Monomeric Myosin Research Articles

Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Parameters of Cell Death and Proliferation of Prostate Cancer Cells with Altered Expression of Myosin 1C Isoforms

Myosin 1C is a monomeric myosin motor with a truncated tail domain. Such motors are referred as slow “tension sensors.” Three isoforms of myosin 1C differ in short N-termed amino acid sequences, the functional differences between isoforms have not been elucidated. Myosin 1C isoform A was described as a diagnostic marker for prostate cancer, but its role in tumor transformation remains unknown. Based on data on the functions of myosin 1C, we hypothesized the potential role of myosin 1C isoforms in maintaining the tumor phenotype of prostate cancer cells. In our work, we showed that a decrease in the expression level of myosin 1C isoform C leads to an increase in the proliferative activity of prostate tumor cells.

Limiting pool and actin architecture controls myosin cluster sizes in adherent cells

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.

Mechanisms of cardiac myosin replacement.

A recent mass spectrometry-based proteomic study from our lab demonstrated that newly synthesized myosin molecules are randomly inserted into preexisting thick filaments in adult mouse hearts in vivo. Therefore, we hypothesized that myosin molecules exist in a dynamic equilibrium between a filamentous pool required for contractility, and a monomeric pool required for myosin replacement. To test this hypothesis, we replaced 27 ± 7% of the cardiac myosin regulatory light chain (RLC) in adult mice with fluorescent RLC (GFP-RLC), via adeno-associated viral transfection. We visualized the random distribution of GFP-RLC within thick filaments in intact hearts with 2-photon microscopy, and the exchange of GFP-RLC between these filaments by quantifying fluorescence recovery after photobleaching (FRAP). Rapid FRAP occurred within minutes, when photobleaching small, ∼1-µm3 areas, of partial sarcomeres. These data demonstrate that the GFP-RLC exchanges between the thick filaments and a monomeric pool on a timescale that is orders of magnitude faster than that of protein replacement (t1/2 = 10.8 days). To determine whether the mechanism of this exchange may involve the folding of monomeric myosin, GFP-labeled myosin molecules were extracted from the transfected hearts and their Stokes radii were measured over a range of ionic strengths, using microfluidic diffusional sizing. At low ionic strengths (150 mM KCl), filament formation was favored, but the monomeric fraction of GFP-labeled myosin demonstrated a radius of 12.6±1.1 nm. At higher ionic strengths (400-600 mM KCl), the radius was increased to 18.0 nm. Similar radii have been reported for smooth muscle myosin monomers in folded and extended conformations. Together, these data suggest that myosin molecules exist in rapid equilibrium between a filamentous and monomeric pool within the sarcomere, and individual monomers may adopt a folded, compact conformation to regulate their exchange into, and out of filaments.

Tension-Dependent Myosin Dynamics on Contractile Actomyosin Structures

Dynamic cellular processes, such as cell migration and division, are powered by mechanical tension generated by the actomyosin cytoskeleton, where myosin molecular motors pull on actin filaments. To maintain proper mechanical tension, myosin dynamics is regulated by several factors, such as phosphorylation on the regulatory light chain. Mechanical tension is also proposed to regulate myosin dynamics. In vitro studies have shown that myosin detaches slower from F-actin under resisting load. However, it is unclear whether mechanical tension plays a role in regulating myosin dynamics in cells. In this work, we assess the role of mechanical tension in myosin turnover dynamics on stress fibers by conducting fluorescence recovery after photobleaching (FRAP) experiments. To perturb cellular tension, cells are treated with non-saturating levels of the Rho-kinase inhibitor Y27632, where the total traction force decreases while the overall actomyosin architecture is preserved. Consistent with past literature, myosin recovers much faster after photobleaching, which is thought to be due to an increase in diffusive myosin monomers. However, the recovery profile suggests a reaction-dominant process, suggesting that myosin dissociates faster due to decreased tension. Furthermore, super-resolution live-cell imaging revealed quasi-sarcomeric myosin bands on individual stress fibers recovering after photobleaching. Under normal levels of cellular tension, myosin always recovers on established myosin bands. In constrast, under reduced tension, myosin bands become more disordered and dynamic. This suggests that myosin turnover by monomers associating with or dissociating from an existing minifilament. The core of myosin bands is stable under tension, resulting in the immobile fraction in FRAP experiments. Upon tension reduction, these cores become mobile and can turn over. Together with experiments that directly modulates tension on stress fibers, such as stress fiber rupturing, our data sheds light on the role of mechanical tension regulating myosin dynamics.

The effect of fish freshness on myosin denaturation in flounder Paralichthys olivaceus muscle during frozen storage

The effect of fish freshness on myosin denaturation during the frozen storage of flounder muscle was studied. The muscle of instantaneously killed flounder was immediately stored at − 20 °C (pre-rigor mortis; day 0), or after storage at 0 °C for 2 days (rigor mortis) or 5 days (post-rigor mortis). Myosin denaturation in frozen stored muscle was studied by measuring Ca2+-ATPase activity, myosin solubility in the presence of salt (salt solubility), and monomeric myosin content. High ATPase activity was maintained in the day-0 samples of instantaneously killed flounder during the early phase of frozen storage. High salt solubility of myosin was also observed in these samples. However, the beneficial effect of frozen storage was lost when the storage period exceeded 2 months. There was no difference in myosin denaturation among the three groups of samples after 2 months of frozen storage. The myosin denaturation profile during the frozen storage of muscle was characterized by high salt solubility in the presence of Mg-ATP, even after ATPase inactivation, especially in the day-0 samples. The salt-soluble fraction contained a large amount of aggregated myosin, as revealed by (NH4)2SO4 fractionation. The monomeric myosin content was explained by Ca2+-ATPase inactivation in the frozen stored meat for all three groups. This study demonstrated that the high quality of flounder meat is maintained during short-term frozen storage, but that the beneficial effects of frozen storage are lost over longer periods of time.

Dictyostelium myosin 1F and myosin 1E inhibit actin waves in a lipid-binding-dependent and motor-independent manner.

Actin waves are F-actin-rich entities traveling on the ventral plasma membrane by the treadmilling mechanism. Actin waves were first discovered and are best characterized in Dictyostelium. Class I myosins are unconventional monomeric myosins that bind lipids through their tails. Dictyostelium has seven class I myosins, six of these have tails (Myo1A-F) while one has a very short tail (Myo1K), and three of them (Myo1D, Myo1E and Myo1F) bind PIP3 with high affinity. Localization of five Dictyostelium Class I myosins synchronizes with localization and propagation of actin waves. Myo1B and Myo1C colocalize with actin in actin waves, whereas Myo1D, E and F localize to the PIP3-rich region surrounded by actin waves. Here, we studied the effect of overexpression of the three PIP3 specific Class I myosins on actin waves. We found that ectopic expression of the short-tail Myo1F inhibits wave formation, short-tail Myo1E has similar but weaker inhibitory effect, but long-tail Myo1D does not affect waves. A study of Myo1F mutants shows that its membrane-binding site is absolutely required for wave inhibition, but the head portion is not. The results suggest that PIP3 specificity and the presence of two membrane-binding sites are required for inhibition of actin waves, and that inhibition may be caused by crosslinking of PIP3 heads groups.

Actin Network Organization by the Monomeric Myosin IXa

Class IX myosins are thought to act as motorized signaling molecules, as they contain a Rho-GTPase-activating domain (RhoGAP) in the tail, which can alter the dynamics of the actin cytoskeleton. Myosin-IXa is localized in acto-myosin networks at cell-cell adhesions, where it was identified as a key requirement for collective cell migration. Many processes depend on the movement of groups of cells; during tumor progression cancer cells invade the tissue surrounding a primary tumor, while in wound healing epithelial cells have to move close to the wound. We have expressed a variety of human myosin-IXa constructs comprising the motor domain and the calmodulin-binding lever arm. Using quantitative analysis of TIRF microscopy data we found that myosin-IXa assembles actin into bundles of up to seven or even more filaments. Electron microscopy combined with single particle image processing revealed that the bundles consisted of highly ordered 2D lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming crosslinks at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Intriguingly, the polarity of the actin filaments in the bundles changed in the presence of calcium. By comparing real EM data with simulated EM of modelled F-actin, we find that approx. 75% of filaments were anti-parallel (in-phase and out-of-phase) at pCa4. This polarity switching is currently investigated in further detail by TIRF microscopy. In addition, we are characterizing the single molecule mechanics of myosin-IXa using an optical-tweezer transducer. Our results indicate that actin lattices induced by the molecular motor myosin-IXa provide orientated actin tracks and a network of regularly spaced platforms for localized Rho-GAP activity in cell polarization and collective cell migration.

Evaluation of Nanoparticle Tracking Analysis for the Detection of Rod-Shaped Particles and Protein Aggregates.

Nanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 μm, and linked to diseases including Alzheimer's and Parkinson's. We used NTA to measure the concentration and hydrodynamic size of gold nanorods (10 nm diameter, 35-250 nm length) and myosin (2 nm diameter, 160 nm length), as models of rod-like particles. Measured hydrodynamic diameters of gold nanorods were consistent with theoretical calculations, as long as particle concentration and solution conditions were controlled. Myosin monomers were invisible by NTA, but a small population of aggregates was detected. We combined NTA results with other light scattering data to gain insight into number and size distribution of protein solutions containing both monomer and aggregates. Finally, we demonstrated the utility of NTA and its limitations by characterizing aggregates of alpha-synuclein. Of note is the use of NTA to detect a change in morphology from compact to elongated by analyzing the ratio of hydrodynamic size to intensity.

Polymerization pathway of mammalian nonmuscle myosin 2s

The three mammalian nonmuscle myosin 2 (NM2) monomers, like all class 2 myosin monomers, are hexamers of two identical heavy (long) chains and two pairs of light (short) chains bound to the heavy chains. The heavy chains have an N-terminal globular motor domain (head) with actin-activated ATPase activity, a lever arm (neck) to which the two light chains bind, and a coiled-coil helical tail. Monomers polymerize into bipolar filaments, with globular heads at each end separated by a bare zone, by antiparallel association of their coiled-coil tails. NM2 filaments are highly dynamic in situ, frequently disassembling and reassembling at different locations within the cell where they are essential for multiple biological functions. Therefore, it is important to understand the mechanisms of filament polymerization and depolymerization. Monomers can exist in two states: folded and unfolded. It has been thought that unfolded monomers form antiparallel dimers that assemble into bipolar filaments. We now show that polymerization in vitro proceeds from folded monomers to folded antiparallel dimers to folded antiparallel tetramers that unfold forming antiparallel bipolar tetramers. Folded dimers and tetramers then associate with the unfolded tetramer and unfold, forming a mature bipolar filament consisting of multiple unfolded tetramers with an entwined bare zone. We also demonstrate that depolymerization is essentially the reverse of the polymerization process. These results will advance our understanding of NM2 filament dynamics in situ.

A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, and Cardiac Muscle Myosin Filaments in Vitro

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/ton), where d equals step size and ton equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

Skeletal Myosin-Binding Protein C Modulates Actomyosin Contractility in an Isoform-Dependent Manner

Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized to discrete regions (C-zones) of myosin thick filaments in all striated muscles. Unlike cardiac muscle with its specific MyBP-C isoform, mass spectrometry shows that rat slow-twitch soleus (SOL) muscle expresses two different slow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-terminal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) expresses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C). Do these MyBP-C isoforms differentially regulate skeletal actomyosin contractility in a muscle-specific manner? To address this, we characterized Ca2+-regulated motion of SOL and EDL native actin-thin filaments (NTFs) over their corresponding native myosin-thick filaments, having the physiological complement of MyBP-C in the C-zone. At low calcium (pCa7.5) where NTFs should be inhibited, motion occurred within the C-zone of both SOL and EDL native thick filaments. Whereas, at high Ca2+ (pCa5), SOL and EDL NTFs had initial fast velocities followed by ∼30% slower velocities within the C-zone. These data suggest that one or more of these MyBP-C can sensitize NTFs to Ca2+ and modulate contractility at high Ca2+. To determine how each isoform contributes to these modulatory capacities, we bacterially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C) and assessed their individual impact on SOL or EDL NTF motility over their respective monomeric myosins. For the EDL where all three MyBP-Cs are expressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while at pCa5 all three reduced velocity by ∼50%. For SOL, where only sMyBP-C isoforms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5 sMyBP-C2 reduced velocity ∼40%. Our data suggest that the distinctive mixture of MyBP-C isoforms expressed in the SOL and EDL is matched to the muscle's physiological demands. Each MyBP-C isoform possesses specific modulatory capacities that may be called upon under different contractile states. Specifically, to fine-tune actomyosin contractility within the C-zone by Ca2+-sensitizing the thin filament and/or modulating velocity at maximal activation.

Targeting of Myosin to Cellular Organelles Results in Dramatic Reorganization based on Cargo/Motor Affinity

Unconventional myosins are vital molecular motors involved in active transport of cargo as well as maintenance of organelle shape. In particular, myosin VI has been shown to be involved in clathrin-mediated endocytosis and maintenance of golgi shape. Though the biophysical characteristics of unconventional myosins have been well studied and numerous cargo-binding partners have been identified, investigation into the interplay of these properties at the cellular level has been limited. Here, we investigate the influence of both monomeric and dimeric conformations of myosin VI, as well as the effects of binding affinity of the motor-cargo interaction may have on both subcellular morphology and cargo transport. We have generated a set of expressible fluorescent protein markers that label individual organelles/compartments. Using these markers combined with the FKBP-FRB system, we are able to selectively target myosin to peroxisomes (Pex3), early endosomes (Rab5), and clathrin-coated vesicles (Dab2) upon rapamycin stimulation. Irreversible localization of myosin to these structures results in disruption of normal compartment shape and size. Specifically, large clusters of peroxisomes and endosomes are observed and Dab2 labeled clathrin coated vesicles are disrupted and dispersed throughout the cell. In addition, these changes drastically remodel the cellular actin cytoskeletal network. These observations are seen using both artificially dimerized myosin VI and, to a lesser extent, monomeric myosin VI. Further, by controlling the strength of the FKBP-FRB interaction through mutagenesis, we have found that the strength of interaction between myosin and cargo plays a vital role in this reorganization.

Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

Cellular mechanisms governing non-muscle myosin 2 (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamella, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibers, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibers to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally-active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

Unfolding of the myosin head by purealin in glycerol.

Purealin is a small bioactive compound obtained from the marine sponge. The compound modulates various types of ATPase activity of myosin from skeletal muscle, cardiac muscle, and smooth muscle. To elucidate the structural basis of these effects of purealin on myosin ATPases, we examined the effect of purealin on the conformation of skeletal muscle myosin in aqueous solution and in glycerol. Analysis of the circular dichroism spectrum of subfragment 1, a single-headed fragment of myosin, revealed that in 10% glycerol purealin decreased the β-sheet content of S1, but in aqueous solution it had little effect on the secondary structure of S1. A myosin monomer conforms to two pear-like globular heads attached to a long tail. Electron microscopy observations with rotary shadowing revealed that purealin unfolded each globular head to an extended single strand. The tips of the unfolded strand bound each other and formed a ring in one molecule. These results suggest that binding of purealin affects the critical parameters of myosin folding.

Smooth muscle function and myosin polymerization

Smooth muscle is able to function over a much broader length range than striated muscle. The ability to maintain contractility after a large length change is thought to be due to an adaptive process involving restructuring of the contractile apparatus to maximize overlap between the contractile filaments. The molecular mechanism for the length-adaptive behavior is largely unknown. In smooth muscle adapted to different lengths we quantified myosin monomers, basal and activation-induced myosin light chain (MLC) phosphorylation, shortening velocity, power output and active force. The muscle was able to generate a constant maximal force over a two fold length range when it was allowed to go through isometric contraction/relaxation cycles after each length change (length adaptation). In the relaxed state, myosin monomer concentration and basal MLC phosphorylation decreased linearly, while in the activated state activation-induced MLC phosphorylation and shortening velocity/power output increased linearly with muscle length. The results suggest that recruitment of myosin monomers and oligomers into the actin filament lattice (where they form force-generating filaments) occurs during muscle adaptation to longer length, with the opposite occurring during adaptation to shorter length.

Self-organization of actin networks by a monomeric myosin

The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase-activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration.

The Inhibited, Interacting-Heads Motif Characterizes Myosin II from the Earliest Animals with Muscles

Electron microscopic studies have shown that the inhibited (relaxed) state of myosin II is characterized by an intramolecular interaction between myosin heads, in both thick filaments and isolated monomers. Interaction inhibits head activity by blocking actin-binding in one head and ATPase activity in the other. Interacting-heads are present in vertebrate smooth muscle and nonmuscle monomers, and in thick filaments of invertebrate smooth muscle (flatworms), invertebrate striated muscle (mollusks, arthropods) and vertebrate striated muscle (zebrafish, mouse and human); furthermore, head interactions underlie the off-state in myosins regulated by both Ca2+ binding and RLC phosphorylation. Head-head interaction has thus been conserved through evolution from mollusks and arthropods to humans. Our goal has been to determine how early this self-inhibiting motif arose, by studying the off-state of myosin monomers and/or filaments from primitive species. Filaments from sea anemones (Cnidaria), the most primitive animals with muscles, have the same 14.5-nm repeat as other muscles, but EM images have not yet been reconstructed. However, sea anemone myosin monomers, under relaxing conditions, have the same folded tail and bent-back, interacting-heads motif as myosins from the higher species, further demonstrating the very early origin of this structure. Interestingly, unlike the animals mentioned above, Acanthamoeba myosin monomers under relaxing conditions appear to lack a folded tail or interacting heads. The sequence of this myosin is strikingly different from the myosin IIs of animals, and its regulatory mechanism is quite different, suggesting that self-inhibition by interacting heads evolved after amoebae and animals diverged. Conservation of interacting heads over time suggests that it is a highly successful mechanism for regulating myosin activity. We are now studying the more primitive sea sponges (Porifera), one of the earliest animal groups, which lack muscles.

Kinetic Characterization of Stabilized Smooth Muscle Myosin Filaments

We have measured kinetic parameters of smooth muscle myosin (SMM) filament interactions with actin, ADP, and ATP using stopped-flow fluorimetry. Our objective was to determine which parameters, if any, are different from those of monomeric myosins, S1 and HMM, using buffers at physiologic ionic strength. Previously we measured the ATP-dependence of SMM filament velocities (using EDC-cross-linked filaments that are stable to ATP-induced depolymerization) in an inverted in vitro motility assay with myosin filaments moving over surface-attached actin. We compared this to the ATP-dependence of solution actin-activated ATPase measurements. The two relationships are very similar, suggesting that they are both attachment-limited and regulated by the same kinetic step, presumably phosphate release (Haldeman et al JBC 2014). This is in contrast to the kinetics of actin moving over a surface of monomeric SMM (standard vitro motility assay), known to be predominately detachment-limited, limited by ADP release. We hypothesized that heads within moving filaments are attached to unhindered and thus flexible S2 regions putting minimal strain on heads put in the drag position. In contrast, the S2 region of monomeric myosin is bound to the surface and not as flexible allowing the relative stiff head domains to become strained, which affects the underlying kinetics of acto-SMM interactions. All kinetic parameters measured so far in solution for myosin or acto-myosin, ADP association, ADP dissociation, ATP binding, ATP-induced dissociation, and acto-myosin affinity, are very similar to those for S1 and HMM. Measurements of Pi release kinetics by limited turnover are planned. If Pi release kinetics is also similar, this will suggest that the attachment-limited kinetics that is unique to filaments is not due to alterations in any fundamental kinetic parameters, but instead is due to differences in the effective head or head/S2 stiffness during the assays.