a more efficient action of left ventricular myosin, or whether it is due to the morphological differences between ventricles. Such a question cannot be answered by studying the entire ventricles or myocytes because any observed differences would be wiped out by averaging the information obtained from trillions of myosin molecules present in a ventricle or myocyte. We therefore searched for the differences between single myosin molecules of the LV and RV of failing hearts In-situ. We show that the parameters that define the mechanical characteristics of working myosin (kinetic rates and the distribution of spatial orientation of myosin lever arm) were the same in both ventricles. These results suggest that there is no difference in the way myosin interacts with thin filaments in myocytes of failing hearts, and suggests that the difference in pumping efficiencies are caused by interactions between muscle proteins other than myosin or that they are purely morphological.

The molecular basis of thin filament activation: from single molecule to muscle

For muscles to effectively power locomotion, trillions of myosin molecules must rapidly attach and detach from the actin thin filament. This is accomplished by precise regulation of the availability of the myosin binding sites on actin (i.e. activation). Both calcium (Ca++) and myosin binding contribute to activation, but both mechanisms are simultaneously active during contraction, making their relative contributions difficult to determine. Further complicating the process, myosin binding accelerates the attachment rate of neighboring myosin molecules, adding a cooperative element to the activation process. To de-convolve these two effects, we directly determined the effect of Ca++ on the rate of attachment of a single myosin molecule to a single regulated actin thin filament, and separately determined the distance over which myosin binding increases the attachment rate of neighboring molecules. Ca++ alone increases myosin’s attachment rate ~50-fold, while myosin binding accelerates attachment of neighboring molecules 400 nm along the actin thin filament.

Effect of Calcium on Myosin Binding to a Regulated Thin Filament from Single Molecule to Ensemble

Muscle contraction is regulated via two processes that both contribute to activation of the thin filament (actin+troponin+tropomyosin). First, calcium (Ca++) displaces tropomyosin, which increases the availability of myosin binding sites on actin. Second, myosin binding accelerates the attachment of neighboring myosin molecules to the thin filament. The relative importance and underlying mechanisms of these two processes remain unclear, therefore we devised assays to separately quantify their contribution . Myosin binding only activates nearby myosins, therefore by varying myosin density we varied the contribution of this process. Myosin-induced activation was eliminated by using a single myosin molecule in the laser trap assay to directly measure Ca++'s effect on myosin's interaction with a thin filament. Slightly increasing the myosin density generated mini ensembles of myosin that experienced weak coupling . Increasing the myosin density further using a motility assay created large myosin ensembles with strong myosin-induced activation, and thin filament speed was measured at increasing ATP and Ca++ levels. The data were analyzed with a mathematical model that de-convolves the effects of Ca++-induced and myosin-induced activation. This analysis revealed (1) how Ca++ affects myosin's attachment to a thin filament; and (2) that myosin molecules are coupled over a Ca++-independent distance L=400nm. Scaling up the model generated accurate predictions of isometric force production and Ca++ binding to the thin filament previously observed in muscle fibers, providing a molecular basis for how Ca++ and myosin contribute to filament activation.

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.