Laser Trap Assay Research Articles

Muscle Fatigue from the Perspective of a Single Crossbridge.

The repeated intense stimulation of skeletal muscle rapidly decreases its force- and motion-generating capacity. This type of fatigue can be temporally correlated with the accumulation of metabolic by-products, including phosphate (Pi) and protons (H). Experiments on skinned single muscle fibers demonstrate that elevated concentrations of these ions can reduce maximal isometric force, unloaded shortening velocity, and peak power, providing strong evidence for a causative role in the fatigue process. This seems to be due, in part, to their direct effect on muscle's molecular motor, myosin, because in assays using isolated proteins, these ions directly inhibit myosin's ability to move actin. Indeed, recent work using a single molecule laser trap assay has revealed the specific steps in the crossbridge cycle affected by these ions. In addition to their direct effects, these ions also indirectly affect myosin by decreasing the sensitivity of the myofilaments to calcium, primarily by altering the ability of the muscle regulatory proteins, troponin and tropomyosin, to govern myosin binding to actin. This effect seems to be partially due to fatigue-dependent alterations in the structure and function of specific subunits of troponin. Parallel efforts to understand the molecular basis of muscle contraction are providing new technological approaches that will allow us to gain unprecedented molecular detail of the fatigue process. This will be crucial to fully understand this ubiquitous phenomenon and develop appropriately targeted therapies to attenuate the debilitating effects of fatigue in clinical populations.

Using Single Myosin and Small Myosin Ensembles to Examine Thin Filament Activation in a Laser Trap Assay

Thin filament activation in muscle requires both Ca++ binding to troponin and myosin strongly binding to actin. Despite extensive study, the relative importance of each of these processes to overall thin filament activation remains unclear. To determine the role of Ca++ we examined the effect of increasing Ca++ concentrations on the frequency of single molecule binding events in a single molecule laser trap assay using reconstituted thin filaments. The frequency of actomyosin binding events increased from 0.03 at low Ca++ (pCa7), to 0.30 at pCa6 to 0.44 events/sec at pCa5. Increasing the myosin concentration so that the regulated filament interacted with a mini-ensemble of myosin (∼6 heads) in the laser trap assay was performed to gain insight into the cooperative nature of actomyosin binding. Binding events were broadly distributed at the highest Ca++ (pCa5), generating forces from 0.2 to 6.75pN, but averaged 1.38±0.05pN. As Ca++ was decreased the high force events were lost, reducing the average force to 0.76pN at pCa6, 0.61 at pCa7 and 0.2pN at pCa9. In addition the total number of actomyosin interactions decreased from 3.7/sec at pCa5 to just 0.05/sec at pCa9. Finally, we measured filament velocity in a motility assay as a function of Ca++ and ATP to distinguish rigor activation vs. Ca++ activation in a large ensemble of myosin. At pCa9 filaments were activated at ATP levels <200 μM while at intermediate Ca++ levels (pCa6.5) filaments achieved only half of the maximum velocity observed at saturating Ca++ (pCa5). Preliminary modeling efforts suggest that these observations are consistent with a model where Ca++ regulates myosin's weak to strong binding transition but once bound strongly an actomyosin interaction activates the filament locally, facilitating neighboring molecules to bind.

The Acidosis-Induced Slowing of Regulated Thin Filament Velocity in a Motility Assay Disappears at Low ATP

Acidosis (low pH) is a putative agent of muscle fatigue that slows contraction velocity but the mechanisms underlying this effect are unclear. It may directly affect myosin by slowing a step associated with detachment or it may act indirectly by affecting the ability of Ca++ to activate the thin filament, through effects on the regulatory proteins troponin/tropmyosin. We measured the effect of manipulating Ca++ and the lifetime rigor binding (by lowering the [ATP]) to examine the relative role of each of these mechanisms. At saturating Ca++ (pCa 4) and ATP concentrations (1mM) decreasing the pH from 7.4 to 6.8 decreased regulated actin filament velocity (Vrtf) by 50%, however at low [ATP] (<200 μM) Vrtf was unaffected by acidosis. Repeating these experiments in the absence of Ca++ (pCa 9) confirmed the phenomenon of rigor activation at normal pH (7.4), with filaments moving fastest at 50 μM ATP (0.5 μm/s). At low pH the filaments moved slower but still demonstrated rigor activation at [ATP] <75 μM. Single molecule laser trap assay experiments are under way to determine whether the slowed velocity was due slowed attachment or detachment kinetics. The acidosis-induced decrease in Vrtf at saturating Ca++ and ATP are consistent with a slowing of the steps in the cross-bridge cycle associated with ADP-release. However this effect disappears at low ATP where the attached lifetime is more dependent on the duration of rigor. Therefore the data suggest that acidosis does not affect the formation of a rigor bond or its ability to activate the thin filament. Inhibition of Ca++ binding to troponin might still contribute to slowing at saturating ATP and submaximal Ca++ levels.

A microfluidic chamber to study the dynamics of muscle-contraction-specific molecular interactions.

In vitro motility and laser trap assays are commonly used for molecular mechanics measurements. However, chemicals cannot be added during these measurements, because they create flows that alter the molecular mechanics. Thus, we designed a microfluidic device that allows the addition of chemicals without creating bulk flows. Biocompatibility of the components of this device was tested. A microchannel chamber was created by photolithography with the patterns transferred to polydimethylsiloxane (PDMS). The PDMS chamber was bound to a polycarbonate membrane, which itself was bound to a molecular mechanics chamber. The microchannels ensured rapid distribution of the chemicals over the membrane, whereas the membrane ensured efficient delivery to the mechanics chamber while preventing bulk flow. The biocompatibility of the materials was tested by comparing the velocity (ν(max)) of propulsion by myosin of fluorescently labeled actin filaments to that of the conventional assay; no difference in ν(max) was observed. To estimate total chemical delivery time, labeled bovine serum albumin was injected in the channel chamber and TIRF was used to determine the time to reach the assay surface (2.7 ± 0.1 s). Furthermore, the standard distance of a trapped microsphere calculated during buffer diffusion using the microfluidic device (14.9 ± 3.2 nm) was not different from that calculated using the conventional assay (15.6 ± 5.3 nm, p = 0.922). Finally, ν(max) obtained by injecting adenosine triphosphate (ATP) in the microchannel chamber (2.37 ± 0.48 μm/s) was not different from that obtained when ATP was delivered directly to the mechanics chamber (2.52 ± 0.42 μm/s, p = 0.822). This microfluidic prototype validates the design for molecular mechanics measurements.

Observing the pCa-Force Relationship with a 3-Bead Laser Trap Assay

Muscular force is highly dependent on the amount of free Ca++ in the myoplasm because actomyosin binding is carefully regulated by troponin (Tn) and tropomyosin (Tm). To gain molecular insight into the Ca++ dependence of force, we directly observed the effect of Ca++ on the force-generating capacity of a mini-ensemble of myosin (∼6 heads) interacting with a single reconstituted thin filament (actin+Tn+Tm) in a three bead laser trap assay at 100uM ATP. Maximum force decreased in a Ca++-dependent manner (6.75, 4.75, 3.25, and 1.85pN at pCa 5, 6, 6.5 and 7 respectively). Furthermore, the average forces were significantly different (p 1.5pN where only 0.1% of the events achieved this force level at pCa7. In addition, the total percent of time the myosin molecules were strongly bound to the thin filament were 41, 21, 16, and 14% at pCa 5, 6, 6.5 and 7 respectively. Overall, these data provide unique insight into the molecular events that underlie the Ca++-dependence of force generation in a regulated system.

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

BackgroundStudies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. MethodsTo measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. ResultsFunb from unregulated actin (0.10±0.01pN) was significantly increased in the presence of CaD (0.17±0.02pN), tropomyosin (0.17±0.02pN) or both regulatory proteins (0.18±0.02pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06±0.01pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. ConclusionsCaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. General significanceThis study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

BackgroundSmooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. MethodsTo measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. ResultsFunb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). ConclusionsThis study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. General significanceThis study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

Direct observation of phosphate inhibiting the force generating capacity of myosin using a laser trap assay

The accumulation of phosphate (Pi) is believed to cause much of the loss in the force during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force‐generating capacity of a mini‐ensemble of skeletal myosin (~8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent, greatly reducing the average force produced. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force‐generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.

The Effect of Phosphate on the Force Generating Capacity of a Mini-Ensemble of Myosin Measured in a Laser Trap Assay

The accumulation of inorganic phosphate (Pi) is believed to cause much of the loss in the force-generating capacity of muscle during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force-generating capacity of a mini-ensemble of skeletal myosin (∼8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force-generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.

Calponin Cross-Links Unphosphorylated Myosin to Actin and Enhances its Binding Force

Smooth muscle is unique in its capability to maintain force for long periods of time at low ATP (energy) consumption. This property, called the latch-state, is hypothesized to occur due to the dephosphorylation of myosin while attached to actin. Alternative theories have proposed that dephosphorylated-detached myosin can also re-attach to actin and contribute to force maintenance. The goal of this study was to investigate the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To confirm the calponin regulation of our actin filaments at physiological concentrations, we used the in-vitro motility assay to measure the rate (1/2max) of actin propulsion by myosin. Our results showed a significant decrease in 1/2max (from 0.54±0.01 μm/s (mean±SE) in the absence to 0.43±0.01 μm/s in the presence of calponin). When calponin was phosphorylated, this inhibitory effect was suppressed (0.50±0.01 μm/s). To measure the force of binding (Funb) of unphosphorylated myosin to actin, we used the laser trap assay. Funb was normalized by the number of myosin molecules estimated per actin filament length. Funb of unregulated actin (0.12±0.01 pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02 pN). When calponin was phosphorylated, this enhancement was lost (0.12±0.01 pN). To verify whether this enhancement of Funb is due to cross-linking of myosinto actin by calponin, we repeated the measurements at high [KCl], as the calponin affinity for myosin decreases at high ionic strength. Indeed, the Funb, in presence of calponin, obtained at a [KCl] of 25mM (0.21±0.02 pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01 pN). These data demonstrate that calponin enhances the force of binding of unphosphorylated myosin to actin by cross-linking them together.

Path-Finding on the Microtubule

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules, mimicking cargo transport as accurately as possible. In stark contrast to kinesin-1, heterodimeric kinesin-2 motors from diverse organisms displayed an astounding range of rotational pitches, expanding the list of torque generating kinesins to include processive representatives of the kinesin-2 family. We provide direct evidence that neck region of kinesin determines the torque generating properties by reversibly manipulating the properties of the neck of the human kinesin-1 and the heterodimeric kinesin-2 from sea urchin. Disrupting the stability of the neck by inserting flexible peptide stretches results in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduces the spiraling of the motor up to the point of protofilament tracking.

Recent Insights into the Molecular Basis of Muscular Fatigue

The cause of muscle fatigue has been studied for more than 100 yr, yet its molecular basis remains poorly understood. Prevailing theories suggest that much of the fatigue-induced loss in force and velocity can be attributed to the inhibitory action of metabolites, principally phosphate (Pi) and hydrogen ions (H, i.e., acidosis), on the contractile proteins, but the precise detail of how this inhibition occurs has been difficult to visualize at the molecular level. However, recent technological developments in the areas of biophysics, molecular biology, and structural biology are enabling researchers to directly observe the function and dysfunction of muscle contractile proteins at the level of a single molecule. In fact, the first direct evidence that high levels of H and Pi inhibit the function of muscle's molecular motor, myosin, has recently been observed in a single molecule laser trap assay. Likewise, advances in structural biology are taking our understanding further, providing detail at the atomic level of how some metabolites might alter the internal motions of myosin and thereby inhibit its ability to generate force and motion. Finally, new insights are also being gained into the indirect role that muscle regulatory proteins troponin (Tn) and tropomyosin (Tn) play in the fatigue process. In vitro studies, incorporating TnTm, suggest that a significant portion of the decreased force and motion during fatigue may be mediated through a disruption of the molecular motions of specific regions within Tn and Tm. These recent advances are providing unprecedented molecular insight into the structure and function of the contractile proteins and, in the process, are reshaping our understanding of the process of fatigue.

The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0-C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1-C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0-C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0-C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0-C3 in proportion to the total number phosphomimetic replacements. Under load, C0-C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0-C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0-C3 on F:V was well fitted by a model that assumed C0-C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.

Torque Generation of Kinesin Motors Is Governed by the Stability of the Neck Domain

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules. In stark contrast to kinesin-1, these motors display a wide range of left-handed spiraling around microtubules and thus generate torque during cargo transport. We provide direct evidence that kinesin's neck region determines the torque-generating properties. A model system based on kinesin-1 corroborates this result: disrupting the stability of the neck by inserting flexible peptide stretches resulted in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduced the spiraling of the motor up to the point of protofilament tracking. Finally, we present a model that explains the physical basis of kinesin's spiraling around the microtubule.

Biophysical Study of Native Yeast Kinetochores Indicates Distinct Roles for Phospho-Regulation of Core Microtubule-Binding Subcomplexes

Chromosome segregation is orchestrated by multi-protein complexes called kinetochores, which initially bind microtubules and later form persistent load-bearing tip attachments. Accurate segregation requires regulation of kinetochores by Aurora B kinase. One popular view is that Aurora acts on kinetochores that have already established erroneous attachments to microtubule tips, phosphorylating two major microtubule-binding subcomplexes, Ndc80 and Dam1, and thereby triggering detachment. It is unknown whether phosphorylation affects initial binding of kinetochores to microtubules. Moreover, the relative importance of phosphoregulation of these subcomplexes in the context of whole kinetochores is unclear. To address these uncertainties, we relied on recently developed laser trap and fluorescence-based assays (Akiyoshi et al. Nature 2010) in which individual native kinetochore particles purified from budding yeast are coupled to single microtubules. Neither phospho-mimetic mutations at kinase target sites on Dam1 nor a mutation that disrupts the Dam1 complex had a strong effect on the initial binding of kinetochore-coated beads to microtubules. By contrast, phospho-mimetic mutations on Ndc80 reduced binding 3-fold. In force-ramp assays with tip-attached kinetochores, phospho-mimetic mutations on either Ndc80 or Dam1 reduced rupture forces roughly 1.5-fold relative to wild type and phospho-deficient controls. Likewise, in force-clamp experiments, phospho-mimetic kinetochores detached up to 8-fold more quickly, depending on the level of force. Taken together, these results indicate that Ndc80 and Dam1 play distinct roles in kinetochore function. Initial binding is dependent mainly on Ndc80 and modulated strongly by phosphorylation of Ndc80. Tip coupling depends on both Ndc80 and Dam1 and it is modulated by phosphorylation of both.

Recent insights into muscle fatigue at the cross-bridge level.

The depression in force and/or velocity associated with muscular fatigue can be the result of a failure at any level, from the initial events in the motor cortex of the brain to the formation of an actomyosin cross-bridge in the muscle cell. Since all the force and motion generated by muscle ultimately derives from the cyclical interaction of actin and myosin, researchers have focused heavily on the impact of the accumulation of intracellular metabolites [e.g., Pi, H+ and adenosine diphoshphate (ADP)] on the function these contractile proteins. At saturating Ca++ levels, elevated Pi appears to be the primary cause for the loss in maximal isometric force, while increased [H+] and possibly ADP act to slow unloaded shortening velocity in single muscle fibers, suggesting a causative role in muscular fatigue. However the precise mechanisms through which these metabolites might affect the individual function of the contractile proteins remain unclear because intact muscle is a highly complex structure. To simplify problem isolated actin and myosin have been studied in the in vitro motility assay and more recently the single molecule laser trap assay with the findings showing that both Pi and H+ alter single actomyosin function in unique ways. In addition to these new insights, we are also gaining important information about the roles played by the muscle regulatory proteins troponin (Tn) and tropomyosin (Tm) in the fatigue process. In vitro studies, suggest that both the acidosis and elevated levels of Pi can inhibit velocity and force at sub-saturating levels of Ca++ in the presence of Tn and Tm and that this inhibition can be greater than that observed in the absence of regulation. To understand the molecular basis of the role of regulatory proteins in the fatigue process researchers are taking advantage of modern molecular biological techniques to manipulate the structure and function of Tn/Tm. These efforts are beginning to reveal the relevant structures and how their functions might be altered during fatigue. Thus, it is a very exciting time to study muscle fatigue because the technological advances occurring in the fields of biophysics and molecular biology are providing researchers with the ability to directly test long held hypotheses and consequently reshaping our understanding of this age-old question.

Phosphorylation of Cardiac Myosin Binding Protein-C Acts as On/Off Switch to Modulate cMyBP-C's Inhibition of Actomyosin Power Generation in the Load-Clamp Laser Trap Assay

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains- C0-C10, binds myosin LMM via its C-terminus, while its N-terminus binds both myosin S2 and actin. These N-terminal interactions can be attenuated by phosphorylation of serines within the C1-C2 linker. In vivo, cMyBP-C exists in a range of phosphorylation states, which may alter its regulation of actomyosin. Therefore, N-terminal fragments (C0-C3) of cMyBP-C were bacterially expressed with one or more phosphorylatable serines (S273, S282, S302) mutated to either alanines to mimic dephosphorylation or aspartic acids as phosphomimetics. Using C0-C3 fragments that were effectively mono-, di- or tri-phosphorylated, we characterized how the extent of cMyBP-C phosphorylation modulated the the force:velocity relationship generated by a small ensemble (∼8 motors) of skeletal muscle myosin in the load-clamped laser trap assay. Mono-phosphorylated C0-C3 inhibited unloaded velocity by 70% as did unphosphorylated C0-C3, while di- and tri-phosphorylated constructs were indistinguishable and less inhibitory (30%). Maximal force was unaffected by the extent of phosphorylation. Thus, the un- and mono-phosphorylated C0-C3 constructs depressed the force-velocity relationship, which was well fitted by a modified Hill equation containing an assumed viscous load due to C0-C3 binding to actin (Weith et al., 2011). In addition, both un- and mono- phosphorylated C0-C3 reduced peak power by ∼40%, while neither di- nor tri-phosphorylated C0-C3 had any effect on peak power. cMyBP-C phosphorylation appears to modulate cMyBP-C's inhibition of actomyosin power generation in an “on/off” manner.

Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20ms) and long (~300ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity