Myosin Turnover Research Articles

UNC-82/NUAK kinase is required by myosin A, but not myosin B, to assemble and function in the thick filament arms of C. elegans striated muscle.

The mechanisms that ensure proper assembly, activity, and turnover of myosin II filaments are fundamental to a diverse range of cellular processes. In Caenorhabditis elegans striated muscle, thick filaments contain two myosins that are functionally distinct and spatially segregated. Using transgenic double mutants, we demonstrate that the ability of increased myosin A expression to restore muscle structure and movement in myosin B mutants requires UNC-82/NUAK kinase activity. Myosin B function appears unaffected in the kinase-impaired unc-82(e1220) mutant: the recessive antimorphic effects on early assembly of paramyosin and myosin A in this mutant are counteracted by increased myosin B expression and exacerbated by loss of myosin B. Using chimeric myosins and motility assays, we mapped the region of myosin A that requires UNC-82 activity to a 531-amino-acid region of the coiled-coil rod. This region includes the 264-amino-acid Region 1, which is sufficient in chimeric myosins to rescue the essential filament-initiation function of myosin A, as well as two sites that interact with myosin head domains in the Interacting Heads Motif. A specific physical interaction between myosin A and UNC-82::GFP is supported by GFP labeling of ectopic myosin A filaments but not thin filaments. We hypothesize that UNC-82 regulates assembly competence of myosin A during parallel assembly in the filament arms.

Myosin turnover controls actomyosin contractile instability

Actomyosin contractile force produced by myosin II molecules that bind and pull actin filaments is harnessed for diverse functions, from cell division by the cytokinetic contractile ring to morphogenesis driven by supracellular actomyosin networks during development. However, actomyosin contractility is intrinsically unstable to self-reinforcing spatial variations that may destroy the actomyosin architecture if unopposed. How cells control this threat is not established, and while large myosin fluctuations and punctateness are widely reported, the full course of the instability in cells has not been observed. Here, we observed the instability run its full course in isolated cytokinetic contractile rings in cell ghosts where component turnover processes are absent. Unprotected by turnover, myosin II merged hierarchically into aggregates with increasing amounts of myosin and increasing separation, up to a maximum separation. Molecularly explicit simulations reproduced the hierarchical aggregation which precipitated tension loss and ring fracture and identified the maximum separation as the length of actin filaments mediating mechanical communication between aggregates. In the final simulated dead-end state, aggregates were morphologically quiescent, including asters with polarity-sorted actin, similar to the dead-end state observed in actomyosin systems invitro. Our results suggest the myosin II turnover time controls actomyosin contractile instability in normal cells, long enough for aggregation to build robust aggregates but sufficiently short to intercept catastrophic hierarchical aggregation and fracture.

A computational modeling of invadopodia protrusion into an extracellular matrix fiber network

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

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.

Elasticity, Stability, and Quasioscillations of Cell-Cell Junctions in Solid Confluent Epithelia

Macroscopic properties and shapes of biological tissues depend on the remodeling of cell-cell junctions at the microscopic scale. We propose a theoretical framework that couples a vertex model of solid confluent tissues with the dynamics describing generation of local force dipoles in the junctional actomyosin. Depending on the myosin turnover rate, junctions either preserve stable length or collapse to initiate cell rearrangements. We find that noise can amplify and sustain transient oscillations to the fixed point, giving rise to quasiperiodic junctional dynamics. We also discover that junctional stability is affected by cell arrangements and junctional rest tensions, which may explain junctional collapse during convergence and extension in embryos.

Detection of Super-Relaxed Myosin in Specific Human Skeletal Muscle Fiber Types

We are studying the super-relaxed state of myosin, with the goal of developing novel therapeutic tools for treatment of contractile and metabolic disorders. In muscle, myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction, and to help maintain basal metabolic rate at rest. The myosin super-relaxed state (SRX) in skeletal muscle is hypothesized to play an important role in regulating muscle cell contractility and thermogenesis, yet its physiological role in skeletal muscle remains elusive. The SRX is a biochemical state of myosin with slowed ATP turnover kinetics, due to auto-inhibition of the catalytic domains. Previous work on myosin SRX focused on samples obtained from rabbits and mice. Here, we have used refined quantitative epifluorescence microscopy of fluorescent MANT-ATP to measure single-nucleotide turnover of myosin in skinned skeletal muscle fibers from human donors for the first time. As with most mammalian muscles, the human vastus lateralis is composed of fibers expressing different myosin heavy chain (MHC) isoforms (I, IIA and IIX), which was determined for each fiber tested using gel electrophoresis. Preliminary results show that human skeletal SRX from MHC I fibers have faster ATP turnover compared to MHC II fibers, as previously observed in rabbit muscle fibers. From this work, we are making significant progress toward understanding the biochemical and physiological significance of myosin SRX in human skeletal muscle.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Myosin Quality Control Systems in Zebrafish Skeletal Muscle Development

In order to maintain homeostasis, defective proteins must be identified, removed, and replaced. In skeletal muscle, failures in this process can lead to a variety of incurable muscle diseases (myopathies), many of which are fatal. While aspects of protein turnover have been studied widely, the protein turnover process in skeletal muscle is still incompletely understood, especially with respect to the motor protein, myosin. By studying the assembly and maintenance of the basic unit of striated muscle, the sarcomere, we aim to elucidate the components involved and the mechanism controlling muscle myosin quality control in skeletal muscle.Zebrafish make an excellent model system to study sarcomere protein turnover due to their ability to survive with myopathies that are fatal in other organisms. Using two different zebrafish lines, steif, and herzschlag, we and others show that steif sarcomeres fail to assemble myosin thick filaments due to a loss of a critical component (Etard et al., 2007), while herzschlag sarcomeres degrade over time as a consequence of muscle contractions. Transmission electron microscopy coupled with H&E staining revealed a slow muscle atrophy specific to herzschlag embryos. To identify factors involved in response to myosin damage during assembly or maintenance, we have screened through candidate genes, myosin chaperones and muscle specific E3 enzymes, using in situ hybridization and qPCR. Our data indicates a division of function where specific E3 enzymes and myosin chaperones respond to myosin damage exclusively during assembly, while other E3 enzymes respond to myosin damage during sarcomere maintenance. Pharmaceutical inhibition of the proteasome shifts this response in steif embryos, but not herzschlag, suggesting an interconnected network of protein quality control during myosin assembly. Together, this work helps highlight potential therapeutic targets to treat myosin turnover related myopathies.Support or Funding InformationNSERCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HSP90 modulates the myosin replacement rate in myofibrils.

Myosin is a major myofibrillar component in skeletal muscles. In myofibrils, ~300 myosin molecules form a single thick filament in which there is constant turnover of myosin. Our previous study demonstrated that the myosin replacement rate is reduced by inhibition of protein synthesis (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015); however, additional factors influencing myosin replacement were unknown. Here, we showed that rapid myosin replacement requires heat shock protein 90 (HSP90) activity. We utilized the fluorescence recovery after photobleaching technique to measure the replacement rate of green fluorescent protein-fused myosin heavy chain (GFP-MYH) in myotubes overexpressing HSP90. Intriguingly, the myosin replacement rate was significantly increased in HSP90-overexpressing myotubes, whereas the myosin replacement rate slowed markedly in the presence of an HSP90-specific inhibitor, indicating that HSP90 activity promotes myosin replacement. To determine the mechanism of this effect, we investigated whether HSP90 activity increased the amount of myosin available for incorporation into myofibrils. Strikingly, the gene expression levels of MYHs were significantly elevated by HSP90 overexpression but downregulated by inhibition of HSP90 activity. Cytosolic myosin content was also increased in myotubes overexpressing HSP90. Taken together, our results demonstrate that HSP90 activity facilitates myosin replacement by upregulating MYH gene expression and thereby increasing cytosolic myosin content.

Tension regulates myosin dynamics during Drosophila embryonic wound repair

Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair.

Tension regulates myosin dynamics during Drosophila embryonic wound repair

Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair.

Local Pulses of Rhoa Activation Assemble Polarized Network Architectures for Efficient Actomyosin Contractility

Spatiotemporal patterning of actomyosin contractility plays a key role in cell and tissue morphogenesis during early development. In embryonic cells, actomyosin arrays are highly dynamic structures that remodel on a time scale of 10s of seconds, through a combination of local actomyosin turnover and rapid spatial redistribution of filaments and motors caused by myosin activity or actin polymerization. Because of these dynamic and active properties, contractility is complex and intrinsically self-organizing.We used the C. elegans early embryo to understand how cells pattern force generation through local modulation of self-organized contractility, focusing on pulsed contractility in the C. elegans embryo.We combined two-color fluorescence imaging, live single-molecule imaging, particle tracking, image analysis, and numerical modeling to tease apart the mechanisms of pulse initiation and termination. Our results demonstrate that the mechanical component (advection) played little role in pulse initiation or termination, and that the process was mostly governed by Actin and Myosin turnover. In our system, autocatalytic RhoA activation/recruitment is responsible for pulse initiation, while the delayed recruitment of a RhoA inactivator (RGA-3/4) onto Actin filaments drives pulse termination. Using single-molecule imaging, we further show that this mode of actin assembly drives the formation of structural units displaying a characteristically polarized architecture with (1) a gradient of myosin accumulation, myosin being recruited at the center of the pulses and (2) a transient anisotropic organization of the actin network, where filaments elongate from the instide of the pulse, barbed ends pointing outwards, as visualized by displacement of the actin-barbed-end-tracking formins that processively elongate actin filaments.Based on these results, we propose here that pulsed contractions represent a mode of actomyosin assembly which structures the actomyosin network in functional modules to drive efficient cell contractility.

Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase.

Dynamics of myosin degradation in intensive care unit-acquired weakness during severe critical illness

Intensive care unit (ICU)-acquired muscle wasting is a devastating complication leading to persistent weakness and functional disability. The mechanisms of this myopathy are unclear, but a disturbed balance of myosin heavy chain (MyHC) is implicated. To investigate pathways of myosin turnover in severe critically ill patients at high risk of ICU-acquired weakness. Prospective, mechanistic, observational study. Interdisciplinary ICUs of a university hospital. Twenty-nine patients with Sequential Organ Failure Assessment (SOFA) scores of at least 8 on three consecutive days within the first 5 days in ICU underwent two consecutive open skeletal muscle biopsies from the vastus lateralis at median days 5 and 15. Control biopsy specimens were from healthy subjects undergoing hip-replacement surgery. None. Time-dependent changes in myofiber architecture, MyHC synthesis, and degradation were determined and correlated with clinical data. ICU-acquired muscle wasting was characterized by early, disrupted myofiber ultrastructure followed by atrophy of slow- and fast-twitch myofibers at later time points. A rapid decrease in MyHC mRNA and protein expression occurred by day 5 and persisted at day 15 (P < 0.05). Expression of the atrophy genes MuRF-1 and Atrogin1 was increased at day 5 (P < 0.05). Early MuRF-1 protein content was closely associated with late myofiber atrophy and the severity of weakness. Decreased synthesis and increased degradation of MyHCs contribute to ICU-acquired muscle wasting. The rates and time frames suggest that pathogenesis of muscle failure is initiated very early during critical illness. The persisting reduction of MyHC suggests that sustained treatment is required.

Mechano-Chemical Coupling Drives Cell Area Oscillations during Morphogenesis

Marie Curie Career Integration Grant (No. PCIG09-GA-2011-293479); Spanish Ministry of Science (No. BFU2011-25828)

Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.

Investigating the Mechanism of Pulsed Actin-Myosin Contraction

During embryonic development, masses of cells undergo dramatic rearrangements to organize into separate layers that will give rise to different parts of the body. This incredibly dynamic process is called gastrulation and is driven by cell shape changes that collectively deform the tissue. Apical constriction is a common cell shape change that converts a columnar shaped cell into a wedge or cone shaped cell. The resulting change in cell geometry facilitates the bending or folding of an epithelial sheet. Previously, we imaged the dynamics of apical constriction during the embryonic development of the fruit fly, Drosophila melanogaster. We found that apical constriction is driven by a ratchet-like contraction of the actin-myosin meshwork across the apical surface of these cells. This involves pulses of myosin driven contraction that are repeated cyclically to constrict the cell. Here, we examine the mechanism of pulsed contractions. It was possible that contraction pulses result from mechanical signaling (i.e. stretching) between neighboring cells. However, using RNAi of adherens junction proteins to disrupt cell-cell adhesion, we find that contraction pulses can occur even when cells are not mechanically coupled. This suggests that contraction pulses in the embryo result from the dynamic properties of actin-myosin networks. We are currently examining the importance of actin and myosin turnover during pulsed contraction in the embryo.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

Yeast UCS proteins promote actomyosin interactions and limit myosin turnover in cells

Two functions are proposed for the conserved family of UCS proteins: helping to fold myosin motor proteins and stimulating the motor function of folded myosins. We examined both functions in yeast. The fission yeast UCS protein (Rng3p) concentrates in nodes containing myosin-II (Myo2) and other proteins that condense into the cytokinetic contractile ring. Both the N-terminal (central) and C-terminal (UCS) domains of Rng3p can concentrate independently in contractile rings, but only full-length Rng3p supports contractile ring function in vivo. The presence of Rng3p in ATPase assays doubles the apparent affinity (K(ATPase)) of both native Myo2 and recombinant heads of Myo2 for actin filaments. Rng3p promotes gliding of actin filaments by full-length Myo2 molecules, but not Myo2 heads alone. Myo2 isolated from mutant strains defective for Rng3p function is soluble and supports actin filament gliding. In budding yeast the single UCS protein (She4p) acts on both myosin-I isoforms (Myo3p and Myo5p) and one of two myosin-V isoforms (Myo4p). Myo5p turns over approximately 10 times faster in she4Delta cells than wild-type cells, reducing the level of Myo5p in cells 10-fold and in cortical actin patches approximately 4-fold. Nevertheless, Myo5p isolated from she4Delta cells has wild-type ATPase and motility activities. Thus, a fraction of this yeast myosin can fold de novo in the absence of UCS proteins, but UCS proteins promote myosin stability and interactions with actin.