Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties.

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in Nc. Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

Molecular mechanics of cardiac myosin-binding protein C in native thick filaments.

The heart's pumping capacity results from highly regulated interactions of actomyosin molecular motors. Mutations in the gene for a potential regulator of these motors, cardiac myosin-binding protein C (cMyBP-C), cause hypertrophic cardiomyopathy. However, cMyBP-C's ability to modulate cardiac contractility is not well understood. Using single-particle fluorescence imaging techniques, transgenic protein expression, proteomics, and modeling, we found that cMyBP-C slowed actomyosin motion generation in native cardiac thick filaments. This mechanical effect was localized to where cMyBP-C resides within the thick filament (i.e., the C-zones) and was modulated by phosphorylation and site-specific proteolytic degradation. These results provide molecular insight into why cMyBP-C should be considered a member of a tripartite complex with actin and myosin that allows fine tuning of cardiac muscle contraction.

Effect of low pH on single skeletal muscle myosin mechanics and kinetics.

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.

Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay.

Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity (F(max)) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. F(max) was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas F(max) was severely depressed for one of the DCM mutants (65% S532P). Although F(max) was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity (V(actin)) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These F(max) data combined with previous V(actin) measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM.

Myosin V exhibits a high duty cycle and large unitary displacement.

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance.

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.

Smooth muscle myosin heavy chain isoforms and their role in muscle physiology.

Unlike vertebrate skeletal muscle, smooth muscle myosin heavy chain isoforms are encoded by a single gene. Alternative splicing of the primary transcript from a single gene generates four smooth muscle myosin heavy chain isoforms. These isoforms differ both at the carboxyl terminus (SM1 and SM2 isoforms) and at the amino terminus (SM-A and SM-B isoforms). The smooth muscle myosin heavy chain isoforms are differentially expressed during smooth muscle development and in different smooth muscle cell types. The mechanical properties of smooth muscle may be correlated with the myosin heavy chain content/isoform expression. However, the precise function of each smooth muscle myosin heavy chain isoform to muscle contraction remains to be determined. This review mainly focuses on the molecular basis of smooth muscle myosin heavy chain isoform diversity, its expression during development and disease, and its role in muscle physiology.

In vivo analysis of an essential myosin light chain mutation linked to familial hypertrophic cardiomyopathy.

Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutations in both the regulatory (Glu22Lys) and essential light chains (Met149Val) result in an unusual pattern of hypertrophy, leading to obstruction of the midventricular cavity. When a human genomic fragment containing the Met149Val essential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguously establish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, we generated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the coding regions of the gene loci. Expression of the proteins did not lead to a hypertrophic response, even in senescent animals. Changes did occur at the myofilament and cellular levels, with the myofibrils showing increased Ca(2+) sensitivity and significant deficits in relaxation in a transgene dose-dependent manner. Clearly, mice do not always recapitulate important aspects of human hypertrophy. However, because of the discordance of these data with data obtained in transgenic mice containing the human genomic fragment, we believe that the concept that these point mutations by themselves can cause hypertrophic cardiomyopathy should be revisited.

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever.

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

Functional consequences of mutations in the smooth muscle myosin heavy chain at sites implicated in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is frequently associated with mutations in the beta-cardiac myosin heavy chain. Many of the implicated residues are located in highly conserved regions of the myosin II class, suggesting that these mutations may impair the basic functions of the molecular motor. To test this hypothesis, we have prepared recombinant smooth muscle heavy meromyosin with mutations at sites homologous to those associated with FHC by using a baculovirus/insect cell expression system. Several of the heavy meromyosin mutants, in particular R403Q, showed an increase in actin filament velocity in a motility assay and an enhanced actin-activated ATPase activity. Single molecule mechanics, using a laser trap, gave unitary displacements and forces for the mutants that were similar to wild type, but the attachment times to actin following a unitary displacement were markedly reduced. These results suggest that the increases in activity are due to a change in kinetics and not due to a change in the intrinsic mechanical properties of the motor. In contrast to earlier reports, we find that mutations in residues implicated in FHC affect motor function by enhancing myosin activity rather than by a loss of function.

Single-molecule mechanics of R403Q cardiac myosin isolated from the mouse model of familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that can result in sudden death in the absence of any overt symptoms. Many of the cases documented to date have been linked with missense mutations in the beta-myosin heavy chain gene. Here we present data detailing the functional impact of one of the most deadly mutations, R403Q, on myosin motor function. Experiments were performed on whole cardiac myosin purified from a mouse model of FHC to eliminate potential uncertainties associated with protein expression systems. The R403Q mutant myosin demonstrated 2.3-fold higher actin-activated ATPase activity, 2.2-fold greater average force generation, and 1.6-fold faster actin filament sliding in the motility assay. The force- and displacement-generating capacities of both the normal and mutant myosin were also characterized at the single molecule level in the laser trap assay. Both control and mutant generated similar unitary forces ( approximately 1 pN) and displacements ( approximately 7 nm) without any differences in event durations. On the basis of the distribution of mean unitary displacements, this mutation may possibly perturb the mechanical coordination between the 2 heads of cardiac myosin. Any of these observations could, alone or possibly in combination, result in abnormal power output and potentially a stimulus for the hypertrophic response.

R403Q and L908V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level.

Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the beta-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (v(actin)). At the single molecule level, v(actin) approximately d/t(on), where d is the myosin unitary step displacement and t(on) is the step duration. Laser trap studies were performed at 10 microM MgATP to estimate d and t(on) for the normal and mutant myosin molecules. The increase in v(actin) can be explained by a significant decrease in the average t(on)'s in both the R403Q and L908V mutants (approximately 30 ms) compared to controls (approximately 40 ms), while d was not different for all myosins tested (approximately 7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosin's inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.

Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament.

Muscle contraction is the result of myosin cross-bridges (XBs) cyclically interacting with the actin-containing thin filament. This interaction is modulated by the thin filament regulatory proteins, troponin and tropomyosin (Tm). With the use of an in vitro motility assay, the role of Tm in myosin's ability to generate force and motion was assessed. At saturating myosin surface densities, Tm had no effect on thin filament velocity. However, below 50% myosin saturation, a significant reduction in actin-Tm filament velocity was observed, with complete inhibition of movement occurring at 12. 5% of saturating surface densities. Under similar conditions, actin filaments alone demonstrated no reduction in velocity. The effect of Tm on force generation was assessed at the level of a single thin filament. In the absence of Tm, isometric force was a linear function of the density of myosin on the motility surface. At 50% myosin surface saturation, the presence of Tm resulted in a 2-fold enhancement of force relative to actin alone. However, no further potentiation of force was observed with Tm at saturating myosin surface densities. These results indicate that, in the presence of Tm, the strong binding of myosin cooperatively activates the thin filament. The inhibition of velocity at low myosin densities and the potentiation of force at higher myosin densities suggest that Tm can directly modulate the kinetics of a single myosin XB and the recruitment of a population of XBs, respectively. At saturating myosin conditions, Tm does not appear to affect the recruitment or the kinetics of myosin XBs.

Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms.

1. Cardiac V3 myosin generates slower actin filament velocities and higher average isometric forces (in an in vitro motility assay) when compared with the V1 isoform. 2. To account for differences in V1 and V3 force and motion generation at the molecular level, we characterized the mechanics and kinetics of single V1 and V3 myosin molecules using a dual laser trap setup. 3. No differences in either unitary displacement (approximately 7 nm) or force (approximately 0.8 pN) were observed between isoforms; however, the duration of unitary displacement events was significantly longer for the V3 isoform at MgATP concentrations > 10 microM. 4. Our results were interpreted on the basis of a cross-bridge model in which displacement event durations were determined by the rates of MgADP release from, and MgATP binding to, myosin. 5. We propose that the release rate of MgADP from V3 myosin is half that of V1 myosin without any difference in their rates of MgATP binding; thus, kinetic differences between the two cardiac myosin isoforms are sufficient to account for their functional diversity.

Two heads of myosin are better than one for generating force and motion.

Several classes of the myosin superfamily are distinguished by their "double-headed" structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth- and skeletal-muscle myosins. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.

The molecular mechanics of smooth muscle myosin.

Smooth muscle cells are capable of generating forces comparable to those of skeletal muscle cells but with far less myosin, the molecular motor that powers muscle contraction. This unique capability may be inherent to the myosin molecule. We have directly characterized the molecular mechanics of smooth muscle myosin using new technologies developed to measure the forces generated by these proteins. The data help explain the differences in force and velocity in whole smooth and skeletal muscles.

Myosin conformational states determined by single fluorophore polarization.

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke.

Fluorescent phalloidin enables visualization of actin without effects on myosin's actin filament sliding velocity and hydrolytic properties in vitro.

Recent reports have demonstrated an activating effect of phalloidin in striated muscle. Furthermore, modeling of X-ray diffraction and crystallographic data suggests that phalloidin binding may induce conformational changes in actin. To determine whether phalloidin affects the mechanics of the actomyosin interaction, the velocity of actin filaments variably labeled with rhodamine-phalloidin was measured. In addition, solution actin-activated myosin subfragment-1 ATPase activity with phalloidin-labeled actin was compared to unlabeled actin. Here we found that phalloidin does not significantly effect actin filament velocity or parameters of ATPase, namely Vmax and K(m). Possible differences between muscle strip data and these in vitro results are discussed.

A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap.

Two smooth muscle myosin heavy chain isoforms differ by a 7-amino-acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((-)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (approximately 10 nm) but the attachment time (t(on) for the (-)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(-insert)/Favg(+insert) = 1.1 +/- 0.2 (mean +/- SE) using the in vitro motility mixture assay, and the unitary force (F approximately 1 pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, t(on) under loaded conditions was longer for the (-)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of t(on) from d measurements to various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.

Molecular mechanics of two smooth muscle heavy meromyosin constructs that differ by an insert in the motor domain.

Phasic and tonic smooth muscles express two myosin heavy chain isoforms that differ by only a seven amino acid insert in a flexible surface loop located near the nucleotide-binding site. The inserted isoform found predominantly in phasic muscle has two times the actin-activated ATPase activity and in vitro actin filament velocity as the non-insert isoform found mainly in tonic muscle (Kelley, C.A., Takahashi, M., Yu, J.H. & Adelstein, R.S. 1993. J Biol Chem 268, 12848, Rovner, A.S., Freyzon, Y. & Trybus, K.M. 1997. J Musc Res Cell Motil 18, 103). We used a laser trap to characterize the molecular mechanics of the inserted isoform [(+)insert] and of a mutant lacking the insert [(-)insert], which is analogous to the isoform found in tonic muscles. The constructs were expressed in the baculovirus/insect cell system. Unitary displacements (Duni) were similar for both the constructs (approximately 10 nm) but the attachment time (ton) for the (-)insert was two times that of the (+)insert. These data suggest that the insert in the nucleotide-binding loop does not affect the inherent mechanics of the myosin molecule but rather the kinetics of the cross-bridge cycle.