Length Myosin Research Articles

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Non-Linear Cross-Bridge Elasticity, ATP-Independent Detachment and ATP-Velocity Relationships for Skeletal Muscle Actomyosin

The idea that contraction of skeletal muscle and heart results from ATP-driven actomyosin cross-bridge cycles is generally accepted. However, operational details remain controversial. For instance, in conflict with most accepted views, evidence was recently presented [1] for appreciably non-linear elasticity with low stiffness for post-power-stroke cross-bridges. Moreover, a non-hyperbolic relationship was observed [2] between MgATP concentration and sliding velocity for actin filaments propelled in vitro by myosin subfragment 1 or full length myosin. Here we present convincing evidence for a hyperbolic [MgATP]-velocity relationship (r2=0.998; Michaelis-Menten constants, Vmax=15.28 ± 0.28 µm/s (mean±SEM) and KM= 0.389 ± 0.023 mM) when actin filaments are propelled by heavy meromyosin from rabbit fast skeletal muscle myosin (28-29oC; >3 independent experiments). Because the hyperbolic [MgATP]-velocity relationship is not readily consistent with inter-head cooperativity the results were interpreted using a cross-bridge model with independent myosin heads. The inter-state transition rates were strain-dependent and the model had one detached state and five attached actomyosin (AM) states with either MgATP (AMATP) or MgADP and/or inorganic phosphate (Pi) or no nucleotide at the active site. The AMADPPi state was a strongly bound pre-power-stroke state whereas the remaining states without Pi were post-power-stroke states required to account for strain-dependent MgADP-release on the one hand and MgATP-dependence of velocity and competitive inhibition of MgATP binding by MgADP (AM, AMADP, AMATP) on the other. The MgATP induced detachment was supplemented by MgATP independent, but strain-dependent, detachment from the rigor (AM) state. This model predicts a hyperbolic [MgATP]-velocity relationship if the cross-bridge elasticity is non-linear but a non-hyperbolic [MgATP]-velocity relationship (cf. [2]) if cross-bridge elasticity is linear.

Exploring Bundle Selectivity of Myosin X Forced Dimer Constructs in vivo

Cells organize their contents and regulate cell shape and mechanics through molecular motors functioning on cytoskeletal filaments. Myosin X, an actin-based motor that concentrates at the distal tips of filopodia of mammalian cells, selects the fascin-actin bundle at the filopodial core for motility. While poorly processive on single actin filaments, it takes processive runs on actin bundled by fascin. Recently we showed that the post-IQ region is the main contributor to myosin X's selectivity. This region contains a charged single alpha-helix (SAH), which may impart unique mechanical or affinity properties to the motor. The structural character of this region was perturbed by insertion of a free swivel (GSGGSG flexible linker) after the SAH domain. The post-SAH swivel mutant showed no preference for bundled actin for motility in vitro, thus providing support to a selectivity model where the search space of the forward head for the next binding site is constrained to neighboring filaments in a bundle. Here we investigate if interrupting the “selectivity module” also has the described effect in live cells.We overexpressed in the U2OS cell line three fluorescently tagged forms of myosin X: the GCN4 forced dimer used in in vivo experiments, the post-SAH swivel mutant of GCN4 dimer and the wild type with a native dimerization domain. GCN4 forced dimer performs in the cell in the same manner as full- length myosin X. Introducing a free swivel in the SAH domain region affects the selective nature of myosin X in the natural cytoskeleton environment of the cell interior as co-localization of GFP labeled protein with filopodial tips does not occur for the Swivel mutant.

Examination of the Kinetics of Myosin VI during Endocytosis

The molecular motor myosin VI has been implicated in endocytosis, a trafficking pathway mediating intracellular uptake of items such as membrane receptors and nutrients. Previous studies demonstrate the localization of the large insert isoform of myosin VI to clathrin-coated vesicles and the no insert isoform of myosin VI to early endosomes. The kinetics of myosin VI functionality on these endocytic structures has not yet been examined, however. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover kinetics of the no insert and large insert isoforms of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures and that different isoforms on different intracellular compartments have distinct turnover rates. The FRAP assay system is then implemented with a novel live cell expression of an artificial dimer of myosin VI to demonstrate the dimeric functionality of myosin VI on endocytic structures in vivo. Further studies of the turnover rates of the myosin VI binding partner Dab2 on clathrin-coated structures demonstrate that Dab2 turns over more rapidly than full length myosin VI, a novel indication of the relative turnover rates of a motor protein versus its binding partner on a given cellular compartment. The turnover kinetics of specific myosin VI motor domain mutants on endocytic structures are also examined, e.g. the D179Y mutant implicated in progressive hearing loss, a study which offers insight into the functional source of myosin VI-related deafness. In addition to providing insight into the endocytic functionality of myosin VI, these FRAP studies offer general insight into the dynamics of myosin motor proteins on intracellular structures, the functional differences between isoforms of given myosin proteins, and the comparative turnover rates of myosin proteins and their binding partners.

Cargo Activation of Full Length Myosin Va by Melanophilin Observed at the Single Molecule Level

Full-length myosin Va (myoVa) is auto-inhibited via a motor domain-globular tail interaction, unlike the truncated constitutively active myoVa-HMM. One potential mechanism to activate the full-length motor is cargo binding to the tail, which would compete with the head-tail interaction and trigger the molecule to extend and be activated for transport.In the absence of cargo, it was recently shown that full-length myoVa has two modes of interaction with actin in the presence of MgATP (Armstrong et al.). Most motors bind to actin but do not move, while the remainder show processive motion, but with a variable stepping pattern and altered gating. Here we investigate how binding of melanophilin (Mlph), which links the melanocyte-specific isoform of myoVa to the Rab27a(GTP)-melanosome complex, affects the properties of myoVa at the single-molecule level.In the absence of Mlph at 150mM KCl, a subset of Quantum dot labeled full-length myoVa moved at a median velocity of 566nm/s with the variable stepping pattern previously described, suggesting altered gating under these conditions. Addition of Mlph recruited 7-times more motors to move processively, consistent with a simple model of cargo activation. The myoVa-Mlph complex also showed increased run lengths, with many traveling to the ends of the actin filament. In the presence of Mlph, myoVa moved much more slowly (median velocity=76nm/s), leading to longer travel times on actin. When Mlph was bound to the motor the step sizes were normally distributed around 60 ± 14nm (SD) steps. Therefore, while myoVa moves more slowly along actin in the presence of the cargo adapter protein Mlph, it covers a greater distance with a more uniform and efficient stepping pattern. This slower processive movement could potentially facilitate binding of the Rab27a(GTP)-melanosome complex.

Myosin VI Comes Together - Structure and Motor Regulation by Binding Partners

Myosin VI is an unconventional myosin motor that moves towards the minus end of actin filaments. The motor is known to interact with various binding partners and function as an anchor and a vesicle carrier in many diverse functions, including endocytosis and exocytosis. The multiple functions are likely to be regulated through the binding partners. The functional units of the motor (monomers/dimers) and the effects of binding partners remain unclear. Here we present a quantitative FRET based assay to measure the association of binding partners and determine the effect upon the oligomeric state of myosin VI. We have shown that myosin VI tail forms relatively tight interactions with the binding partners NDP52 (Kd 1 μM) and Dab2 (Kd 5 μM). We also found that these binding partners oligomerize myosin VI, increasing the affinity of oligomerization from >80 μM to 4 μM. We purpose this is achieved by the binding partners relieving auto-inhibition of the cargo-binding domain. Furthermore, we have characterized the interactions of binding partners with full length myosin VI using size-exclusion chromatography and sucrose density gradients. Single molecule photobleaching assays have been performed to determine the stoichiometry of the complexes. Functional measurements have been carried out using ATPase and single molecule motility assays in the presence of the binding partners to determine effects upon the motor activity. Supported by DFG, SFB-863, Freidrich Baur-Stiftung and EMBO.

One-Step Construction of Lentiviral Reporter Using Red-Mediated Recombination

Current approaches to generate lentiviral vectors, which have been used extensively for gene therapy, are time consuming and require a large expenditure. Here, we directly clone the full length myosin light chain kinase cDNA into enhanced green fluorescence protein (EGFP)-fused pLenti6/V5 expression vector in just one step with the use of Red-mediated recombination system, allowing for rapid and effective cloning of lentiviral expression vectors. In addition, the simultaneous expression of EGFP reporter provides a convenient monitoring mean for host cell infection and for localization of the target proteins.

Similar Regions of Instability in Tropomyosin and Myosin Coiled Coils

Tropomyosin shares sequence similarities with the N-terminal portion of the striated muscle myosin subfragment-2 (S2) coiled coil domain. Hypotheses of the instability of tropomyosin coiled coils may also be relevant to the myosin S2 coiled coil. Gravitational force spectroscopy indicates that the S1/S2 junction can be separated by forces applied through the rigor actomyosin bond which illustrates the low stability of this region of myosin. A comparison of the Langevian dynamics simulations on these two long coiled coils of tropomyosin and myosin suggests some similarities in the dynamics of these structures as well. An atomic model of full length tropomyosin was constructed by fusing existing crystallographic structures of tropomyosin fragments. Dynamics simulations of up to 10 nanoseconds at physiological temperatures yielded traces demonstrating regions where the coiled coil structure readily separates. Similar regions of instability are observed during dynamics simulations of human myosin S2 both in the existing crystallographic structure and in a full length myosin S2 predicted atomic model. Interestingly, mutations that give rise to familial hypertrophic cardiomyopathies appear to cluster in some of these regions of instability. Dynamics simulations on mutated atomic models of both tropomyosin and myosin S2 indicate that the mutations can impact the structure of these unstable regions. In myosin, a deletion mutation, del930, has a particularly strong structural impact and is known to cause a high incidence of sudden death clinically. These data suggest that such structural instabilities of homologous regions of tropomyosin and myosin might be a target for disease causing polymorphisms and could well be of important functional significance to the contractile functions in muscle.

Processive Runs of Full Length Myosin VA Are Interrupted by Pauses and Dwells

Full length myosin Va (FL-MyoVa) forms an inhibited, folded conformation at low salt, stabilized by interactions between the globular tails and the heads. High ionic strength disrupts this interaction, resulting in an extended, active processive motor. In vivo, it has been postulated that cargo binding disrupts the folded conformation and activates the motor. It is possible that splice variations in the tail (-B+D+F, melanocyte; +B-D-F, brain) could modify the ability of myosin Va to form the inhibited state. Two FL-MyoVa splice variants and an HMM-MyoVa, with biotin tags for Qdot labeling, were expressed in Sf9 cells. Sedimentation velocity experiments showed similar transitions from the folded-to-extended conformation for the two splice variants as a function of salt. TIRF microscopy was then used to observe processive runs on actin. The velocities of both FL-MyoVa splice variants were similar, and increased 270% (171-460nm/sec) with increasing KCl concentration (25-200mM). In contrast, the velocity of HMM-MyoVa increased by a more modest 50% (381-586nm/sec). The trajectories of the FL-MyoVa and HMM-MyoVa were also strikingly different. Both FL-MyoVa splice variants underwent processive runs that were interrupted by periods during which the motor dwelled at fixed points on the actin filament, presumably in the folded, inhibited state. At lower KCl concentration, FL-MyoVa dwelled approximately half of the total trajectory duration. Increasing ionic strength decreased duration of the dwells. HMM-MyoVa was fully active and maintained continuous processive movement at all KCl concentrations. The slower overall velocities for the FL-MyoVa splice variants, compared to HMM-MyoVa, results from inclusion of the dwell periods. We propose that during a processive run, a single FL-MyoVa can switch between an active and inhibited state without dissociating from actin, and that this phenomenon is independent of splice variations in the tail domain.

A Comparison of Polymer Blocking Agents in the in vitro Motility Assay

Blocking agents are used in in vitro motility assays to stabilize the motor proteins myosin or heavy meromyosin (HMM) and to prevent non-specific binding of actin to regions of microscope coverslip that are devoid of motors. Bovine serum albumin (BSA) or casein is typically used for blocking, but there is occasional need for non-protein blocking agents. We compared skeletal myosin and HMM function in motility assays as a function of the blocking agent that was used; these blocking agents included polysorbate (Tween) 20 and six different molecular weights of polyvinyl pyrrolidone (PVP) ranging from 10 kDa to 1.3 MDa, as well as BSA and β-casein as controls. In vitro motility assays were preformed and actin filament movement was quantified using automated particle tracking algorithms. PVPs of all molecular weights supported the motility of both HMM and myosin, though there was a slight downward trend in mobility at the highest molecular weights. When HMM was used in the motility assay, Tween showed poor mobility (1.7 um/s) compared to BSA (9.4 um/s). In contrast, full length myosin showed high mobility when blocked with Tween (8.3 um/s) compared to BSA (6.6 um/s). To determine whether Tween is a direct inhibitor of HMM function, NH4-activated ATPase assays were performed in solution with either BSA or Tween. There was no significant difference in ATPase rates between these two conditions. However, when the NH4-activated ATPase assay was repeated with HMM bound to a flow cell, Tween inhibited the ATPase activity. Thus while Tween does not directly inhibit the motor domains of HMM, it may adversely alter its binding to hydrophobic surfaces. In conclusion, low- to mid-molecular weight PVPs are excellent polymer blocking agents for actin-myosin or actin-HMM motility assays.

The Ups and Downs of Smooth Muscle Myosin Regulation

Since discovery that regulatory light chain (RLC) phosphorylation was the primary method of regulation of smooth and non-muscle myosin II, the structure of the phosphorylated and dephosphorylated forms has been a goal only partially realized. Regulation in both smooth muscle and the related scallop striated muscle myosin requires a 2-headed myosin species; single headed species are unregulated. Thus head to head interactions are key to achieving the inhibited state. A folded conformation of full length myosin, generally referred to as the 10S conformation was discovered early by conventional electron microscopy and in 1999, a structural explanation for the head-head interactions was obtained from 2-D arrays of dephosphorylated smooth muscle heavy meromyosin formed on lipid monolayers and imaged in 3-D by cryoEM. The structure was later confirmed by 2-D arrays of the 10S conformation of whole myosin. While this structure explained inhibition of the solubilized form of regulated myosins, it had not been observed in filaments. Surprisingly, the first observation of smooth muscle myosin-like head-head interactions in a thick filament was obtained from tarantula striated muscle, not from smooth muscle myosin. Even more surprising was the observation of a similar conformation in relaxed cardiac muscle thick filaments. Thus, these head-head interactions observed first in smooth muscle HMM, appear to be ubiquitous in relaxed muscle although still not confirmed for thick filaments in relaxed smooth muscle. Still to be determined is an explanation of the factors that can lead to solubilization and the location of the N-terminus of the RLC, whose location and structure has yet to be revealed, in the phosphorylated and dephosphorylated state. Theoretical modeling has provided possible explanations for several factors that affect regulation but has not yet yielded a coherent theory. Supported by NIAMSD.

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

BackgroundMyosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).Methodology/Principal Findings Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.Conclusions/SignificanceIn summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.

Myosin VI: cellular functions and motor properties

Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed.

Cooperativity between Two Heads of Dictyostelium Myosin II in in Vitro Motility and ATP Hydrolysis

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 μM actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax. These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity.