Laser Trap Assay Research Articles

One must reconstitute the functions of interest from purified proteins.

I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the in vitro motility assay and the dual-beam single-molecule laser trap assay for myosin-driven actin filament movement, used widely as key assays for understanding how both muscle and nonmuscle myosin molecular motors work. As for all discoveries, the history of the development of the myosin assays involves many people who are not authors of the final publications, but without whom the assays would not have been developed as they are. Also, early experiences shape how one develops ideas and experiments, and influence future discoveries in major ways. I am pleased here to trace my own path and acknowledge the many individuals involved and my early science experiences that led to the work I and my students, postdoctoral fellows, and sabbatical visitors did to develop these assays. Mentors are too often overlooked in historical descriptions of discoveries, and my story starts with those who mentored me.

Mechanical coupling coordinates microtubule growth

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.

Mechanical coupling coordinates microtubule growth.

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.

A mutation in switch I alters the load-dependent kinetics of myosin Va

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (Pi) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated Pi on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of Pi-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM Pi, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of Pi from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the Pi-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of Pi-rebinding.

Cardiac myosin velocity and force are dramatically improved with an alternative triphosphate substrate.

Cardiac myosin is the molecular motor that drives the pumping action of the heart; therefore, there is strong interest in gaining control over its function in an effort to develop more effective treatments for various forms of heart disease. We sought to determine if the force and motion generating capacity of β-cardiac myosin could be altered in vitro by replacing ATP with different isomers of an alternative energy source, azobenzene triphosphate (AzoTP). Using the ortho-isomer of AzoTP, cardiac myosin moved actin filaments 62% faster than ATP in a motility assay (p<0.001). Subsequent experiments suggested that the increased velocity may be the result of an accelerated rate of ortho-AzoDP-release from myosin. Cardiac myosin also generated 10% more force when using ortho-AzoTP compared to ATP in a laser trap assay (p<0.001), which was likely due to an increased rate of attachment to actin because the frequency of single actomyosin binding events observed in a three-bead laser trap assay was double that observed for ortho-AzoTP vs. ATP (p<0.001). In contrast to ortho-AzoTP, with the meta isomer of AzoTP cardiac myosin translocated actin filaments 90% slower than with ATP (p<0.001). This finding is likely do to a slowed rate of detachment from actin, based on preliminary single molecule findings. With the para-isomer of AzoTP no movement was detected in the motility assay, suggesting it may not bind to the nucleotide-binding site. Thus, these findings demonstrate that the function of beta-cardiac myosin can be manipulated using structural isomers of an alternative energy substrate (AzoTP). These findings therefore may provide important new insights into how to alter the function of cardiac myosin in vivo, which could help in the development of novel therapies to different forms of heart disease.

A point mutation in switch 1 alters the load dependence of phosphate rebinding to actomyosin.

Myosin transduces chemical energy from ATP into mechanical work to generate force and/or motion. Key to this process is the coupling of the powerstroke and the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved remain unclear. We determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10 pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30 mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7 nm for the WT myosin. Repeating these experiments using a construct with a mutation in the Switch I region of the active site (S217A) increased myosin's sensitivity to load five-fold (d = 3.5 nm). Thus, these findings provide a quantitative measure of the force-dependence of Pi-rebinding to myosin and suggest that this effect involves the switch I element of the nucleotide-binding pocket.

A mutation in the switch 1 region of myosin VA alters the load-dependence of phosphate rebinding

The load-dependence of reversible product release in myosins is key to understanding how it converts chemical energy into mechanical force. Following the powerstroke and phosphate (Pi) release, the rebinding of Pi to the actomyosin⋅ADP complex is highly load-dependent, but the underlying mechanisms/structures involved remain unclear. Therefore, we introduced a mutation (S217A) into the switch I region of the active site to examine its impact on the load dependence of Pi rebinding to single-headed myosin Va (MV) in a mini-ensemble (∼10 heads) three-bead laser trap assay at 100 uM ATP.

Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disassembling Microtubule Tips.

Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or RNA. Often the filamentous substrates serve merely as passive tracks for mechanoenzymes but microtubules and F-actin are themselves dynamic protein polymers, capable of generating movement and force independently of conventional motors. Microtubule-driven forces are particularly important during mitosis, when they align duplicated chromosomes at the metaphase plate and then pull them apart during anaphase. These vital movements depend on specialized protein assemblies called kinetochores that couple the chromosomes to the tips of dynamic microtubule filaments, thereby allowing filament shortening to produce pulling forces. Although great strides have been made toward understanding the structures and functions of many kinetochore subcomplexes, the biophysical basis for their coupling to microtubule tips remains unclear. During tip disassembly, strain energy is released when straight protofilaments in the microtubule lattice curl outward, creating a conformational wave that propagates down the microtubule. A popular viewpoint is that the protofilaments as they curl outward hook elements of the kinetochore and tug on them, transferring some of their curvature strain energy to the kinetochore. As a first step toward testing this idea, we recently developed a laser trap assay to directly measure the working strokes generated by curling protofilaments. Our "wave" assay is based on an earlier pioneering study, with improvements that allow measurement of curl-driven movements as functions of force and quantification of their conformational strain energy. In this chapter, we provide a detailed protocol for our assay and describe briefly our instrument setup and data analysis methods.

Recent insights into the relative timing of myosin's powerstroke and release of phosphate.

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force-generating powerstroke, and the release of phosphate (Pi ) from the nucleotide-binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High-resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi -release precedes the powerstroke. However, advances in functional assays, enabling sub-millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Myosin's powerstroke occurs prior to the release of phosphate from the active site.

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (Pi ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining Pi in its active site, by either elevating Pi in solution or by introducing a mutation in myosin's active site (S217A) to slow Pi -release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s-1 ) and without a detectable delay, both in the absence and presence of 30 mM Pi . The elevated levels of Pi did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that Pi rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower Pi release rate. These findings provide direct evidence that myosin Va generates a powerstroke with Pi still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of Pi from the active site.

Tirasemtiv and dATP Synergistically Reverse the Acidosis-induced Depression of Myosin’s Force and Motion Generating Capacity

In response to repeated, intense contractions muscle reduces its ability to generate force and velocity, characterizing fatigue. This reduction is due, in part, to the depressive direct effects of acidosis on myosin and thin-filament function, particularly at sub-saturating calcium concentrations. We sought to reverse these effects in vitro by 1) replacing ATP with 2-deoxy ATP (dATP), and 2) the addition of the troponin activator tirasemtiv (TR). We used a mini-ensemble laser trap assay to measure the force generating capacity with a regulated thin filament at pCa 7. Acidosis (pH 6.8 vs 7.4) significantly decreased myosin's force-generating capacity (0.80 ± 0.23 vs. 0.93 ± 0.29 pN; p < 0.05, at 6.8 and 7.4 respectively) as measured in a mini-ensemble laser trap assay. However, replacing the ATP with dATP and adding TR at pH 6.8 caused force to not only recover but increase above the control value at pH 7.4 (0.93 ± 0.007 vs. 1.32 ± 0.001; p < 0.05, for 7.4 and 6.8 respectively). The increased force at low pH (6.8) appeared to be due to an increased rate of myosin's attachment to the thin filament because the frequency of binding events was significantly increased under acidic conditions with dATP and TR (2.34 ± 2.94) added compared to ATP (0.47 ± 1.16; p < 0.05). Overall, these findings suggest that acidosis directly inhibits myosin's ability to bind and translocate a regulated actin filament, and that these agents that increase myosin's rate of attachment to actin can reverse these effects in vitro. Thus, these results suggest that these agents might offer hope in mitigating the deleterious effects of muscle fatigue in clinical populations.

Myosin'S Powerstroke Occurs with Phosphate Still in the Active Site

A fundamental property of myosin is its ability to harness the power of ATP hydrolysis to generate force. A key unanswered question is the timing of the force-generating powerstroke relative to phosphate (Pi) -release from its active site. We examined the ability of single-headed myosin Va (MV) construct to bind to actin and generate a powerstroke in a single molecule laser trap assay in the presence of elevated Pi. We examined both WT MV and the impact of a mutation (S217A) in the conserved switch I region of the active site which slows actin-activated Pi-release. Upon binding to actin WT myosin rapidly (>500/s) generated a 7±0.5 nm powerstroke. In the presence of 30mM Pi, no decrease in the size (7±0.4 nm) or delay in the rate of the development of the powerstroke (>500/s) was observed, however the average duration of strong actomyosin binding (ton) was reduced in the presence of Pi (240±20 vs 113± ms, p<0.05). The reduced ton is consistent with Pi rebinding to the active site and accelerating detachment from actin. Therefore, this suggests myosin generates the powerstroke with Pi still in the active site. S217A generated a powerstroke with a similar rate (>500/s) and size (9±0.9 nm) to the WT, but ton was shorter, but not significantly different, than WT (143±20 ms, p=0.22 and 62±5 ms, p<0.05 for S217A in the absence and presence of Pi, respectively). Since the S217A mutant slows actin-activated Pi-release from the active site our results fit best to a model in which the powerstroke occurs with Pi still in the active site. Together these findings suggest that actin-binding, and not the release of Pi from the active site, trigger the conformational changes that lead to the powerstroke, and thus force-generation by myosin.

FRET and Optical Trapping Measurements Reveal Relationship between Phosphate Release and the Power Stroke in Myosin V

Myosin V is able to processively walk along actin filaments by coordinating its mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. Our previous FRET measurements of the power stroke in myosin V containing a single IQ domain (MV 1IQ) revealed a fast rotation of the lever arm prior to the phosphate release step and a slower rotation of the lever that occurs during the transition between actomyosin.ADP states. Here we introduce a mutation in the active site (S217A) that is proposed to slow entry of phosphate into the exit tunnel. The mutation causes both fast lever arm rotation and phosphate release to occur with the same rate constant (slowing the maximum rate 10-fold), suggesting entry of phosphate into the exit tunnel gates the lever arm swing. In single molecule laser trapping studies we found the step size of S217A was similar to WT (4nm). Furthermore, raising the Pi concentration in the laser trap assay, which should increase the population of the actomyosin.ADP.Pi state, also did not affect the step size of S217A MV 1IQ. Interestingly, the attached time increased dramatically in the presence of 15 mM Pi. We also found that Pi can inhibit actin gliding in the vitro motility assay in both S217A and WT MV 1IQ in a dose-dependent manner, which is consistent with the optical trapping studies, while Pi did not impact steady-state ATPase activity. Our results suggest a model in which attachment to actin and the power stroke (lever arm swing) occur rapidly before phosphate is released in myosin V. In addition, Pi rebinding to the actomyosin.ADP state may be capable of reversing the power stroke without altering the actin binding interface.

Timing and Load-dependence of the Powerstroke and Pi-release in Skeletal Muscle Myosin

Muscle myosin converts chemical energy from ATP into mechanical work to power contraction, however it is still unclear how this transduction occurs at the molecular level. A key unanswered question is whether phosphate release precedes the powerstroke or if the powerstroke occurs first. This has been a challenging question to answer because these events occur faster than the time resolution of most instruments. Therefore, we used an ultrafast single molecule laser trap assay, with sub-millisecond time resolution, to directly visualize the timing of the powerstorke in fast skeletal muscle myosin. By elevating the level of phosphate in the buffer (15 mM) under varying ATP concentration, we ensured that myosin remained in a phosphate (Pi) bound state. We observed that myosin generated a powerstroke in the presence of phosphate that was similar in size (∼5 nm) and rate (∼5000 s−1 at 3 pN resistive force) to that observed in the absence of phosphate. This suggests that myosin generates its powerstroke prior to the release of phosphate. Further analyses are under way to determine if elevated levels of phosphate induced reversal of the powerstroke later in the binding event. Additional experiments were done over a range of assistive and resistive loads, from −6pN to +6pN, to determine the load-dependence of the powerstroke and the effect on myosin detachment rate from different chemomechanical states. Thus these data provide novel insights into the fundamental nature of force generation by muscle myosin.

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.

Mechanisms Through Which Agents Of Muscle Fatigue, Acidosis And Phosphate, Inhibit Muscle Myosin Function.

PURPOSE: During muscle fatigue from intense activity, elevated concentrations of hydrogen ions (acidosis) and inorganic phosphate (Pi) inhibit muscle’s ability to generate force and motion. However, it is not clear exactly how these metabolic by-products reduce the force and enzymatic function of muscle’s molecular motor, myosin. METHODS: To determine these mechanisms we directly measured the effect of these fatigue agents on the force generating capacity of isolated myosin in a laser trap assay and on its ability to hydrolyze ATP in an ATPase assay. RESULTS: Acidosis (pH 7.4 vs. 6.5) in a mini-ensemble laser trap assay reduced myosin’s average force production by 20% (p < 0.05) due to a slowed rate of actomyosin binding. This conclusion was supported by the observation that acidosis slowed myosin’s ability to hydrolyze ATP by roughly 90% (p < 0.05) in a solution assay. By contrast elevated levels of Pi (0 vs. 10-15mM), in the presence of low pH (6.5), caused a similar reduction in force. However, this was likely due to an accelerated rate of myosin’s detachment from actin, because myosin’s ATPase rate also recovered back toward the control value (pH 7.4, no Pi) when Pi was added. CONCLUSION: Thus, these data provide unique insight into the molecular mechanisms that underlie the loss of muscle function during fatigue. In our current work we are using these findings to explore methods to mitigate these effects in vitro in a first step toward attenuating fatigue in diseases such as chronic heart failure.

An estimate to the first approximation of microtubule rupture force.

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.

Acidosis affects muscle contraction by slowing the rates myosin attaches to and detaches from actin.

The loss of muscle force and power during fatigue from intense contractile activity is associated with, and likely caused by, elevated levels of phosphate ([Formula: see text]) and hydrogen ions (decreased pH). To understand how these deficits in muscle performance occur at the molecular level, we used direct measurements of mini-ensembles of myosin generating force in the laser trap assay at pH 7.4 and 6.5. The data are consistent with a mechanochemical model in which a decrease in pH reduces myosin's detachment from actin (by slowing ADP release), increases non-productive myosin binding (by detached myosin rebinding without a powerstroke), and reduces myosin's attachment to actin (by slowing the weak-to-strong binding transition). Additional support of this mechanism is found by incorporating it into a branched pathway model for the effects of [Formula: see text] on myosin's interaction with actin. Including pH-dependence in one additional parameter (acceleration of [Formula: see text]-induced detachment), the model reproduces experimental measurements at high and low pH, and variable [Formula: see text], from the single molecule to large ensemble levels. Furthermore, when scaled up, the model predicts force-velocity relationships that are consistent with muscle fiber measurements. The model suggests that reducing pH has two opposing effects, a decrease in attachment favoring a decrease in muscle force and a decrease in detachment favoring an increase in muscle force. Depending on experimental details, the addition of [Formula: see text] can strengthen one or the other effect, resulting in either synergistic or antagonistic effects. This detailed molecular description suggests a molecular basis for contractile failure during muscle fatigue.

Acidosis and Phosphate Directly Reduce Myosin's Force-Generating Capacity Through Distinct Molecular Mechanisms.

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H+]) and phosphate (Pi) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding Pi to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and Pi also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of Pi under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of Pi and H+ on muscle contraction during fatigue.

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.