Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

PubMed Central

Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

2015-01-01

Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

PubMed Central

Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J.; Polo, Simona

2016-01-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

PubMed

Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

2016-04-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Long-range self-organization of cytoskeletal myosin II filament stacks.

PubMed

Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

2017-02-01

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

PubMed Central

Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E.; Valladares, Basilio; Maciver, Sutherland K.

2014-01-01

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles*

PubMed Central

Nagy, Stanislav; Rock, Ronald S.

2010-01-01

Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses. PMID:20538587

Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts.

PubMed

An, Yanru; Xue, Guosheng; Shaobo, Yang; Mingxi, Deng; Zhou, Xiaowei; Yu, Weichuan; Ishibashi, Toyotaka; Zhang, Lei; Yan, Yan

2017-06-15

Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used Drosophila embryonic neuroblasts as a model to study the apical constriction process during cell delamination. We observe dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex, the nonjunctional myosin forms flows and pulses, which are termed medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are crucial to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of a neuroblast is set apart from that of its neighbors by Notch signaling-mediated lateral inhibition. When we inhibit Notch signaling activity in the embryo, we observe that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, these results demonstrate that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts. © 2017. Published by The Company of Biologists Ltd.

Human Myo19 is a novel myosin that associates with mitochondria

PubMed Central

Quintero, Omar A.; DiVito, Melinda M.; Adikes, Rebecca C.; Kortan, Melisa B.; Case, Lindsay B.; Lier, Audun J.; Panaretos, Niki S.; Slater, Stephanie Q.; Rengarajan, Michelle; Feliu, Marianela; Cheney, Richard E.

2009-01-01

Summary Mitochondria are pleomorphic organelles [1, 2] that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease [3-5]. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport [6]. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues and antibodies to human Myo19 detect a ∼109kD band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain-of-function where the majority of the mitochondria move continuously. Moving mitochondria travel for many microns with an obvious leading end and distorted shape. The motility and shape-change are sensitive to latrunculin B, indicating that both are actin-dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells. PMID:19932026

Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

NASA Astrophysics Data System (ADS)

Nie, Wei

The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

Myosin II dynamics are regulated by tension in intercalating cells.

PubMed

Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

2009-11-01

Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine.

PubMed

Hayakawa, T; Yoshida, Y; Yasui, M; Ito, T; Iwasaki, T; Wakamatsu, J; Hattori, A; Nishimura, T

2012-01-01

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing L-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing L-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50°C, while myosin in a high ionic strength solution formed opaque gels at 60-70°C. The gel of myosin in a low ionic strength solution with L-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50°C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing L-histidine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Remote control of molecular motors using light-activated gearshifting

NASA Astrophysics Data System (ADS)

Bryant, Zev

2013-03-01

Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in vivo and provide sophisticated components for directed nanoscale transport in vitro. We previously constructed myosin motors that respond to a change in [Ca++] by reversing their direction of motion along the polarized actin filament. To expand the potential applications of controllable molecular motors, we have now developed myosins that shift gears in response to blue light illumination. Light is a versatile control signal that can be readily modulated in time and space, and is generally orthogonal to cellular signaling. Using structure-guided protein engineering, we have incorporated LOV photoreceptor domains into the lever arms of chimeric myosins, resulting in motors that robustly speed up, slow down, or switch directions upon illumination. These genetically encoded motors should be directly deployable inside living cells. Our successful designs include constructs based on two different myosin classes, and we show that optical velocity control can be implemented in motors that move at microns/sec speeds, enabling practical biological and bioengineering applications.

Load and Pi control flux through the branched kinetic cycle of myosin V.

PubMed

Kad, Neil M; Trybus, Kathleen M; Warshaw, David M

2008-06-20

Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (P(i)), both forward and backward steps become less load-dependent. From these data, we infer that P(i) release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of P(i). We propose a branched kinetic model to explain these data. Our model includes P(i) release prior to the most load-dependent step in the cycle, implying that P(i) release and load both act as checkpoints that control the flux through two parallel pathways.

Human Usher 1B/mouse shaker-1: the retinal phenotype discrepancy explained by the presence/absence of myosin VIIA in the photoreceptor cells.

PubMed

el-Amraoui, A; Sahly, I; Picaud, S; Sahel, J; Abitbol, M; Petit, C

1996-08-01

Usher syndrome type 1 (USH1) associates severe congenital deafness, vestibular dysfunction and progressive retinitis pigmentosa leading to blindness. The gene encoding myosin VIIA is responsible for USH1B. Mutations in the murine orthologous gene lead to the shaker-1 phenotype, which manifests cochlear and vestibular dysfunction, without any retinal defect. To address this phenotypic discrepancy, the expression of myosin VIIA in retinal cells was analyzed in human and mouse during embryonic development and adult life. In the human embryo, myosin VIIA was present first in the pigment epithelium cells, and later in these cells as well as in the photoreceptor cells. In the adult human retina, myosin VIIA was present in both cell types. In contrast, in mouse, only pigment epithelium cells expressed the protein throughout development and adult life. Myosin VIIA was also found to be absent in the photoreceptor cells of other rodents (rat and guinea-pig), whereas these cells expressed the protein in amphibians, avians and primates. These observations suggest that retinitis pigmentosa of USH1B results from a primary rod and cone defect. The USH1B/shaker-1 paradigm illustrates a species-specific cell pattern of gene expression as a possible cause for the discrepancy between phenotypes involving defective orthologous genes in man and mouse. Interestingly, in the photoreceptor cells, myosin VIIA is mainly localized in the inner and base of outer segments as well as in the synaptic ending region where it is co-localized with the synaptic vesicles. Therefore, we suggest that myosin VIIA might play a role in the trafficking of ribbon-synaptic vesicle complexes and the renewal processes of the outer photoreceptor disks.

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

PubMed

Zhang, Wenwu; Gunst, Susan J

2017-07-01

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. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

The Effect of Experimental Hyperthyroidism on Characteristics of Actin-Myosin Interaction in Fast and Slow Skeletal Muscles.

PubMed

Kopylova, G V; Shchepkin, D V; Bershitsky, S Y

2018-05-01

The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin-myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.

Dynamics of myosin II organization into cortical contractile networks and fibers

NASA Astrophysics Data System (ADS)

Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

2014-03-01

The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

Myosin conformational states determined by single fluorophore polarization

PubMed Central

Warshaw, David M.; Hayes, Eric; Gaffney, Donald; Lauzon, Anne-Marie; Wu, Junru; Kennedy, Guy; Trybus, Kathleen; Lowey, Susan; Berger, Christopher

1998-01-01

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. PMID:9653135

Single Molecule Stepping and Structural Dynamics of Myosin X

PubMed Central

Sun, Yujie; Sato, Osamu; Ruhnow, Felix; Arsenault, Mark E.; Ikebe, Mitsuo; Goldman, Yale E.

2010-01-01

Myosin X is an unconventional myosin with puzzling motility properties. We studied the motility of dimerized myosin X using single molecule fluorescence techniques – polTIRF, FIONA, and Parallax to measure rotation angles and 3-dimensional position of the molecule during its walk. It was found that Myosin X steps processively in a hand-over-hand manner following a left-handed helical path along both single actin filaments and bundles. Its step size and velocity are smaller on actin bundles than individual filaments, suggesting myosin X often steps onto neighboring filaments in a bundle. The data suggest that a previously postulated single α-helical domain mechanically extends the 3-IQ motif lever arm and either the neck-tail hinge or the tail is flexible. These structural features, in conjunction with the membrane and microtubule binding domains, enable myosin X to perform multiple functions on varied actin structures in cells. PMID:20364131

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

PubMed Central

Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

1989-01-01

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs.

PubMed

Lucero, Amy; Stack, Christianna; Bresnick, Anne R; Shuster, Charles B

2006-09-01

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

PubMed Central

Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

2006-01-01

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

PARAMETERS OF TEXTURE CHANGE IN PROCESSED FISH: MYOSIN DENATURATION.

PubMed

Chu, George Hao; Sterling, Clarence

1970-03-01

The white muscle of the Sacramento blackfish (Orthodon microlepidotus) was processed by freezing, dehydration, and cooking. Myosin was extracted immediately afterwards or following a period of storage in order to examine evidence for denaturation. The tests used were the solubility of whole muscle protein and the intrinsic viscosity, isoelectric point, ATPase activity, ultra-violet absorption spectrum, and optical rotatory dispersion of purified myosin extract. Almost all measures used showed that denaturation increased in the order: fresh < frozen < frozen-stored < dehydrated < dehydrated-stored < cooked.

Scale dependence of the mechanics of active gels with increasing motor concentration.

PubMed

Sonn-Segev, Adar; Bernheim-Groswasser, Anne; Roichman, Yael

2017-10-18

Actin is a protein that plays an essential role in maintaining the mechanical integrity of cells. In response to strong external stresses, it can assemble into large bundles, but it grows into a fine branched network to induce cell motion. In some cases, the self-organization of actin fibers and networks involves the action of bipolar filaments of the molecular motor myosin. Such self-organization processes mediated by large myosin bipolar filaments have been studied extensively in vitro. Here we create active gels, composed of single actin filaments and small myosin bipolar filaments. The active steady state in these gels persists long enough to enable the characterization of their mechanical properties using one and two point microrheology. We study the effect of myosin concentration on the mechanical properties of this model system for active matter, for two different motor assembly sizes. In contrast to previous studies of networks with large motor assemblies, we find that the fluctuations of tracer particles embedded in the network decrease in amplitude as motor concentration increases. Nonetheless, we show that myosin motors stiffen the actin networks, in accordance with bulk rheology measurements of networks containing larger motor assemblies. This implies that such stiffening is of universal nature and may be relevant to a wider range of cytoskeleton-based structures.

A model of muscle contraction based on the Langevin equation with actomyosin potentials.

PubMed

Tamura, Youjiro; Ito, Akira; Saito, Masami

2017-02-01

We propose a muscle contraction model that is essentially a model of the motion of myosin motors as described by a Langevin equation. This model involves one-dimensional numerical calculations wherein the total force is the sum of a viscous force proportional to the myosin head velocity, a white Gaussian noise produced by random forces and other potential forces originating from the actomyosin structure and intra-molecular charges. We calculate the velocity of a single myosin on an actin filament to be 4.9-49 μm/s, depending on the viscosity between the actomyosin molecules. A myosin filament with a hundred myosin heads is used to simulate the contractions of a half-sarcomere within the skeletal muscle. The force response due to a quick release in the isometric contraction is simulated using a process wherein crossbridges are changed forcibly from one state to another. In contrast, the force response to a quick stretch is simulated using purely mechanical characteristics. We simulate the force-velocity relation and energy efficiency in the isotonic contraction and adenosine triphosphate consumption. The simulation results are in good agreement with the experimental results. We show that the Langevin equation for the actomyosin potentials can be modified statistically to become an existing muscle model that uses Maxwell elements.

Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion

PubMed Central

Zihni, Ceniz; Harris, Andrew R.; Bailly, Maryse; Charras, Guillaume T.; Balda, Maria S.; Matter, Karl

2012-01-01

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells. PMID:23185572

Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro

PubMed Central

Kitamura, Kazuo; Tokunaga, Makio; Esaki, Seiji; Iwane, Atsuko Hikikoshi; Yanagida, Toshio

2005-01-01

We have previously measured the process of displacement generation by a single head of muscle myosin (S1) using scanning probe nanometry. Given that the myosin head was rigidly attached to a fairly large scanning probe, it was assumed to stably interact with an underlying actin filament without diffusing away as would be the case in muscle. The myosin head has been shown to step back and forth stochastically along an actin filament with actin monomer repeats of 5.5 nm and to produce a net movement in the forward direction. The myosin head underwent 5 forward steps to produce a maximum displacement of 30 nm per ATP at low load (<1 pN). Here, we measured the steps over a wide range of forces up to 4 pN. The size of the steps (∼5.5 nm) did not change as the load increased whereas the number of steps per displacement and the stepping rate both decreased. The rate of the 5.5-nm steps at various force levels produced a force-velocity curve of individual actomyosin motors. The force-velocity curve from the individual myosin heads was comparable to that reported in muscle, suggesting that the fundamental mechanical properties in muscle are basically due to the intrinsic stochastic nature of individual actomyosin motors. In order to explain multiple stochastic steps, we propose a model arguing that the thermally-driven step of a myosin head is biased in the forward direction by a potential slope along the actin helical pitch resulting from steric compatibility between the binding sites of actin and a myosin head. Furthermore, computer simulations show that multiple cooperating heads undergoing stochastic steps generate a long (>60 nm) sliding distance per ATP between actin and myosin filaments, i.e., the movement is loosely coupled to the ATPase cycle as observed in muscle. PMID:27857548

Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro.

PubMed

Kitamura, Kazuo; Tokunaga, Makio; Esaki, Seiji; Iwane, Atsuko Hikikoshi; Yanagida, Toshio

2005-01-01

We have previously measured the process of displacement generation by a single head of muscle myosin (S1) using scanning probe nanometry. Given that the myosin head was rigidly attached to a fairly large scanning probe, it was assumed to stably interact with an underlying actin filament without diffusing away as would be the case in muscle. The myosin head has been shown to step back and forth stochastically along an actin filament with actin monomer repeats of 5.5 nm and to produce a net movement in the forward direction. The myosin head underwent 5 forward steps to produce a maximum displacement of 30 nm per ATP at low load (<1 pN). Here, we measured the steps over a wide range of forces up to 4 pN. The size of the steps (∼5.5 nm) did not change as the load increased whereas the number of steps per displacement and the stepping rate both decreased. The rate of the 5.5-nm steps at various force levels produced a force-velocity curve of individual actomyosin motors. The force-velocity curve from the individual myosin heads was comparable to that reported in muscle, suggesting that the fundamental mechanical properties in muscle are basically due to the intrinsic stochastic nature of individual actomyosin motors. In order to explain multiple stochastic steps, we propose a model arguing that the thermally-driven step of a myosin head is biased in the forward direction by a potential slope along the actin helical pitch resulting from steric compatibility between the binding sites of actin and a myosin head. Furthermore, computer simulations show that multiple cooperating heads undergoing stochastic steps generate a long (>60 nm) sliding distance per ATP between actin and myosin filaments, i.e., the movement is loosely coupled to the ATPase cycle as observed in muscle.

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

PubMed Central

Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

2016-01-01

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

PubMed Central

Bird, Jonathan E.; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R.; Friedman, Thomas B.

2014-01-01

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes*

PubMed Central

Bultema, Jarred J.; Boyle, Judith A.; Malenke, Parker B.; Martin, Faye E.; Dell'Angelica, Esteban C.; Cheney, Richard E.; Di Pietro, Santiago M.

2014-01-01

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. PMID:25324551

Electrostatic origin of the unidirectionality of walking myosin V motors.

PubMed

Mukherjee, Shayantani; Warshel, Arieh

2013-10-22

Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ∼2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors.

The fraction of myosin motors that participate in isometric contraction of rabbit muscle fibers at near-physiological temperature.

PubMed

Tsaturyan, Andrey K; Bershitsky, Sergey Y; Koubassova, Natalia A; Fernandez, Manuel; Narayanan, Theyencheri; Ferenczi, Michael A

2011-07-20

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca(2+) at 4°C were heated to 31-34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41-43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ~6.3 pN. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

PubMed Central

Stark, Benjamin C.; James, Michael L.; Pollard, Luther W.; Sirotkin, Vladimir; Lord, Matthew

2013-01-01

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors. PMID:24244528

Myosin V is a biological Brownian machine.

PubMed

Fujita, Keisuke; Iwaki, Mitsuhiro

2014-01-01

Myosin V is a vesicle transporter that unidirectionally walks along cytoskeletal actin filaments by converting the chemical energy of ATP into mechanical work. Recently, it was found that myosin V force generation is a composition of two processes: a lever-arm swing, which involves a conformational change in the myosin molecule, and a Brownian search-and-catch, which involves a diffusive "search" by the motor domain that is followed by an asymmetric "catch" in the forward actin target such that Brownian motion is rectified. Here we developed a system that combines optical tweezers with DNA nano-material to show that the Brownian search-and-catch mechanism is the energetically dominant process at near stall force, providing 13 kBT of work compared to just 3 kBT by the lever-arm swing. Our result significantly reconsiders the lever-arm swinging model, which assumes the swing dominantly produces work (>10 kBT), and sheds light on the Brownian search-and-catch as a driving process.

Myosin V is a biological Brownian machine

PubMed Central

Fujita, Keisuke; Iwaki, Mitsuhiro

2014-01-01

Myosin V is a vesicle transporter that unidirectionally walks along cytoskeletal actin filaments by converting the chemical energy of ATP into mechanical work. Recently, it was found that myosin V force generation is a composition of two processes: a lever-arm swing, which involves a conformational change in the myosin molecule, and a Brownian search-and-catch, which involves a diffusive “search” by the motor domain that is followed by an asymmetric “catch” in the forward actin target such that Brownian motion is rectified. Here we developed a system that combines optical tweezers with DNA nano-material to show that the Brownian search-and-catch mechanism is the energetically dominant process at near stall force, providing 13 kBT of work compared to just 3 kBT by the lever-arm swing. Our result significantly reconsiders the lever-arm swinging model, which assumes the swing dominantly produces work (>10 kBT), and sheds light on the Brownian search-and-catch as a driving process. PMID:27493501

Left-Right Asymmetry: Myosin 1D at the Center.

PubMed

Yuan, Shiaulou; Brueckner, Martina

2018-05-07

While a ciliated organizer generates vertebrate left-right asymmetry, most invertebrates lack an organizer and instead utilize a myosin-based mechanism. A recent study now reveals that this myosin mechanism is conserved in the vertebrate organizer and functions to regulate cilia. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

PubMed

Fulcher, F Kent; Smith, Bethany T; Russ, Misty; Patel, Yashomati M

2008-10-15

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty

2008-10-15

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform viamore » MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.« less

Regulation of intracellular trafficking and secretion of adiponectin by myosin II.

PubMed

Bedi, Deepa; Dennis, John C; Morrison, Edward E; Braden, Tim D; Judd, Robert L

2017-08-19

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB. Copyright © 2017 Elsevier Inc. All rights reserved.

X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns

PubMed Central

Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto; Suggs, Jennifer A.; Bernstein, Sanford I.

2014-01-01

Fruit fly (Drosophila melanogaster) is one of the most useful animal models to study the causes and effects of hereditary diseases because of its rich genetic resources. It is especially suitable for studying myopathies caused by myosin mutations, because specific mutations can be induced to the flight muscle-specific myosin isoform, while leaving other isoforms intact. Here we describe an X-ray-diffraction-based method to evaluate the structural effects of mutations in contractile proteins in Drosophila indirect flight muscle. Specifically, we describe the effect of the headless myosin mutation, Mhc10-Y97, in which the motor domain of the myosin head is deleted, on the X-ray diffraction pattern. The loss of general integrity of the filament lattice is evident from the pattern. A striking observation, however, is the prominent meridional reflection at d = 14.5 nm, a hallmark for the regularity of the myosin-containing thick filament. This reflection has long been considered to arise mainly from the myosin head, but taking the 6th actin layer line reflection as an internal control, the 14.5-nm reflection is even stronger than that of wild-type muscle. We confirmed these results via electron microscopy, wherein image analysis revealed structures with a similar periodicity. These observations have major implications on the interpretation of myosin-based reflections. PMID:25400584

Genome-wide identification, splicing, and expression analysis of the myosin gene family in maize (Zea mays)

PubMed Central

Wang, Guifeng; Zhong, Mingyu; Wang, Gang; Song, Rentao

2014-01-01

The actin-based myosin system is essential for the organization and dynamics of the endomembrane system and transport network in plant cells. Plants harbour two unique myosin groups, class VIII and class XI, and the latter is structurally and functionally analogous to the animal and fungal class V myosin. Little is known about myosins in grass, even though grass includes several agronomically important cereal crops. Here, we identified 14 myosin genes from the genome of maize (Zea mays). The relatively larger sizes of maize myosin genes are due to their much longer introns, which are abundant in transposable elements. Phylogenetic analysis indicated that maize myosin genes could be classified into class VIII and class XI, with three and 11 members, respectively. Apart from subgroup XI-F, the remaining subgroups were duplicated at least in one analysed lineage, and the duplication events occurred more extensively in Arabidopsis than in maize. Only two pairs of maize myosins were generated from segmental duplication. Expression analysis revealed that most maize myosin genes were expressed universally, whereas a few members (XI-1, -6, and -11) showed an anther-specific pattern, and many underwent extensive alternative splicing. We also found a short transcript at the O1 locus, which conceptually encoded a headless myosin that most likely functions at the transcriptional level rather than via a dominant-negative mechanism at the translational level. Together, these data provide significant insights into the evolutionary and functional characterization of maize myosin genes that could transfer to the identification and application of homologous myosins of other grasses. PMID:24363426

Myosin vc interacts with Rab32 and Rab38 proteins and works in the biogenesis and secretion of melanosomes.

PubMed

Bultema, Jarred J; Boyle, Judith A; Malenke, Parker B; Martin, Faye E; Dell'Angelica, Esteban C; Cheney, Richard E; Di Pietro, Santiago M

2014-11-28

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

PubMed Central

Cammarato, Anthony; Dambacher, Corey M.; Knowles, Aileen F.; Kronert, William A.; Bodmer, Rolf

2008-01-01

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. PMID:18045988

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

PubMed

Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

2017-06-30

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s -1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s -1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of myosin light chain in shrimp hemocytic phagocytosis.

PubMed

Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

2010-11-01

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

Participation of Myosin Va and Pka Type I in the Regeneration of Neuromuscular Junctions

PubMed Central

Röder, Ira Verena; Strack, Siegfried; Reischl, Markus; Dahley, Oliver; Khan, Muzamil Majid; Kassel, Olivier; Zaccolo, Manuela; Rudolf, Rüdiger

2012-01-01

Background The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration. Methodology/Principal Findings To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology. Conclusions/Significance Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle. PMID:22815846

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

PubMed

Yang, Yuting; Gourinath, S; Kovács, Mihály; Nyitray, László; Reutzel, Robbie; Himmel, Daniel M; O'Neall-Hennessey, Elizabeth; Reshetnikova, Ludmilla; Szent-Györgyi, Andrew G; Brown, Jerry H; Cohen, Carolyn

2007-05-01

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

A model of stereocilia adaptation based on single molecule mechanical studies of myosin I.

PubMed Central

Batters, Christopher; Wallace, Mark I; Coluccio, Lynne M; Molloy, Justin E

2004-01-01

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical 'T2' behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear. PMID:15647165

The post-rigor structure of myosin VI and implications for the recovery stroke

PubMed Central

Ménétrey, Julie; Llinas, Paola; Cicolari, Jérome; Squires, Gaëlle; Liu, Xiaoyan; Li, Anna; Sweeney, H Lee; Houdusse, Anne

2008-01-01

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP. PMID:18046460

Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed

PubMed Central

Shaffer, Justin F.; Kier, William M.

2012-01-01

SUMMARY The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds. PMID:22189767

Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture.

PubMed

Kagawa, Maiko; Sato, Naruki; Obinata, Takashi

2006-11-01

Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.

Whole genome duplication events in plant evolution reconstructed and predicted using myosin motor proteins

PubMed Central

2013-01-01

Background The evolution of land plants is characterized by whole genome duplications (WGD), which drove species diversification and evolutionary novelties. Detecting these events is especially difficult if they date back to the origin of the plant kingdom. Established methods for reconstructing WGDs include intra- and inter-genome comparisons, KS age distribution analyses, and phylogenetic tree constructions. Results By analysing 67 completely sequenced plant genomes 775 myosins were identified and manually assembled. Phylogenetic trees of the myosin motor domains revealed orthologous and paralogous relationships and were consistent with recent species trees. Based on the myosin inventories and the phylogenetic trees, we have identified duplications of the entire myosin motor protein family at timings consistent with 23 WGDs, that had been reported before. We also predict 6 WGDs based on further protein family duplications. Notably, the myosin data support the two recently reported WGDs in the common ancestor of all extant angiosperms. We predict single WGDs in the Manihot esculenta and Nicotiana benthamiana lineages, two WGDs for Linum usitatissimum and Phoenix dactylifera, and a triplication or two WGDs for Gossypium raimondii. Our data show another myosin duplication in the ancestor of the angiosperms that could be either the result of a single gene duplication or a remnant of a WGD. Conclusions We have shown that the myosin inventories in angiosperms retain evidence of numerous WGDs that happened throughout plant evolution. In contrast to other protein families, many myosins are still present in extant species. They are closely related and have similar domain architectures, and their phylogenetic grouping follows the genome duplications. Because of its broad taxonomic sampling the dataset provides the basis for reliable future identification of further whole genome duplications. PMID:24053117

Whole genome duplication events in plant evolution reconstructed and predicted using myosin motor proteins.

PubMed

Mühlhausen, Stefanie; Kollmar, Martin

2013-09-22

The evolution of land plants is characterized by whole genome duplications (WGD), which drove species diversification and evolutionary novelties. Detecting these events is especially difficult if they date back to the origin of the plant kingdom. Established methods for reconstructing WGDs include intra- and inter-genome comparisons, KS age distribution analyses, and phylogenetic tree constructions. By analysing 67 completely sequenced plant genomes 775 myosins were identified and manually assembled. Phylogenetic trees of the myosin motor domains revealed orthologous and paralogous relationships and were consistent with recent species trees. Based on the myosin inventories and the phylogenetic trees, we have identified duplications of the entire myosin motor protein family at timings consistent with 23 WGDs, that had been reported before. We also predict 6 WGDs based on further protein family duplications. Notably, the myosin data support the two recently reported WGDs in the common ancestor of all extant angiosperms. We predict single WGDs in the Manihot esculenta and Nicotiana benthamiana lineages, two WGDs for Linum usitatissimum and Phoenix dactylifera, and a triplication or two WGDs for Gossypium raimondii. Our data show another myosin duplication in the ancestor of the angiosperms that could be either the result of a single gene duplication or a remnant of a WGD. We have shown that the myosin inventories in angiosperms retain evidence of numerous WGDs that happened throughout plant evolution. In contrast to other protein families, many myosins are still present in extant species. They are closely related and have similar domain architectures, and their phylogenetic grouping follows the genome duplications. Because of its broad taxonomic sampling the dataset provides the basis for reliable future identification of further whole genome duplications.

Motor Force Homeostasis in Skeletal Muscle Contraction

PubMed Central

Chen, Bin; Gao, Huajian

2011-01-01

In active biological contractile processes such as skeletal muscle contraction, cellular mitosis, and neuronal growth, an interesting common observation is that multiple motors can perform coordinated and synchronous actions, whereas individual myosin motors appear to randomly attach to and detach from actin filaments. Recent experiment has demonstrated that, during skeletal muscle shortening at a wide range of velocities, individual myosin motors maintain a force of ∼6 pN during a working stroke. To understand how such force-homeostasis can be so precisely regulated in an apparently chaotic system, here we develop a molecular model within a coupled stochastic-elastic theoretical framework. The model reveals that the unique force-stretch relation of myosin motor and the stochastic behavior of actin-myosin binding cause the average number of working motors to increase in linear proportion to the filament load, so that the force on each working motor is regulated at ∼6 pN, in excellent agreement with experiment. This study suggests that it might be a general principle to use catch bonds together with a force-stretch relation similar to that of myosin motors to regulate force homeostasis in many biological processes. PMID:21767492

Thick Filament Length and Isoform Composition Determine Self-Organized Contractile Units in Actomyosin Bundles

PubMed Central

Thoresen, Todd; Lenz, Martin; Gardel, Margaret L.

2013-01-01

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. PMID:23442916

Robust mechanobiological behavior emerges in heterogeneous myosin systems.

PubMed

Egan, Paul F; Moore, Jeffrey R; Ehrlicher, Allen J; Weitz, David A; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

2017-09-26

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Robust mechanobiological behavior emerges in heterogeneous myosin systems

NASA Astrophysics Data System (ADS)

Egan, Paul F.; Moore, Jeffrey R.; Ehrlicher, Allen J.; Weitz, David A.; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

2017-09-01

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Tropomyosins as discriminators of myosin function.

PubMed

Ostap, E Michael

2008-01-01

Vertebrate nonmuscle cells express multiple tropomyosin isoforms that are sorted to subcellular compartments that have distinct morphological and dynamic properties. The creation of these compartments has a role in controlling cell morphology, cell migration and polarization of cellular components. There is increasing evidence that nonmuscle myosins are regulated by tropomyosin in these compartments via the regulation of actin attachment, ATPase kinetics, or by stabilization of cytoskeletal tracks for myosin-based transport. In this chapter, I review the literature describing the regulation of various myosins by tropomyosins and consider the mechanisms for this regulation.

A missense mutation in myosin VIIA prevents aminoglycoside accumulation in early postnatal cochlear hair cells.

PubMed

Richardson, G P; Forge, A; Kros, C J; Marcotti, W; Becker, D; Williams, D S; Thorpe, J; Fleming, J; Brown, S D; Steel, K P

1999-11-28

Myosin VIIA is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the myosin VIIA molecule we show that this myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for myosin VIIA in the process of aminoglycoside accumulation are discussed.

Myosin II Activity Softens Cells in Suspension.

PubMed

Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

2015-04-21

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

PubMed Central

2012-01-01

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

PubMed Central

1992-01-01

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1469047

Motor-motor interactions in ensembles of muscle myosin: using theory to connect single molecule to ensemble measurements

NASA Astrophysics Data System (ADS)

Walcott, Sam

2013-03-01

Interactions between the proteins actin and myosin drive muscle contraction. Properties of a single myosin interacting with an actin filament are largely known, but a trillion myosins work together in muscle. We are interested in how single-molecule properties relate to ensemble function. Myosin's reaction rates depend on force, so ensemble models keep track of both molecular state and force on each molecule. These models make subtle predictions, e.g. that myosin, when part of an ensemble, moves actin faster than when isolated. This acceleration arises because forces between molecules speed reaction kinetics. Experiments support this prediction and allow parameter estimates. A model based on this analysis describes experiments from single molecule to ensemble. In vivo, actin is regulated by proteins that, when present, cause the binding of one myosin to speed the binding of its neighbors; binding becomes cooperative. Although such interactions preclude the mean field approximation, a set of linear ODEs describes these ensembles under simplified experimental conditions. In these experiments cooperativity is strong, with the binding of one molecule affecting ten neighbors on either side. We progress toward a description of myosin ensembles under physiological conditions.

Stochastic dynamics of small ensembles of non-processive molecular motors: The parallel cluster model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erdmann, Thorsten; Albert, Philipp J.; Schwarz, Ulrich S.

2013-11-07

Non-processive molecular motors have to work together in ensembles in order to generate appreciable levels of force or movement. In skeletal muscle, for example, hundreds of myosin II molecules cooperate in thick filaments. In non-muscle cells, by contrast, small groups with few tens of non-muscle myosin II motors contribute to essential cellular processes such as transport, shape changes, or mechanosensing. Here we introduce a detailed and analytically tractable model for this important situation. Using a three-state crossbridge model for the myosin II motor cycle and exploiting the assumptions of fast power stroke kinetics and equal load sharing between motors inmore » equivalent states, we reduce the stochastic reaction network to a one-step master equation for the binding and unbinding dynamics (parallel cluster model) and derive the rules for ensemble movement. We find that for constant external load, ensemble dynamics is strongly shaped by the catch bond character of myosin II, which leads to an increase of the fraction of bound motors under load and thus to firm attachment even for small ensembles. This adaptation to load results in a concave force-velocity relation described by a Hill relation. For external load provided by a linear spring, myosin II ensembles dynamically adjust themselves towards an isometric state with constant average position and load. The dynamics of the ensembles is now determined mainly by the distribution of motors over the different kinds of bound states. For increasing stiffness of the external spring, there is a sharp transition beyond which myosin II can no longer perform the power stroke. Slow unbinding from the pre-power-stroke state protects the ensembles against detachment.« less

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro*

PubMed Central

Haldeman, Brian D.; Brizendine, Richard K.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

2014-01-01

Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ∼0.63 μm long and contain ∼176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function*

PubMed Central

Kronert, William A.; Melkani, Girish C.; Melkani, Anju; Bernstein, Sanford I.

2014-01-01

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

Actomyosin-based tissue folding requires a multicellular myosin gradient

PubMed Central

Miller, Pearson W.; Chanet, Soline; Stoop, Norbert; Dunkel, Jörn

2017-01-01

Tissue folding promotes three-dimensional (3D) form during development. In many cases, folding is associated with myosin accumulation at the apical surface of epithelial cells, as seen in the vertebrate neural tube and the Drosophila ventral furrow. This type of folding is characterized by constriction of apical cell surfaces, and the resulting cell shape change is thought to cause tissue folding. Here, we use quantitative microscopy to measure the pattern of transcription, signaling, myosin activation and cell shape in the Drosophila mesoderm. We found that cells within the ventral domain accumulate different amounts of active apical non-muscle myosin 2 depending on the distance from the ventral midline. This gradient in active myosin depends on a newly quantified gradient in upstream signaling proteins. A 3D continuum model of the embryo with induced contractility demonstrates that contractility gradients, but not contractility per se, promote changes to surface curvature and folding. As predicted by the model, experimental broadening of the myosin domain in vivo disrupts tissue curvature where myosin is uniform. Our data argue that apical contractility gradients are important for tissue folding. PMID:28432215

Dynamics of myosin-driven skeletal muscle contraction: I. Steady-state force generation.

PubMed

Lan, Ganhui; Sun, Sean X

2005-06-01

Skeletal muscle contraction is a canonical example of motor-driven force generation. Despite the long history of research in this topic, a mechanistic explanation of the collective myosin force generation is lacking. We present a theoretical model of muscle contraction based on the conformational movements of individual myosins and experimentally measured chemical rate constants. Detailed mechanics of the myosin motor and the geometry of the sarcomere are taken into account. Two possible scenarios of force generation are examined. We find only one of the scenarios can give rise to a plausible contraction mechanism. We propose that the synchrony in muscle contraction is due to a force-dependent ADP release step. Computational results of a half sarcomere with 150 myosin heads can explain the experimentally measured force-velocity relationship and efficiency data. We predict that the number of working myosin motors increases as the load force is increased, thus showing synchrony among myosin motors during muscle contraction. We also find that titin molecules anchoring the thick filament are passive force generators in assisting muscle contraction.

Dynamics of Myosin-Driven Skeletal Muscle Contraction: I. Steady-State Force Generation

PubMed Central

Lan, Ganhui; Sun, Sean X.

2005-01-01

Skeletal muscle contraction is a canonical example of motor-driven force generation. Despite the long history of research in this topic, a mechanistic explanation of the collective myosin force generation is lacking. We present a theoretical model of muscle contraction based on the conformational movements of individual myosins and experimentally measured chemical rate constants. Detailed mechanics of the myosin motor and the geometry of the sarcomere are taken into account. Two possible scenarios of force generation are examined. We find only one of the scenarios can give rise to a plausible contraction mechanism. We propose that the synchrony in muscle contraction is due to a force-dependent ADP release step. Computational results of a half sarcomere with 150 myosin heads can explain the experimentally measured force-velocity relationship and efficiency data. We predict that the number of working myosin motors increases as the load force is increased, thus showing synchrony among myosin motors during muscle contraction. We also find that titin molecules anchoring the thick filament are passive force generators in assisting muscle contraction. PMID:15778440

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

PubMed

Kim, Kyoungtae; Keller, Thomas C S

2002-01-07

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis

PubMed Central

Wang, Yan; Xu, Yingqiong; Liu, Qian; Zhang, Yuanyuan; Gao, Zhen; Yin, Mingzhu; Jiang, Nan; Cao, Guosheng; Yu, Boyang; Cao, Zhengyu; Kou, Junping

2017-01-01

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases. PMID:28352215

Disordered Actomyosin Is Sufficient to Promote Cooperative and Telescopic Contractility

NASA Astrophysics Data System (ADS)

Murrell, Michael; Linsmeier, Ian; Banerjee, Shiladitya; Kim, Tae Yoon; Jung, Wonyeong; Oakes, Patrick

While the molecular interactions between myosin motors and F-actin are well known, the relationship between F-actin organization and myosin-mediated force generation remains poorly understood. Here, we explore the accumulation of myosin-induced stresses within a 2D biomimetic model of the actomyosin cortex, where myosin activity is controlled spatially and temporally using light. By controlling the geometry and the duration of myosin activation, we show that contraction of disordered actomyosin is highly cooperative, telescopic with the activation area and generates a pattern of mechanical stresses consistent with those observed in contractile cells. We quantitatively reproduce these properties using an in vitro isotropic model of the actomyosin cytoskeleton, and explore the physical origins of telescopic contractility in disordered networks using agent-based simulations. NSF CMMI-1525316.

Thick filament length and isoform composition determine self-organized contractile units in actomyosin bundles.

PubMed

Thoresen, Todd; Lenz, Martin; Gardel, Margaret L

2013-02-05

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

PubMed

Calábria, Luciana Karen; Peixoto, Pablo Marco Veras; Passos Lima, Andreia Barcelos; Peixoto, Leonardo Gomes; de Moraes, Viviane Rodrigues Alves; Teixeira, Renata Roland; Dos Santos, Claudia Tavares; E Silva, Letícia Oliveira; da Silva, Maria de Fátima Rodrigues; dos Santos, Ana Alice Diniz; Garcia-Cairasco, Norberto; Martins, Antônio Roberto; Espreafico, Enilza Maria; Espindola, Foued Salmen

2011-09-01

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration.

PubMed

Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S

2016-11-01

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

PubMed Central

Preller, Matthias

2017-01-01

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures. PMID:29256864

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction*

PubMed Central

Zhang, Wen-Cheng; Peng, Ya-Jing; Zhang, Gen-Sheng; He, Wei-Qi; Qiao, Yan-Ning; Dong, Ying-Ying; Gao, Yun-Qian; Chen, Chen; Zhang, Cheng-Hai; Li, Wen; Shen, Hua-Hao; Ning, Wen; Kamm, Kristine E.; Stull, James T.; Gao, Xiang; Zhu, Min-Sheng

2010-01-01

Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K+-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance. PMID:20018858

Myosin VIIa as a common component of cilia and microvilli.

PubMed

Wolfrum, U; Liu, X; Schmitt, A; Udovichenko, I P; Williams, D S

1998-01-01

The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

Mechanochemical models of processive molecular motors

NASA Astrophysics Data System (ADS)

Lan, Ganhui; Sun, Sean X.

2012-05-01

Motor proteins are the molecular engines powering the living cell. These nanometre-sized molecules convert chemical energy, both enthalpic and entropic, into useful mechanical work. High resolution single molecule experiments can now observe motor protein movement with increasing precision. The emerging data must be combined with structural and kinetic measurements to develop a quantitative mechanism. This article describes a modelling framework where quantitative understanding of motor behaviour can be developed based on the protein structure. The framework is applied to myosin motors, with emphasis on how synchrony between motor domains give rise to processive unidirectional movement. The modelling approach shows that the elasticity of protein domains are important in regulating motor function. Simple models of protein domain elasticity are presented. The framework can be generalized to other motor systems, or an ensemble of motors such as muscle contraction. Indeed, for hundreds of myosins, our framework can be reduced to the Huxely-Simmons description of muscle movement in the mean-field limit.

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

PubMed Central

Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

2015-01-01

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

Mechanism of the calcium-regulation of muscle contraction--in pursuit of its structural basis.

PubMed

Wakabayashi, Takeyuki

2015-01-01

The author reviewed the research that led to establish the structural basis for the mechanism of the calcium-regulation of the contraction of striated muscles. The target of calcium ions is troponin on the thin filaments, of which the main component is the double-stranded helix of actin. A model of thin filament was generated by adding tropomyosin and troponin. During the process to provide the structural evidence for the model, the troponin arm was found to protrude from the calcium-depleted troponin and binds to the carboxyl-terminal region of actin. As a result, the carboxyl-terminal region of tropomyosin shifts and covers the myosin-binding sites of actin to block the binding of myosin. At higher calcium concentrations, the troponin arm changes its partner from actin to the main body of calcium-loaded troponin. Then, tropomyosin shifts back to the position near the grooves of actin double helix, and the myosin-binding sites of actin becomes available to myosin resulting in force generation through actin-myosin interactions.

Coupling of Lever Arm Swing and Biased Brownian Motion in Actomyosin

PubMed Central

Nie, Qing-Miao; Togashi, Akio; Sasaki, Takeshi N.; Takano, Mitsunori; Sasai, Masaki; Terada, Tomoki P.

2014-01-01

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5–11 nm displacement due to the biased Brownian motion and the 3–5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family. PMID:24762409

Coupling of lever arm swing and biased Brownian motion in actomyosin.

PubMed

Nie, Qing-Miao; Togashi, Akio; Sasaki, Takeshi N; Takano, Mitsunori; Sasai, Masaki; Terada, Tomoki P

2014-04-01

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.

Kinetic Adaptations of Myosins for their Diverse Cellular Functions

PubMed Central

Heissler, Sarah M.; Sellers, James R.

2016-01-01

Members of the myosin superfamily are involved in all aspects of eukaryotic life. Their function ranges from the transport of organelles and cargos to the generation of membrane tension, and the contraction of muscle. The diversity of physiological functions is remarkable, given that all enzymatically active myosins follow a conserved mechanoenzymatic cycle in which the hydrolysis of ATP to ADP and inorganic phosphate is coupled to either actin-based transport or tethering of actin to defined cellular compartments. Kinetic capacities and limitations of a myosin are determined by the extent to with actin can accelerate the hydrolysis of ATP and the release of the hydrolysis products and are indispensably linked to its physiological tasks. This review focuses on kinetic competencies that – together with structural adaptations – result in myosins with unique mechanoenzymatic properties targeted to their diverse cellular function. PMID:26929436

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE PAGES

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

2014-12-02

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

PubMed Central

Milton, Deanna L.; Schneck, Amy N.; Ziech, Dominique A.; Ba, Mariam; Facemyer, Kevin C.; Halayko, Andrew J.; Baker, Jonathan E.; Gerthoffer, William T.; Cremo, Christine R.

2011-01-01

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber–associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells. PMID:21205888

Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor*

PubMed Central

Lu, Zekuan; Ma, Xiao-Nan; Zhang, Hai-Man; Ji, Huan-Hong; Ding, Hao; Zhang, Jie; Luo, Dan; Sun, Yujie; Li, Xiang-dong

2014-01-01

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament. PMID:24825904

Characterization of Amoeba proteus myosin VI immunoanalog.

PubMed

Dominik, Magdalena; Kłopocka, Wanda; Pomorski, Paweł; Kocik, Elzbieta; Redowicz, Maria Jolanta

2005-07-01

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

PubMed Central

Clayton, Joseph E.; Pollard, Luther W.; Sckolnick, Maria; Bookwalter, Carol S.; Hodges, Alex R.; Trybus, Kathleen M.; Lord, Matthew

2014-01-01

A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. PMID:24196839

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level*

PubMed Central

Nagy, Attila; Takagi, Yasuharu; Billington, Neil; Sun, Sara A.; Hong, Davin K. T.; Homsher, Earl; Wang, Aibing; Sellers, James R.

2013-01-01

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20–25%) motor. The ADP release step (∼0.35 s−1) of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s−1). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s−1). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments. PMID:23148220

Myosin Va Bound to Phagosomes Binds to F-Actin and Delays Microtubule-dependent Motility

PubMed Central

Al-Haddad, Ahmed; Shonn, Marion A.; Redlich, Bärbel; Blocker, Ariel; Burkhardt, Janis K.; Yu, Hanry; Hammer, John A.; Weiss, Dieter G.; Steffen, Walter; Griffiths, Gareth; Kuznetsov, Sergei A.

2001-01-01

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome–F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an “antagonistic/cooperative mechanism” to explain the saltatory phagosome movement toward the cell center in normal macrophages. PMID:11553713

Sharing is Caring: The Role of Actin/Myosin-V in Synaptic Vesicle Transport between Synapses in vivo

NASA Astrophysics Data System (ADS)

Gramlich, Michael

Inter-synaptic vesicle sharing is an important but not well understood process of pre-synaptic function. Further, the molecular mechanisms that underlie this inter-synaptic exchange are not well known, and whether this inter-synaptic vesicle sharing is regulated by neural activity remains largely unexplored. I address these questions by studying CA1/CA3 Hippocampal neurons at the single synaptic vesicle level. Using high-resolution tracking of individual vesicles that have recently undergone endocytosis, I observe long-distance axonal transport of synaptic vesicles is partly mediated by the actin network. Further, the actin-dependent transport is predominantly carried out by Myosin-V. I develop a correlated-motion analysis to characterize the mechanics of how actin and Myosin-V affect vesicle transport. Lastly, I also observe that vesicle exit rates from the synapse to the axon and long-distance vesicle transport are both regulated by activity, but Myosin-V does not appear to mediate the activity dependence. These observations highlight the roles of the axonal actin network, and Myosin-V in particular, in regulating inter-synaptic vesicle exchange.

Mechanism of the calcium-regulation of muscle contraction — In pursuit of its structural basis —

PubMed Central

WAKABAYASHI, Takeyuki

2015-01-01

The author reviewed the research that led to establish the structural basis for the mechanism of the calcium-regulation of the contraction of striated muscles. The target of calcium ions is troponin on the thin filaments, of which the main component is the double-stranded helix of actin. A model of thin filament was generated by adding tropomyosin and troponin. During the process to provide the structural evidence for the model, the troponin arm was found to protrude from the calcium-depleted troponin and binds to the carboxyl-terminal region of actin. As a result, the carboxyl-terminal region of tropomyosin shifts and covers the myosin-binding sites of actin to block the binding of myosin. At higher calcium concentrations, the troponin arm changes its partner from actin to the main body of calcium-loaded troponin. Then, tropomyosin shifts back to the position near the grooves of actin double helix, and the myosin-binding sites of actin becomes available to myosin resulting in force generation through actin-myosin interactions. PMID:26194856

Role of Heavy Meromyosin in Heat-Induced Gelation in Low Ionic Strength Solution Containing L-Histidine.

PubMed

Hayakawa, Toru; Yoshida, Yuri; Yasui, Masanori; Ito, Toshiaki; Wakamatsu, Jun-ichi; Hattori, Akihito; Nishimura, Takanori

2015-08-01

The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing L-histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without L-histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing L-histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l-histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat-induced globular masses of HMM in low ionic strength solution containing L-histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing L-histidine, resulting in formation of the unique gel. © 2015 Institute of Food Technologists®

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility.

PubMed

Brotto, Marco A; Biesiadecki, Brandon J; Brotto, Leticia S; Nosek, Thomas M; Jin, Jian-Ping

2006-02-01

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

PubMed Central

BROTTO, MARCO A.; BIESIADECKI, BRANDON J.; BROTTO, LETICIA S.; NOSEK, THOMAS M; JIN, J.-P.

2005-01-01

(Summary) Brotto, Marco A., Brandon J. Biesiadecki, Leticia S. Brotto, Thomas M. Nosek, and J.-P. Jin. Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca2+ via the troponin complex. Slow and fast twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin and troponin T (TnT) and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton skinned single fibers from soleus, diaphragm, gastrocnemius and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of TnT and TnI isoform to investigate their role in determining contractility. Type IIa, IIx and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca2+ sensitivity than that of the fast troponin fibers, while fibers containing fast troponin showed a higher cooperativity of Ca2+ activation than that of the slow troponin fibers. The results demonstrate distinctive, but coordinated, regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties. PMID:16192301

Physical driving force of actomyosin motility based on the hydration effect.

PubMed

Suzuki, Makoto; Mogami, George; Ohsugi, Hideyuki; Watanabe, Takahiro; Matubayasi, Nobuyuki

2017-12-01

We propose a driving force hypothesis based on previous thermodynamics, kinetics and structural data as well as additional experiments and calculations presented here on water-related phenomena in the actomyosin systems. Although Szent-Györgyi pointed out the importance of water in muscle contraction in 1951, few studies have focused on the water science of muscle because of the difficulty of analyzing hydration properties of the muscle proteins, actin, and myosin. The thermodynamics and energetics of muscle contraction are linked to the water-mediated regulation of protein-ligand and protein-protein interactions along with structural changes in protein molecules. In this study, we assume the following two points: (1) the periodic electric field distribution along an actin filament (F-actin) is unidirectionally modified upon binding of myosin subfragment 1 (M or myosin S1) with ADP and inorganic phosphate Pi (M.ADP.Pi complex) and (2) the solvation free energy of myosin S1 depends on the external electric field strength and the solvation free energy of myosin S1 in close proximity to F-actin can become the potential force to drive myosin S1 along F-actin. The first assumption is supported by integration of experimental reports. The second assumption is supported by model calculations utilizing molecular dynamics (MD) simulation to determine solvation free energies of a small organic molecule and two small proteins. MD simulations utilize the energy representation method (ER) and the roughly proportional relationship between the solvation free energy and the solvent-accessible surface area (SASA) of the protein. The estimated driving force acting on myosin S1 is as high as several piconewtons (pN), which is consistent with the experimentally observed force. © 2017 Wiley Periodicals, Inc.

Myosin light chains: Teaching old dogs new tricks

PubMed Central

Heissler, Sarah M; Sellers, James R

2014-01-01

The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.

PubMed

Fotopoulos, N; Wernike, D; Chen, Y; Makil, N; Marte, A; Piekny, A

2013-11-01

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects

PubMed Central

Suggs, Jennifer A.; Melkani, Girish C.; Glasheen, Bernadette M.; Detor, Mia M.; Melkani, Anju; Marsan, Nathan P.; Swank, Douglas M.

2017-01-01

ABSTRACT Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in individuals with IBM3. PMID:28258125

The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

PubMed Central

Melkani, Girish C.; Bodmer, Rolf; Ocorr, Karen; Bernstein, Sanford I.

2011-01-01

UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin. PMID:21799905

Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension

PubMed Central

Tetley, Robert J; Blanchard, Guy B; Fletcher, Alexander G; Adams, Richard J; Sanson, Bénédicte

2016-01-01

Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. Here, we develop novel computational methods to analyse the spatiotemporal dynamics of Myosin II during GBE, at the scale of the tissue. We show that initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. Our results are consistent with recent work showing that a combinatorial code of Toll-like receptors downstream of pair-rule genes contributes to Myosin II polarization via local cell-cell interactions. We propose an updated cell-cell interaction model for Myosin II polarization that we tested in a vertex-based simulation. DOI: http://dx.doi.org/10.7554/eLife.12094.001 PMID:27183005

Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

PubMed Central

Carew, Josephine A.; Goyal, Raj K.; Sullivan, Maryrose P.

2014-01-01

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. PMID:24516539

Myosin concentration underlies cell size–dependent scalability of actomyosin ring constriction

PubMed Central

Wright, Graham D.; Leong, Fong Yew; Chiam, Keng-Hwee; Chen, Yinxiao; Jedd, Gregory; Balasubramanian, Mohan K.

2011-01-01

In eukaryotes, cytokinesis is accomplished by an actomyosin-based contractile ring. Although in Caenorhabditis elegans embryos larger cells divide at a faster rate than smaller cells, it remains unknown whether a similar mode of scalability operates in other cells. We investigated cytokinesis in the filamentous fungus Neurospora crassa, which exhibits a wide range of hyphal circumferences. We found that N. crassa cells divide using an actomyosin ring and larger rings constricted faster than smaller rings. However, unlike in C. elegans, the total amount of myosin remained constant throughout constriction, and there was a size-dependent increase in the starting concentration of myosin in the ring. We predict that the increased number of ring-associated myosin motors in larger rings leads to the increased constriction rate. Accordingly, reduction or inhibition of ring-associated myosin slows down the rate of constriction. Because the mechanical characteristics of contractile rings are conserved, we predict that these findings will be relevant to actomyosin ring constriction in other cell types. PMID:22123864

Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution

PubMed Central

Hu, Zhongjun; Taylor, Dianne W.; Reedy, Michael K.; Edwards, Robert J.; Taylor, Kenneth A.

2016-01-01

We describe a cryo–electron microscopy three-dimensional image reconstruction of relaxed myosin II–containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin’s long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for Lethocerus. Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation. PMID:27704041

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis.

PubMed

Dong, Bo; Deng, Wei; Jiang, Di

2011-04-01

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

PAR proteins regulate maintenance-phase myosin dynamics during Caenorhabditis elegans zygote polarization

PubMed Central

Small, Lawrence E.; Dawes, Adriana T.

2017-01-01

Establishment of anterior–posterior polarity in the Caenorhabditis elegans zygote requires two different processes: mechanical activity of the actin–myosin cortex and biochemical activity of partitioning-defective (PAR) proteins. Here we analyze how PARs regulate the behavior of the cortical motor protein nonmuscle myosin (NMY-2) to complement recent efforts that investigate how PARs regulate the Rho GTPase CDC-42, which in turn regulates the actin-myosin cortex. We find that PAR-3 and PAR-6 concentrate CDC-42–dependent NMY-2 in the anterior cortex, whereas PAR-2 inhibits CDC-42–dependent NMY-2 in the posterior domain by inhibiting PAR-3 and PAR-6. In addition, we find that PAR-1 and PAR-3 are necessary for inhibiting movement of NMY-2 across the cortex. PAR-1 protects NMY-2 from being moved across the cortex by forces likely originating in the cytoplasm. Meanwhile, PAR-3 stabilizes NMY-2 against PAR-2 and PAR-6 dynamics on the cortex. We find that PAR signaling fulfills two roles: localizing NMY-2 to the anterior cortex and preventing displacement of the polarized cortical actin–myosin network. PMID:28615321

Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

PubMed

Canclini, Lucía; Wallrabe, Horst; Di Paolo, Andrés; Kun, Alejandra; Calliari, Aldo; Sotelo-Silveira, José Roberto; Sotelo, José Roberto

2014-03-15

Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail. Copyright © 2013 Elsevier Inc. All rights reserved.

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

PubMed

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M; Ichimura, Taro

2016-07-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

PubMed Central

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M.; Ichimura, Taro

2016-01-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery. PMID:27446684

Principles of Unconventional Myosin Function and Targeting

PubMed Central

Hartman, M. Amanda; Finan, Dina; Sivaramakrishnan, Sivaraj; Spudich, James A.

2016-01-01

Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins’ roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels. PMID:21639800

Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin.

PubMed

Shiroguchi, Katsuyuki; Chin, Harvey F; Hannemann, Diane E; Muneyuki, Eiro; De La Cruz, Enrique M; Kinosita, Kazuhiko

2011-04-01

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.

Final Progress Report for FG02-89ER14030

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanson, Maureen R

2011-10-26

Intracellular Dynamics of Energy-Transducing Organelles. The location and interaction of intracellular organelles is important for exchange of substrate and product between compartments for optimum functioning of biochemical pathways and energy transduction. Plastids and stromules, tubular plastid extensions, are highly dynamic in many plant tissues. Stromules can connect two or more plastids and proteins and macromolecular complexes can be transferred between them. Stromules have been observed to form close contacts with other organelles, the plasma membrane, and can pass through channels in the nucleus. Chloroplasts move in response to light and mechanical stimulus. Especially in non-green cells, plastids change shape andmore » position, and stromules extend and retract. Stromules appear to be involved in recycling of chloroplast proteins when photosynthesis is limited, through an autophagic process that results in degradation of portions of the stromal contents without complete destruction of the chloroplast. Mutations in several genes known to mediate chloroplast division result in altered stromule morphology in some cells. Plastid and stromule motility is mediated by the actin cytoskeleton. The possible role of myosins in chloroplast movement was investigated by labeling the cargo-binding tails of six Arabidopsis myosin XI proteins with yellow fluorescent protein (YFP). The fluorescent proteins were found to localize to vesicles and peroxisomes. The portion of the myosin tail domain fused to YFP affected whether specific or non-specific localization was observed. In contrast to experiments in animal cells, movement of labeled organelles was not entirely inhibited by expression of defective myosin in which the motor domain was replaced with a fluorescent protein. None of the six myosin proteins tested labeled plastids or chloroplasts. However, the Arabidopsis myosin XI gene family expresses an additional seven myosins that await further examination. These experiments have provided more information on stromule formation and function and the actin-myosin machinery that mediates the intracellular trafficking that is required for photosynthesis and metabolism to operate efficiently within the plant cell.« less

Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

PubMed

Kuroda, Taruho S; Fukuda, Mitsunori

2005-01-01

Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina

2013-02-15

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sitesmore » (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.« less

Functional adaptation between yeast actin and its cognate myosin motors.

PubMed

Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

2011-09-02

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

PubMed

Sugi, Haruo; Chaen, Shigeru; Kobayashi, Takakazu; Abe, Takahiro; Kimura, Kazushige; Saeki, Yasutake; Ohnuki, Yoshiki; Miyakawa, Takuya; Tanokura, Masaru; Sugiura, Seiryo

2014-01-01

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Definite Differences between In Vitro Actin-Myosin Sliding and Muscle Contraction as Revealed Using Antibodies to Myosin Head

PubMed Central

Sugi, Haruo; Chaen, Shigeru; Kobayashi, Takakazu; Abe, Takahiro; Kimura, Kazushige; Saeki, Yasutake; Ohnuki, Yoshiki; Miyakawa, Takuya; Tanokura, Masaru; Sugiura, Seiryo

2014-01-01

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures. PMID:24918754

Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization

PubMed Central

Badirou, Idinath; Pan, Jiajia; Legrand, Céline; Wang, Aibing; Lordier, Larissa; Boukour, Siham; Roy, Anita; Vainchenker, William

2014-01-01

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization. PMID:25185263

Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

PubMed

Badirou, Idinath; Pan, Jiajia; Legrand, Céline; Wang, Aibing; Lordier, Larissa; Boukour, Siham; Roy, Anita; Vainchenker, William; Chang, Yunhua

2014-10-16

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

Simulating the dynamics of the mechanochemical cycle of myosin-V

PubMed Central

Mukherjee, Shayantani; Alhadeff, Raphael; Warshel, Arieh

2017-01-01

The detailed dynamics of the cycle of myosin-V are explored by simulation approaches, examining the nature of the energy-driven motion. Our study started with Langevin dynamics (LD) simulations on a very coarse landscape with a single rate-limiting barrier and reproduced the stall force and the hand-over-hand dynamics. We then considered a more realistic landscape and used time-dependent Monte Carlo (MC) simulations that allowed trajectories long enough to reproduce the force/velocity characteristic sigmoidal correlation, while also reproducing the hand-over-hand motion. Overall, our study indicated that the notion of a downhill lever-up to lever-down process (popularly known as the powerstroke mechanism) is the result of the energetics of the complete myosin-V cycle and is not the source of directional motion or force generation on its own. The present work further emphasizes the need to use well-defined energy landscapes in studying molecular motors in general and myosin in particular. PMID:28193897

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures

NASA Technical Reports Server (NTRS)

Muller, S. A.; Haner, M.; Ortiz, I.; Aebi, U.; Epstein, H. F.

2001-01-01

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Unrevealed part of myosin's powerstroke accounts for high efficiency of muscle contraction.

PubMed

Bibó, András; Károlyi, György; Kovács, Mihály

2017-09-01

Myosin II, the motor protein driving muscle contraction, uses energy of ATP hydrolysis to produce movement along actin. The key step of energy transduction is the powerstroke, involving rotation of myosin's lever while myosin is attached to actin. Macroscopic measurements indicated high thermodynamic efficiency for energy conversion. However, single-molecule experiments indicated lower efficiency, provoking a long-standing discrepancy. Based on the Fluctuation-Dissipation Theorem, we built a sufficiently detailed but low degree-of-freedom model reconstructing the entire mechanoenzymatic cycle. We show that a high axial stiffness of the lever during an initial, experimentally yet unrevealed part of the powerstroke results in a short-time, ratchet-like Kramers effect, and is responsible for the missing efficiency. The second part of the powerstroke is an Eyring-like relaxation that dominantly contributes to lever rotation, but produces only a minor part of the work. The model reveals the structural background of myosin's capability to function as a robust molecular engine and a very precise load sensor as well. Our model also suggests an explanation for the malfunction of myosins harboring mutations that lead to hypertrophic cardiomyopathies with most severe clinical prognosis. The model explains how a force-transmitting device within a biological motor can enable high energetic efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects.

PubMed

Walklate, Jonathan; Vera, Carlos; Bloemink, Marieke J; Geeves, Michael A; Leinwand, Leslie

2016-05-06

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The actin-activated ATPase of co-polymer filaments of myosin and myosin-rod.

PubMed Central

Stepkowski, D; Orlova, A A; Moos, C

1994-01-01

The actin activated ATPase of myosin at low ionic strength shows a complex dependence on actin concentration, in contrast with the simple hyperbolic actin activation kinetics of heavy meromyosin and subfragment-1. To investigate how the aggregation of myosin influences the actomyosin ATPase kinetics, we have studied the actin-activated ATPase of mixed filaments in which the myosin molecules are separated from each other by copolymerization with myosin rod. Electron microscopy of copolymer filaments, alone and bound to actin, indicates that the myosin heads are distributed randomly along the co-polymer filaments. The actin-activated ATPase of myosin decreases with increasing rod, approaching a plateau of about 30% of the control at a rod/myosin molar ratio of 4:1. The decrease in ATPase persists even at Vmax, the extrapolated limit at infinite actin, indicating that it is not due merely to the loss of cooperative actin binding. Furthermore, the actin dependence of the ATPase still shows a biphasic character like that of control myosin, even at rod/myosin ratio of 12:1, so this complexity is not probably due solely to the structural proximity of myosin molecules, but may involve a non-equivalence of myosin heads or myosin molecules in the filament environment. Images Figure 1 Figure 2 PMID:8198528

Changes in proteins during the ripening of Spanish dried beef 'Cecina'.

PubMed

García, I; Díez, V; Zumalacárregui, J M

1997-08-01

Changes in the solubility of sarcoplasmic and myofibrillar proteins were tracked in Semitendinosus and Rectus femoris muscles during the ripening process of Spanish 'Cecina'. The extractability of both types of proteins decreased during the ripening. This phenomenon was more marked in the initial stages of processing. Electrophoretic studies of the myofibrillar proteins showed the virtual disappearance of the myosin heavy chain, troponin C and myosin light chain 2 from the smoking phase onward and the appearance of three components of molecular weight of about 65, 70 and 75 kda during ripening. The remaining proteins did not suffer appreciable changes.

Optical Traps to Study Properties of Molecular Motors

PubMed Central

Spudich, James A.; Rice, Sarah E.; Rock, Ronald S.; Purcell, Thomas J.; Warrick, Hans M.

2016-01-01

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility. PMID:22046048

Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle

PubMed Central

Tanner, Bertrand C.W.; McNabb, Mark; Palmer, Bradley M.; Toth, Michael J.; Miller, Mark S.

2014-01-01

Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either i) uniformly, from the Z-line end of thick-filaments, or ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulation also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere. PMID:24486373

Size and speed of the working stroke of cardiac myosin in situ

PubMed Central

Caremani, Marco; Pinzauti, Francesca; Reconditi, Massimo; Piazzesi, Gabriella; Stienen, Ger J. M.; Lombardi, Vincenzo; Linari, Marco

2016-01-01

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T (0.8–0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s−1 at high load to ∼8 nm⋅hs−1 and 6,000 s−1 at low load. Increases in sarcomere length (1.9–2.2 μm) and external [Ca2+]o (1–2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca2+-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis

PubMed Central

Zhou, Qiongqiong; Kee, Yee-Seir; Poirier, Christopher C.; Jelinek, Christine; Osborne, Jonathan; Divi, Srikanth; Surcel, Alexandra; Will, Marie E.; Eggert, Ulrike S.; Müller-Taubenberger, Annette; Iglesias, Pablo A.; Cotter, Robert J.; Robinson, Douglas N.

2010-01-01

Summary Background During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. Results To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain and is needed to promote myosin II bipolar thick filament remodeling. Conclusion 14-3-3 connects microtubules, Rac and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Further, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes cytokinesis shape change and cell mechanics. PMID:20951045

Myosin Motors: The Chemical Restraints Imposed by ATP

NASA Astrophysics Data System (ADS)

Rayment, I.; Allingham, J.

Most molecular motors use ATP. This is not surprising since this is the universal currency of energy for most of life's processes. The question to be addressed in this chapter is how does the fundamental chemistry of ATP hydrolysis in- fluence the observed organization of linear molecular motors seen today with a focus on myosin. This chapter is written to ask in simple terms what can be gained by reconsidering the chemistry of ATP hydrolysis.

Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

NASA Technical Reports Server (NTRS)

Baldwin, Kenneth M.; Haddad, Fadia

2002-01-01

The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

Myosin storage myopathy: slow skeletal myosin (MYH7) mutation in two isolated cases.

PubMed

Laing, N G; Ceuterick-de Groote, C; Dye, D E; Liyanage, K; Duff, R M; Dubois, B; Robberecht, W; Sciot, R; Martin, J-J; Goebel, H H

2005-02-08

Myosin storage myopathy is a congenital myopathy characterized by subsarcolemmal hyaline bodies in type 1 muscle fibers, which are ATPase positive and thus contain myosin. Mutations recently were identified in the type 1 muscle fiber myosin gene (MYH7) in Swedish and Saudi families with myosin storage myopathy. The authors have identified the arginine 1845 tryptophan mutation found in the Swedish families in two isolated Belgian cases, indicating a critical role for myosin residue arginine 1845.

An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

2009-01-01

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins

PubMed Central

Li, Jianchao; He, Yunyun; Weck, Meredith L.; Lu, Qing; Tyska, Matthew J.; Zhang, Mingjie

2017-01-01

Unconventional myosin 7a (Myo7a), myosin 7b (Myo7b), and myosin 15a (Myo15a) all contain MyTH4-FERM domains (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in their cargo binding tails and are essential for the growth and function of microvilli and stereocilia. Numerous mutations have been identified in the MyTH4-FERM tandems of these myosins in patients suffering visual and hearing impairment. Although a number of MF domain binding partners have been identified, the molecular basis of interactions with the C-terminal MF domain (CMF) of these myosins remains poorly understood. Here we report the high-resolution crystal structure of Myo7b CMF in complex with the extended PDZ3 domain of USH1C (a.k.a., Harmonin), revealing a previously uncharacterized interaction mode both for MyTH4-FERM tandems and for PDZ domains. We predicted, based on the structure of the Myo7b CMF/USH1C PDZ3 complex, and verified that Myo7a CMF also binds to USH1C PDZ3 using a similar mode. The structure of the Myo7b CMF/USH1C PDZ complex provides mechanistic explanations for >20 deafness-causing mutations in Myo7a CMF. Taken together, these findings suggest that binding to PDZ domains, such as those from USH1C, PDZD7, and Whirlin, is a common property of CMFs of Myo7a, Myo7b, and Myo15a. PMID:28439001

A technique for studying cardiac myosin dynamics using optical tweezers

NASA Astrophysics Data System (ADS)

Paolino, Michael; Migirditch, Sam; Nesmelov, Yuri; Hester, Brooke; Appalachian State Biophysics; Optical Sciences Facility Team

A primary protein involved in human muscle contraction is myosin, which exists in α- and β- isoforms. Myosin exerts forces on actin filaments when ATP is present, driving muscle contraction. A significant decrease in the population of cardiac α-myosin has been linked to heart failure. It is proposed that slow β-myosin in a failing heart could, through introduction of a drug, be made to mimic the action of α-myosin, thereby improving cardiac muscle performance. In working towards testing this hypothesis, the focus of this work is to develop a technique to measure forces exerted by myosin on actin using optical tweezers. An actin-myosin arrangement is constructed between two optically trapped polystyrene microspheres. The displacement of a microsphere is monitored when ATP is introduced, and the force responsible is measured. With this achieved, we can then modify the actin-myosin arrangement, for example with varying amounts of α- and β- myosin and test the effects on forces exerted. In this work, assemblies of actin and myosin molecules and preliminary force measurements are discussed. North Carolina Space Grant.

Recovering actives in multi-antitarget and target design of analogs of the myosin II inhibitor blebbistatin

NASA Astrophysics Data System (ADS)

Roman, Bart I.; Guedes, Rita C.; Stevens, Christian V.; García-Sosa, Alfonso T.

2018-05-01

In multitarget drug design, it is critical to identify active and inactive compounds against a variety of targets and antitargets. Multitarget strategies thus test the limits of available technology, be that in screening large databases of compounds versus a large number of targets, or in using in silico methods for understanding and reliably predicting these pharmacological outcomes. In this paper, we have evaluated the potential of several in silico approaches to predict the target, antitarget and physicochemical profile of (S)-blebbistatin, the best-known myosin II ATPase inhibitor, and a series of analogs thereof. Standard and augmented structure-based design techniques could not recover the observed activity profiles. A ligand-based method using molecular fingerprints was, however, able to select actives for myosin II inhibition. Using further ligand- and structure-based methods, we also evaluated toxicity through androgen receptor binding, affinity for an array of antitargets and the ADME profile (including assay-interfering compounds) of the series. In conclusion, in the search for (S)-blebbistatin analogs, the dissimilarity distance of molecular fingerprints to known actives and the computed antitarget and physicochemical profile of the molecules can be used for compound design for molecules with potential as tools for modulating myosin II and motility-related diseases.

Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications.

PubMed

Sugi, Haruo; Chaen, Shigeru; Akimoto, Tsuyoshi

2018-05-04

The most straightforward way to get information on the performance of individual myosin heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC). The EC enables us to visualize and record ATP-induced myosin head movement in hydrated skeletal muscle myosin filaments. When actin filaments are absent, myosin heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, myosin heads are found to move away from, but not towards, the bare region, indicating that myosin heads perform a recovery stroke (average amplitude, 6 nm). After exhaustion of ATP, myosin heads return to their neutral position. In the actin⁻myosin filament mixture, myosin heads form rigor actin myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the myosin head catalytic domain (CAD), respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca 2+ -activated force in muscle. In both the power and recovery strokes, myosin heads return to their neutral position after exhaustion of ATP.

Separation of large mammalian ventricular myosin differing in ATPase activity.

PubMed

Rupp, Heinz; Maisch, Bernhard

2007-01-01

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.

Characterization of myosin heavy chain and its gene in Amoeba proteus.

PubMed

Oh, S W; Jeon, K W

1998-01-01

Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNAs encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, as checked by indirect immunofluorescence microscopy and by immunoelectron microscopy. The open reading frame of a cloned myosin cDNA contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While the amino-acid sequence of the globular head region of amoeba's myosin had a high degree of similarity with that of myosins from various organisms, the tail region building a coiled-coil structure did not show a significant sequence similarity. There appeared to be at least three different isoforms of myosins in amoebae, with closely related amino acids in the globular head region.

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo.

PubMed

Morita, Toshiyuki; Tsuchiya, Akiko; Sugimoto, Masazumi

2011-09-01

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

PubMed

Orfanos, Zacharias; Sparrow, John C

2013-01-01

During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

PubMed Central

Rao, Mala V.; Engle, Linda J.; Mohan, Panaiyur S.; Yuan, Aidong; Qiu, Dike; Cataldo, Anne; Hassinger, Linda; Jacobsen, Stephen; Lee, Virginia M-Y.; Andreadis, Athena; Julien, Jean-Pierre; Bridgman, Paul C.; Nixon, Ralph A.

2002-01-01

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons. PMID:12403814

Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration.

PubMed

Croft, Daniel R; Coleman, Mathew L; Li, Shuixing; Robertson, David; Sullivan, Teresa; Stewart, Colin L; Olson, Michael F

2005-01-17

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

The regulation of smooth muscle contractility by zipper-interacting protein kinase.

PubMed

Ihara, Eikichi; MacDonald, Justin A

2007-01-01

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

Cross-reactivity of termite myosin; a potential allergen

USDA-ARS?s Scientific Manuscript database

Myosin and myosin isoforms are common food allergens in crustaceans; such as, shrimp, lobster, and crab. Allergy to Shellfish is a prevalent and potentially long lasting disorder that can severely affect health and quality of life. Myosin and myosin isoforms of dust mites and cockroaches are simil...

The myosin ID pathway and left-right asymmetry in Drosophila.

PubMed

Géminard, Charles; González-Morales, Nicanor; Coutelis, Jean-Baptiste; Noselli, Stéphane

2014-06-01

Drosophila is a classical model to study body patterning, however left-right (L/R) asymmetry had remained unexplored, until recently. The discovery of the conserved myosin ID gene as a major determinant of L/R asymmetry has revealed a novel L/R pathway involving the actin cytoskeleton and the adherens junction. In this process, the HOX gene Abdominal-B plays a major role through the control of myosin ID expression and therefore symmetry breaking. In this review, we present organs and markers showing L/R asymmetry in Drosophila and discuss our current understanding of the underlying molecular genetic mechanisms. Drosophila represents a valuable model system revealing novel strategies to establish L/R asymmetry in invertebrates and providing an evolutionary perspective to the problem of laterality in bilateria. © 2014 Wiley Periodicals, Inc.

Structural Insights into Functional Overlapping and Differentiation among Myosin V Motors*

PubMed Central

Nascimento, Andrey F. Z.; Trindade, Daniel M.; Tonoli, Celisa C. C.; de Giuseppe, Priscila O.; Assis, Leandro H. P.; Honorato, Rodrigo V.; de Oliveira, Paulo S. L.; Mahajan, Pravin; Burgess-Brown, Nicola A.; von Delft, Frank; Larson, Roy E.; Murakami, Mario T.

2013-01-01

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors. PMID:24097982

Continued Expression of Neonatal Myosin Heavy Chain in Adult Dystrophic Skeletal Muscle

NASA Astrophysics Data System (ADS)

Bandman, Everett

1985-02-01

The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.

Structure of the Rigor Actin-Tropomyosin-Myosin Complex

PubMed Central

Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan

2014-01-01

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895

Isolation, properties and P content of the human brain myosin.

PubMed

Fazekas, S; Ováry, I; Horváth, E; Székessy-Hermann, V; Juhász, P

1982-01-01

KCl-, and NaCl-myosins were prepared from different parts of the central nervous system (CNS). Throughout these experiments P and lipid contents were higher in NaCl-myosins than in KCl-preparations. Both KCl-, and NaCl-myosins have increased lipid and P contents compared with skeletal muscle myosins. When the specimens were separated by a molecular sieve, it was found by chromatographic technique on Sepharose 4B column that the cerebral and cerebellar myosins were composed of two fractions of different molecular mass while the brain stem and spinal cord myosins revealed only a single peak. The myosin fractions' Ca-ATPase activity could be augmented by rabbit muscle actin. The myosin preparations developed filamentous systems and aggregates which could be shown by scanning electron microscopy. All the CNS-myosin preparations could be phosphorylated; however, they were saturated to a different degree and were influenced by the presence or absence of serotonin. The kinetic studies revealed that the phosphate saturation of the brain stem, cerebellar and cerebral myosins depended on the ATP concentration and incubation time. The alkaline hydrolysates of lipid-free human brain myosin preparations contained amino acid phosphates, P-Arg, P-Lys and P-His in different amounts depending on their sources. In response to a phosphorylating mixture only the amount of P-Arg was elevated in the cerebral myosins, P-Arg and P-His in the brain stem preparations, and P-Arg, P-His and the amounts of unidentified compounds in the cerebellar ones.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

PubMed

Miserey-Lenkei, Stéphanie; Bousquet, Hugo; Pylypenko, Olena; Bardin, Sabine; Dimitrov, Ariane; Bressanelli, Gaëlle; Bonifay, Raja; Fraisier, Vincent; Guillou, Catherine; Bougeret, Cécile; Houdusse, Anne; Echard, Arnaud; Goud, Bruno

2017-11-01

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

PubMed Central

Fazal, Fabeha; Gu, Lianzhi; Ihnatovych, Ivanna; Han, YooJeong; Hu, WenYang; Antic, Nenad; Carreira, Fernando; Blomquist, James F.; Hope, Thomas J.; Ucker, David S.; de Lanerolle, Primal

2005-01-01

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis. PMID:15988034

Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

PubMed

Weil, D; Levy, G; Sahly, I; Levi-Acobas, F; Blanchard, S; El-Amraoui, A; Crozet, F; Philippe, H; Abitbol, M; Petit, C

1996-04-16

The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

PubMed

Novak, K D; Peterson, M D; Reedy, M C; Titus, M A

1995-12-01

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Myrip couples the capture of secretory granules by the actin-rich cell cortex and their attachment to the plasma membrane.

PubMed

Huet, Sébastien; Fanget, Isabelle; Jouannot, Ouardane; Meireles, Patricia; Zeiske, Tim; Larochette, Nathanaël; Darchen, François; Desnos, Claire

2012-02-15

Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis

PubMed Central

Raghavan, Srivatsan; Shen, Colette J.; Desai, Ravi A.; Sniadecki, Nathan J.; Nelson, Celeste M.; Chen, Christopher S.

2010-01-01

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments. PMID:20682635

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness.

PubMed

Wang, Yang; Melkani, Girish C; Suggs, Jennifer A; Melkani, Anju; Kronert, William A; Cammarato, Anthony; Bernstein, Sanford I

2012-06-01

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.

Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

PubMed

Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The fungal myosin I is essential for Fusarium toxisome formation.

PubMed

Tang, Guangfei; Chen, Yun; Xu, Jin-Rong; Kistler, H Corby; Ma, Zhonghua

2018-01-01

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

The fungal myosin I is essential for Fusarium toxisome formation

PubMed Central

Xu, Jin-Rong

2018-01-01

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi. PMID:29357387

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoenitzer, Veronika; Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg; Eichner, Norbert

Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report themore » heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.« less

Protein Phosphatase 1ß Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

PubMed Central

Yamamoto, Shinya; Bayat, Vafa; Bellen, Hugo J.; Tan, Change

2013-01-01

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well. PMID:23936219

The Myosin IXb Motor Activity Targets the Myosin IXb RhoGAP Domain as Cargo to Sites of Actin Polymerization

PubMed Central

van den Boom, Frank; Düssmann, Heiko; Uhlenbrock, Katharina; Abouhamed, Marouan

2007-01-01

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties. PMID:17314409

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

PubMed Central

Lapierre, Lynne A.; Kumar, Ravindra; Hales, Chadwick M.; Navarre, Jennifer; Bhartur, Sheela G.; Burnette, Jason O.; Provance, D. William; Mercer, John A.; Bähler, Martin; Goldenring, James R.

2001-01-01

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems. PMID:11408590

Evolution and Classification of Myosins, a Paneukaryotic Whole-Genome Approach

PubMed Central

Sebé-Pedrós, Arnau; Grau-Bové, Xavier; Richards, Thomas A.; Ruiz-Trillo, Iñaki

2014-01-01

Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animal multicellularity. Indeed, single-celled holozoans have the most complex myosin complement among eukaryotes, with paralogs of most myosins previously considered animal specific. We recover a dynamic evolutionary history, with several lineage-specific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family. PMID:24443438

Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

PubMed Central

Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

1989-01-01

Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

Bud detachment in hydra requires activation of fibroblast growth factor receptor and a Rho–ROCK–myosin II signaling pathway to ensure formation of a basal constriction

PubMed Central

Holz, Oliver; Apel, David; Steinmetz, Patrick; Lange, Ellen; Hopfenmüller, Simon; Ohler, Kerstin; Sudhop, Stefanie

2017-01-01

Background: Hydra propagates asexually by exporting tissue into a bud, which detaches 4 days later as a fully differentiated young polyp. Prerequisite for detachment is activation of fibroblast growth factor receptor (FGFR) signaling. The mechanism which enables constriction and tissue separation within the monolayered ecto‐ and endodermal epithelia is unknown. Results: Histological sections and staining of F‐actin by phalloidin revealed conspicuous cell shape changes at the bud detachment site indicating a localized generation of mechanical forces and the potential enhancement of secretory functions in ectodermal cells. By gene expression analysis and pharmacological inhibition, we identified a candidate signaling pathway through Rho, ROCK, and myosin II, which controls bud base constriction and rearrangement of the actin cytoskeleton. Specific regional myosin phosphorylation suggests a crucial role of ectodermal cells at the detachment site. Inhibition of FGFR, Rho, ROCK, or myosin II kinase activity is permissive for budding, but represses myosin phosphorylation, rearrangement of F‐actin and constriction. The young polyp remains permanently connected to the parent by a broad tissue bridge. Conclusions: Our data suggest an essential role of FGFR and a Rho‐ROCK‐myosin II pathway in the control of cell shape changes required for bud detachment. Developmental Dynamics 246:502–516, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists PMID:28411398

Bud detachment in hydra requires activation of fibroblast growth factor receptor and a Rho-ROCK-myosin II signaling pathway to ensure formation of a basal constriction.

PubMed

Holz, Oliver; Apel, David; Steinmetz, Patrick; Lange, Ellen; Hopfenmüller, Simon; Ohler, Kerstin; Sudhop, Stefanie; Hassel, Monika

2017-07-01

Hydra propagates asexually by exporting tissue into a bud, which detaches 4 days later as a fully differentiated young polyp. Prerequisite for detachment is activation of fibroblast growth factor receptor (FGFR) signaling. The mechanism which enables constriction and tissue separation within the monolayered ecto- and endodermal epithelia is unknown. Histological sections and staining of F-actin by phalloidin revealed conspicuous cell shape changes at the bud detachment site indicating a localized generation of mechanical forces and the potential enhancement of secretory functions in ectodermal cells. By gene expression analysis and pharmacological inhibition, we identified a candidate signaling pathway through Rho, ROCK, and myosin II, which controls bud base constriction and rearrangement of the actin cytoskeleton. Specific regional myosin phosphorylation suggests a crucial role of ectodermal cells at the detachment site. Inhibition of FGFR, Rho, ROCK, or myosin II kinase activity is permissive for budding, but represses myosin phosphorylation, rearrangement of F-actin and constriction. The young polyp remains permanently connected to the parent by a broad tissue bridge. Our data suggest an essential role of FGFR and a Rho-ROCK-myosin II pathway in the control of cell shape changes required for bud detachment. Developmental Dynamics 246:502-516, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals.

PubMed

Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

2016-06-22

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin

PubMed Central

Newman-Smith, Erin; Kourakis, Matthew J; Reeves, Wendy; Veeman, Michael; Smith, William C

2015-01-01

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. DOI: http://dx.doi.org/10.7554/eLife.05361.001 PMID:25866928

Myosin motor function: the ins and outs of actin-based membrane protrusions

PubMed Central

Nambiar, Rajalakshmi; McConnell, Russell E.

2011-01-01

Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion. PMID:20107861

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

PubMed

Andruchov, Oleg; Galler, Stefan

2008-03-01

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila

PubMed Central

Achal, Madhulika; Trujillo, Adriana S.; Melkani, Girish C.; Farman, Gerrie P.; Ocorr, Karen; Viswanathan, Meera C.; Kaushik, Gaurav; Newhard, Christopher S.; Glasheen, Bernadette M.; Melkani, Anju; Suggs, Jennifer A.; Moore, Jeffrey R.; Swank, Douglas M.; Bodmer, Rolf; Cammarato, Anthony; Bernstein, Sanford I.

2016-01-01

An “invariant proline” separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138–43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc5, an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue. PMID:27107639

The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

PubMed

Zhang, Wen-Bo; Yao, Lin-Lin; Li, Xiang-Dong

2016-06-24

Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

PubMed

Viswanathan, Meera C; Tham, Rick C; Kronert, William A; Sarsoza, Floyd; Trujillo, Adriana S; Cammarato, Anthony; Bernstein, Sanford I

2017-12-15

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the β-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MyTH4-FERM myosins have an ancient and conserved role in filopod formation

PubMed Central

Goodson, Holly V.; Arthur, Ashley L.; Luxton, G. W. Gant; Houdusse, Anne; Titus, Margaret A.

2016-01-01

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation. PMID:27911821

Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.

PubMed

Seebohm, B; Matinmehr, F; Köhler, J; Francino, A; Navarro-Lopéz, F; Perrot, A; Ozcelik, C; McKenna, W J; Brenner, B; Kraft, T

2009-08-05

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the beta-myosin heavy chain, 723Arg --> Gly (R723G) and 736Ile --> Thr (I736T), in comparison to 719Arg --> Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S( *)(rigor R723G) = 0.84 pN/nm S( *)(rigor WT) = 0.29 pN/nm); and ii), a significant increase in force per head (F( *)(10 degrees C), 1.99 pN vs. 1.49 pN = 1.3-fold increase; F( *)(20 degrees C), 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S( *)(active10 degrees C), 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S( *)(rigor); 1.8-fold increase in F( *)(10 degrees C), 1.6-fold in F( *)(20 degrees C); twofold increase in S( *)(active10 degrees C)). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous findings that for the slow myosin isoform S( *) and F( *) are significantly lower than for fast myosin e.g., of rabbit psoas muscle. The data indicate that two mutations, R723G and R719W, are associated with an increase in resistance to elastic distortion of the individual mutated myosin heads whereas mutation I736T has essentially no effect. The data strongly support the notion that major elastic distortion occurs within the converter itself. Apparently, the compliance depends on specific residues, e.g., R719 and R723, presumably located at strategic positions near the long alpha-helix of the light chain binding domain. Because amino acids 719 and 723 are nonconserved residues, cross-bridge stiffness may well be specifically tuned for different myosins.

Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke.

PubMed

Rohde, John A; Thomas, David D; Muretta, Joseph M

2017-03-07

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

PubMed Central

Sun, Xiaojing; Ekker, Stephen C.; Shelden, Eric A.; Takubo, Naoko; Wang, Yihua; Burghardt, Thomas P.

2014-01-01

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo. PMID:25229148

Myosin II Dynamics during Embryo Morphogenesis

NASA Astrophysics Data System (ADS)

Kasza, Karen

2013-03-01

During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals

PubMed Central

Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

2016-01-01

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs. DOI: http://dx.doi.org/10.7554/eLife.15258.001 PMID:27331610

The closed MTIP-MyosinA-tail complex from the malaria parasite invasion machinery

PubMed Central

Bosch, Jürgen; Turley, Stewart; Roach, Claudia M.; Daly, Thomas M.; Bergman, Lawrence W.; Hol, Wim G. J.

2009-01-01

The Myosin A-tail Interacting Protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites. PMID:17628590

Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion

PubMed Central

Michalek, Arthur J.; Kennedy, Guy G.; Warshaw, David M.; Ali, M. Yusuf

2015-01-01

Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network. PMID:26770194

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

PubMed Central

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

2010-01-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study.

PubMed

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir; Mogilner, Alex

2010-12-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.

Myosin: A Link between Streptococci and Heart

NASA Astrophysics Data System (ADS)

Krisher, Karen; Cunningham, Madeleine W.

1985-01-01

Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure.

PubMed

Manickam, Manoj; Jalani, Hitesh B; Pillaiyar, Thanigaimalai; Sharma, Niti; Boggu, Pulla Reddy; Venkateswararao, Eeda; Lee, You-Jung; Jeon, Eun-Seok; Jung, Sang-Hun

2017-07-07

A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Myosin Vb mediates Cu+ export in polarized hepatocytes

PubMed Central

Gupta, Arnab; Schell, Michael J.; Bhattacharjee, Ashima; Lutsenko, Svetlana; Hubbard, Ann L.

2016-01-01

ABSTRACT The cellular machinery responsible for Cu+-stimulated delivery of the Wilson-disease-associated protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the Cu+-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B-myosin Vb interaction reduces the apical surface expression of ATP7B. Overexpression of the myosin Vb tail, which competes for binding of subapical cargos to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular Cu+ export. The myosin-Vb-dependent targeting step occurred in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on γ-tubulin. ATP7B became selectively arrested in this compartment at high [Cu+] in the presence of myosin Vb tail, suggesting that these compartments are precursors of donor–acceptor transfer stations for apically targeted cargos of myosin Vb. Our data suggest that reduced hepatic Cu+ clearance in idiopathic non-Wilsonian types of disease might be associated with the loss of function of myosin Vb. PMID:26823605

ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

PubMed Central

Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

2014-01-01

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

aPKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation

PubMed Central

Petrov, Daria; Dahan, Inbal; Cohen-Kfir, Einav; Ravid, Shoshana

2017-01-01

ABSTRACT Cell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCζ for cell motility, there is little understanding of the mechanism by which aPKCζ mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCζ is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCζ mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCζ mediates EGF-dependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCζ is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCζ mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCζ is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton. PMID:27541056

ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

PubMed

Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

2014-04-01

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

3D Model of Cytokinetic Contractile Ring Assembly: Node-Mediated and Backup Pathways

NASA Astrophysics Data System (ADS)

Bidone, Tamara; Vavylonis, Dimitrios

Cytokinetic ring assembly in model organism fission yeast is a dynamic process, involving condensation of a network of actin filaments and myosin motors bound to the cell membrane through cortical nodes. A 3D computational model of ring assembly illustrates how the combined activities of myosin motors, filament crosslinkers and actin turnover lead to robust ring formation [Bidone et al. Biophys. J, 2014]. We modeled the importance of the physical properties of node movement along the cell membrane and of myosin recruitment to nodes. Experiments by D. Zhang (Temasek Life Sciences) show that tethering of the cortical endoplasmic reticulum (ER) to the plasma membrane modulates the speed of node condensation and the degree of node clumping. We captured the trend observed in these experiments by changes in the node drag coefficient and initial node distribution in simulations PM. The model predicted that reducing crosslinking activities in ER tethering mutants with faster node speed enhances actomyosin clumping. We developed a model of how tilted and/or misplaced rings assemble in cells that lack the node structural component anillin-like Mid1 and thus fail to recruit myosin II to nodes independently of actin. If actin-dependent binding of diffusive myosin to the cortex is incorporated into the model, it generates progressively elongating cortical actomyosin strands with fluctuating actin bundles at the tails. These stands often close into a ring, similar to observations by the group of J.Q. Wu (The Ohio State University). NIH R01GM098430.

Fiber transformation and replacement in low-frequency stimulated rabbit fast-twitch muscles.

PubMed

Schuler, M; Pette, D

1996-08-01

The fast-to-slow conversion of rabbit skeletal muscles by chronic low-frequency (10 Hz, 12 h daily) stimulation involves (1) sequential fast-to-slow fiber-type transitions in the order of type IID-->type IIA-->type I, and (2) the replacement of deteriorating fast-twitch glycolytic fibers by new fibers derived from satellite cells and myotubes. These two processes were analyzed in 30- and 60-day stimulated extensor digitorum longus and tibialis anterior muscles. Fast-to-slow transforming fibers were identified by myofibrillar actomyosin histochemistry as type C fibers and immunohistochemically by their reaction with monoclonal antibodies specific to slow and fast myosin heavy chain isoforms. In situ hybridization of mRNA specific to the myosin heavy chain I isoform identified all fibers expressing slow myosin, i.e., type I and C fibers. The fraction of transforming fibers ranged between 35% and 50% in 30-day stimulated muscles. The percentage of type I fibers (20%) was threefold elevated in extensor digitorum longus muscle, but unaltered (3.5%) in tibialis anterior muscle, suggesting that fast-to-slow fiber conversion was more advanced in the former than in the latter. Fiber replacement was indicated by the finding that the fiber populations of both muscles contained 15% myotubes or small fibers with central nuclei. In situ hybridization revealed that myotubes and small regenerating fibers uniformly expressed myosin heavy chain I mRNA. Similarly, high percentages of slow-myosin-expressing myotubes and small fibers were found in 60-day stimulated muscles.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

2013-11-29

Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143more » flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.« less

Theoretical kinetic studies of models for binding myosin subfragment-1 to regulated actin: Hill model versus Geeves model.

PubMed Central

Chen , Y; Yan, B; Chalovich, J M; Brenner, B

2001-01-01

It was previously shown that a one-dimensional Ising model could successfully simulate the equilibrium binding of myosin S1 to regulated actin filaments (T. L. Hill, E. Eisenberg and L. Greene, Proc. Natl. Acad. Sci. U.S.A. 77:3186-3190, 1980). However, the time course of myosin S1 binding to regulated actin was thought to be incompatible with this model, and a three-state model was subsequently developed (D. F. McKillop and M. A. Geeves, Biophys. J. 65:693-701, 1993). A quantitative analysis of the predicted time course of myosin S1 binding to regulated actin, however, was never done for either model. Here we present the procedure for the theoretical evaluation of the time course of myosin S1 binding for both models and then show that 1) the Hill model can predict the "lag" in the binding of myosin S1 to regulated actin that is observed in the absence of Ca++ when S1 is in excess of actin, and 2) both models generate very similar families of binding curves when [S1]/[actin] is varied. This result shows that, just based on the equilibrium and pre-steady-state kinetic binding data alone, it is not possible to differentiate between the two models. Thus, the model of Hill et al. cannot be ruled out on the basis of existing pre-steady-state and equilibrium binding data. Physical mechanisms underlying the generation of the lag in the Hill model are discussed. PMID:11325734

Autoimmune Myocarditis, Valvulitis, and Cardiomyopathy

PubMed Central

Myers, Jennifer M.; Cunningham, Madeleine W.; Fairweather, DeLisa; Huber, Sally A.

2013-01-01

Cardiac myosin-induced autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells (Smith and Allen 1991; Li, Heuser et al. 2004). It is a paradigm of the immune-mediated cardiac damage believed to play a role in the pathogenesis of a subset of postinfectious human cardiomyopathies (Rose, Herskowitz et al. 1993). Myocarditis is induced in susceptible mice by immunization with purified cardiac myosin (Neu, Rose et al. 1987) or specific peptides derived from cardiac myosin (Donermeyer, Beisel et al. 1995; Pummerer, Luze et al. 1996) (see Basic Protocol 1), or by adoptive transfer of myosin-reactive T cells (Smith and Allen 1991) (see Alternate Protocol). Myocarditis has been induced in Lewis rats by immunization with purified rat or porcine cardiac myosin (Kodama, Matsumoto et al. 1990; Li, Heuser et al. 2004) (see Basic Protocol 2) or S2-16 peptide (Li, Heuser et al. 2004), or by adoptive transfer of T cells stimulated by specific peptides derived from cardiac myosin (Wegmann, Zhao et al. 1994). Myocarditis begins 12 to 14 days after the first immunization, and is maximal after 21 days. Other animal models commonly used to study myocarditis development include the pathogen-induced models in which disease is initiated by viral infection. The first murine model of acute viral myocarditis causes sudden death via viral damage to cardiomyocytes (Huber, Gauntt et al. 1998; Horwitz, La Cava et al. 2000; Fong 2003; Fuse, Chan et al. 2005; Fairweather and Rose 2007; Cihakova and Rose 2008) whereas the second model is based on inoculation with heart-passaged coxsackievirus B3 (CVB3) that includes damaged heart proteins (Fairweather, Frisancho-Kiss et al. 2004; Fairweather D 2004; Fairweather and Rose 2007; Cihakova and Rose 2008) In addition to the protocols used to induce EAM in mice and rats, support protocols are included for preparing purified cardiac myosin using mouse or rat heart tissue (see Support Protocol 1), preparing purified cardiac myosin for injection (see Support Protocol 2), and collecting and assessing hearts by histopathological means (see Support Protocol 3). PMID:23564686

In Vitro and In Vivo Single Myosin Step-Sizes in Striated Muscle a

PubMed Central

Burghardt, Thomas P.; Sun, Xiaojing; Wang, Yihua; Ajtai, Katalin

2016-01-01

Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a “second characterization” is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover “bottom-up” and “top-down” assaying of myosin characteristics. PMID:26728749

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads

PubMed Central

Iwaki, M.; Wickham, S. F.; Ikezaki, K.; Yanagida, T.; Shih, W. M.

2016-01-01

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes. PMID:27941751

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

PubMed

Iwaki, M; Wickham, S F; Ikezaki, K; Yanagida, T; Shih, W M

2016-12-12

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells

PubMed Central

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations. PMID:28125727

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells.

PubMed

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc; Tajsharghi, Homa

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations.

Non-muscle myosin IIB is critical for nuclear translocation during 3D invasion

PubMed Central

Yenepalli, Aishwarya; Denais, Celine Marie; Rape, Andrew; Beach, Jordan R.; Wang, Yu-li; Schiemann, William P.; Baskaran, Harihara; Lammerding, Jan

2015-01-01

Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing. PMID:26261182

Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

PubMed Central

Vasquez, Claudia G; Heissler, Sarah M; Billington, Neil; Sellers, James R; Martin, Adam C

2016-01-01

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos. DOI: http://dx.doi.org/10.7554/eLife.20828.001 PMID:28035903

Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance.

PubMed

Wang, Fei; Kovacs, Mihaly; Hu, Aihua; Limouze, John; Harvey, Estelle V; Sellers, James R

2003-07-25

Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.

Second harmonic generation polarization properties of myofilaments

NASA Astrophysics Data System (ADS)

Samim, Masood; Prent, Nicole; Dicenzo, Daniel; Stewart, Bryan; Barzda, Virginijus

2014-05-01

Second harmonic generation (SHG) polarization microscopy was used to investigate the organization of myosin nanomotors in myofilaments of muscle cells. The distribution of the second-order nonlinear susceptibility component ratio χzzz(2)/χzxx(2) along anisotropic bands of sarcomeres revealed differences between the headless and head-containing regions of myofilaments. The polarization-in polarization-out SHG measurements of headless myosin mutants of indirect flight muscle in Drosophila melanogaster confirmed a lower susceptibility component ratio compared to the head-containing myocytes with wild-type myosins. The increase in the ratio is assigned to the change in the deflection angle of the myosin S2 domain and possible contribution of myosin heads. The nonlinear susceptibility component ratio is a sensitive indicator of the myosin structure, and therefore, it can be used for conformational studies of myosin nanomotors. The measured ratio values can also be used as the reference for ab initio calculations of nonlinear optical properties of different parts of myosins.

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.

2011-12-16

Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chainmore » kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.« less

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

PubMed

Spudich, James A; Aksel, Tural; Bartholomew, Sadie R; Nag, Suman; Kawana, Masataka; Yu, Elizabeth Choe; Sarkar, Saswata S; Sung, Jongmin; Sommese, Ruth F; Sutton, Shirley; Cho, Carol; Adhikari, Arjun S; Taylor, Rebecca; Liu, Chao; Trivedi, Darshan; Ruppel, Kathleen M

2016-01-01

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. © 2016. Published by The Company of Biologists Ltd.

Myosin VI facilitates connexin 43 gap junction accretion.

PubMed

Waxse, Bennett J; Sengupta, Prabuddha; Hesketh, Geoffrey G; Lippincott-Schwartz, Jennifer; Buss, Folma

2017-03-01

In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI. © 2017. Published by The Company of Biologists Ltd.

Stiffness, working stroke, and force of single-myosin molecules in skeletal muscle: elucidation of these mechanical properties via nonlinear elasticity evaluation.

PubMed

Kaya, Motoshi; Higuchi, Hideo

2013-11-01

In muscles, the arrays of skeletal myosin molecules interact with actin filaments and continuously generate force at various contraction speeds. Therefore, it is crucial for myosin molecules to generate force collectively and minimize the interference between individual myosin molecules. Knowledge of the elasticity of myosin molecules is crucial for understanding the molecular mechanisms of muscle contractions because elasticity directly affects the working and drag (resistance) force generation when myosin molecules are positively or negatively strained. The working stroke distance is also an important mechanical property necessary for elucidation of the thermodynamic efficiency of muscle contractions at the molecular level. In this review, we focus on these mechanical properties obtained from single-fiber and single-molecule studies and discuss recent findings associated with these mechanical properties. We also discuss the potential molecular mechanisms associated with reduction of the drag effect caused by negatively strained myosin molecules.

A novel actin binding site of myosin required for effective muscle contraction.

PubMed

Várkuti, Boglárka H; Yang, Zhenhui; Kintses, Bálint; Erdélyi, Péter; Bárdos-Nagy, Irén; Kovács, Attila L; Hári, Péter; Kellermayer, Miklós; Vellai, Tibor; Málnási-Csizmadia, András

2012-02-12

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Involvement of myosin light-chain kinase in endothelial cell retraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wysolmerski, R.B.; Lagunoff, D.

Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylationmore » of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.« less

In vivo myosin step-size from zebrafish skeletal muscle

PubMed Central

Ajtai, Katalin; Sun, Xiaojing; Takubo, Naoko; Wang, Yihua

2016-01-01

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle. PMID:27249818

Single cardiac ventricular myosins are autonomous motors

PubMed Central

Wang, Yihua; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta

2018-01-01

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule. PMID:29669825

Nonmuscle myosin is regulated during smooth muscle contraction.

PubMed

Yuen, Samantha L; Ogut, Ozgur; Brozovich, Frank V

2009-07-01

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.

2009-07-01

The subfragment 2/light meromyosin 'hinge' region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifyingmore » this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.« less

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

NASA Astrophysics Data System (ADS)

Reinach, Fernando C.; Nagai, Kiyoshi; Kendrick-Jones, John

1986-07-01

The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation. The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains1, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them. Here, we describe the use of site-directed mutagenesis to study the function of that site. We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site. When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity. Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it. Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.

Non-weight bearing-induced muscle weakness: the role of myosin quantity and quality in MHC type II fibers.

PubMed

Kim, Jong-Hee; Thompson, LaDora V

2014-07-15

We tested the hypothesis that non-weight bearing-induced muscle weakness (i.e., specific force) results from decreases in myosin protein quantity (i.e., myosin content per half-sarcomere and the ratio of myosin to actin) and quality (i.e., force per half-sarcomere and population of myosin heads in the strong-binding state during muscle contraction) in single myosin heavy chain (MHC) type II fibers. Fisher-344 rats were assigned to weight-bearing control (Con) or non-weight bearing (NWB). The NWB rats were hindlimb unloaded for 2 wk. Diameter, force, and MHC content were determined in permeabilized single fibers from the semimembranosus muscle. MHC isoform and the ratio of MHC to actin in each fiber were determined by gel electrophoresis and silver staining techniques. The structural distribution of myosin from spin-labeled fiber bundles during maximal isometric contraction was evaluated using electron paramagnetic resonance spectroscopy. Specific force (peak force per cross-sectional area) in MHC type IIB and IIXB fibers from NWB was significantly reduced by 38% and 18%, respectively. MHC content per half-sarcomere was significantly reduced by 21%. Two weeks of hindlimb unloading resulted in a reduced force per half-sarcomere of 52% and fraction of myosin strong-binding during contraction of 34%. The results suggest that reduced myosin and actin content (quantity) and myosin quality concomitantly contribute to non-weight bearing-related muscle weakness. Copyright © 2014 the American Physiological Society.

Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation

NASA Astrophysics Data System (ADS)

Nucciotti, V.; Stringari, C.; Sacconi, L.; Vanzi, F.; Linari, M.; Piazzesi, G.; Lombardi, V.; Pavone, F. S.

2009-02-01

Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

14-3-3 proteins tune non-muscle myosin II assembly.

PubMed

West-Foyle, Hoku; Kothari, Priyanka; Osborne, Jonathan; Robinson, Douglas N

2018-05-04

The 14-3-3 family comprises a group of small proteins that are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3 proteins σ, ϵ, ζ, and η correlates with high metastatic potential in multiple cancer types. In Dictyostelium , 14-3-3 promotes myosin II turnover in the cell cortex and modulates cortical tension, cell shape, and cytokinesis. In light of the important roles of 14-3-3 proteins across a broad range of eukaryotic species, we sought to determine how 14-3-3 proteins interact with myosin II. Here, conducting in vitro and in vivo studies of both Dictyostelium (one 14-3-3 and one myosin II) and human proteins (seven 14-3-3s and three nonmuscle myosin IIs), we investigated the mechanism by which 14-3-3 proteins regulate myosin II assembly. Using in vitro assembly assays with purified myosin II tail fragments and 14-3-3, we demonstrate that this interaction is direct and phosphorylation-independent. All seven human 14-3-3 proteins also altered assembly of at least one paralog of myosin II. Our findings indicate a mechanism of myosin II assembly regulation that is mechanistically conserved across a billion years of evolution from amebas to humans. We predict that altered 14-3-3 expression in humans inhibits the tumor suppressor myosin II, contributing to the changes in cell mechanics observed in many metastatic cancers. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mediation of acetylcholine and substance P induced contractions by myosin light chain phosphorylation in feline colonic smooth muscle.

PubMed

Washabau, Robert J; Holt, David E; Brockman, Daniel J

2002-05-01

To determine the role of myosin light chain phosphorylation in feline colonic smooth muscle contraction. Colonic tissue was obtained from eight 12- to 24-month-old cats. Colonic longitudinal smooth muscle strips were attached to isometric force transducers for measurements of isometric stress. Myosin light chain phosphorylation was determined by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stress and phosphorylation were determined following stimulation with ACh or SP, in the absence or presence of a calmodulin antagonist (W-7; 0.1 to 1.0 mM), myosin light chain kinase inhibitor (ML-9; 1 to 10 microM), or extracellular calcium free solutions. Unstimulated longitudinal colonic smooth muscle contained low amounts (6.9+/-3.2%) of phosphorylated myosin light chain. Phosphorylation of the myosin light chains was dose and time dependent with maximal values of 58.5% at 30 seconds of stimulation with 100 microM Ach and 60.2% at 45 seconds of stimulation with 100 nM SP Active isometric stress development closely paralleled phosphorylation of the myosin light chains in ACh- or SP-stimulated muscle. W-7 and ML-9 dose dependently inhibited myosin light chain phosphorylation and isometric stress development associated with ACh or SP stimulation. Removal of extracellular calcium inhibited myosin light chain phosphorylation and isometric stress development in ACh-stimulated smooth muscle. Feline longitudinal colonic smooth muscle contraction is calcium-, calmodulin-, and myosin light chain kinase-dependent. Myosin light chain phosphorylation is necessary for the initiation of contraction in feline longitudinal colonic smooth muscle. These findings may prove useful in determining the biochemical and molecular defects that accompany feline colonic motility disorders.

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

PubMed Central

Tang, Wanjian; Blair, Cheavar A.; Walton, Shane D.; Málnási-Csizmadia, András; Campbell, Kenneth S.; Yengo, Christopher M.

2017-01-01

Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation. PMID:28119616

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

PubMed Central

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minoda, Hiroki; CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012; Okabe, Tatsuhiro

2011-02-25

Research highlights: {yields} We succeeded in recording structural changes of hydrated myosin cross-bridges. {yields} We succeeded in position-marking the cross-bridges with site-directed antibodies. {yields} We recorded cross-bridge movement at different regions in individual cross-bridge. {yields} The movement was smallest at the cross-bridge-subfragment two boundary. {yields} The results provide evidence for the cross-bridge lever arm mechanism. -- Abstract: Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release ofmore » hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.« less

EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

PubMed

Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

2012-06-01

Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships. © 2012 WILEY PERIODICALS, INC.

Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

PubMed

Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

2018-05-10

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We find that ROCK regulates airway smooth muscle contraction by mediating activation of the serine-threonine kinase, Pak, to promote actin polymerization. Pak catalyzes paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signaling module to paxillin, which activates the GEF activity βPIX towards cdc42. Cdc42 is required for the activation of Neuronal Wiskott-Aldrich Syndrome protein (N-WASp), which transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Mechanical stress and network structure drive protein dynamics during cytokinesis.

PubMed

Srivastava, Vasudha; Robinson, Douglas N

2015-03-02

Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1-4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5-7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium, this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Abnormalities in early markers of muscle involvement support a delay in myogenesis in spinal muscular atrophy.

PubMed

Martínez-Hernández, Rebeca; Bernal, Sara; Alias, Laura; Tizzano, Eduardo F

2014-06-01

Spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the spinal cord that results in muscle denervation and profound weakness in affected patients. We sought evidence for primary muscle involvement in the disease during human development by analyzing the expression of several muscle cytoskeletal components (i.e. slow, fast, and developmental myosin, desmin, and vimentin) in fetal or postnatal skeletal muscle samples from 5 SMA cases and 6 controls. At 14 weeks' gestation, SMA samples had higher percentages of myotubes expressing fast myosin and lower percentages of myotubes expressing slow myosin versus control samples. Desmin and vimentin were highly expressed at prenatal stages without notable differences between control and SMA samples, although both proteins showed persistent immunostaining in atrophic fibers in postnatal SMA samples. We also studied the expression of Pax7-positive nuclei as a marker of satellite cells and found no differences between control and SMA prenatal samples. There was, however, a significant increase in satellite cells in postnatal atrophic SMA fibers, suggesting an abnormal myogenic process. Together, these results support the hypothesis of a delay in muscle maturation as one of the primary pathologic components of SMA. Furthermore, myosins and Pax7 may be useful research markers of muscle involvement in this disease.

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers.

PubMed

Prochniewicz, Ewa; Lowe, Dawn A; Spakowicz, Daniel J; Higgins, LeeAnn; O'Conor, Kate; Thompson, LaDora V; Ferrington, Deborah A; Thomas, David D

2008-02-01

To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.

Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

PubMed Central

Homburger, Julian R.; Green, Eric M.; Caleshu, Colleen; Sunitha, Margaret S.; Taylor, Rebecca E.; Ruppel, Kathleen M.; Metpally, Raghu Prasad Rao; Colan, Steven D.; Michels, Michelle; Day, Sharlene M.; Olivotto, Iacopo; Bustamante, Carlos D.; Dewey, Frederick E.; Ho, Carolyn Y.; Spudich, James A.; Ashley, Euan A.

2016-01-01

Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

Differential Expression of Unconventional Myosins in Apoptotic and Regenerating Chick Hair Cells Confirms Two Regeneration Mechanisms

PubMed Central

DUNCAN, LUKE J.; MANGIARDI, DOMINIC A.; MATSUI, JONATHAN I.; ANDERSON, JULIA K.; McLAUGHLIN-WILLIAMSON, KATE; COTANCHE, DOUGLAS A.

2008-01-01

Hair cells of the inner ear are damaged by intense noise, aging, and aminoglycoside antibiotics. Gentamicin causes oxidative damage to hair cells, inducing apoptosis. In mammals, hair cell loss results in a permanent deficit in hearing and balance. In contrast, avians can regenerate lost hair cells to restore auditory and vestibular function. This study examined the changes of myosin VI and myosin VIIa, two unconventional myosins that are critical for normal hair cell formation and function, during hair cell death and regeneration. During the late stages of apoptosis, damaged hair cells are ejected from the sensory epithelium. There was a 4–5-fold increase in the labeling intensity of both myosins and a redistribution of myosin VI into the stereocilia bundle, concurrent with ejection. Two separate mechanisms were observed during hair cell regeneration. Proliferating supporting cells began DNA synthesis 60 hours after gentamicin treatment and peaked at 72 hours postgentamicin treatment. Some of these mitotically produced cells began to differentiate into hair cells at 108 hours after gentamicin (36 hours after bromodeoxyuridine (BrdU) administration), as demonstrated by the colabeling of myosin VI and BrdU. Myosin VIIa was not expressed in the new hair cells until 120 hours after gentamicin. Moreover, a population of supporting cells expressed myosin VI at 78 hours after gentamicin treatment and myosin VIIa at 90 hours. These cells did not label for BrdU and differentiated far too early to be of mitotic origin, suggesting they arose by direct transdifferentiation of supporting cells into hair cells. PMID:17048225

Calmodulin Bound to the First IQ Motif Is Responsible for Calcium-dependent Regulation of Myosin 5a*

PubMed Central

Lu, Zekuan; Shen, Mei; Cao, Yang; Zhang, Hai-Man; Yao, Lin-Lin; Li, Xiang-dong

2012-01-01

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca2+-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca2+ and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca2+ regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca2+-dependent regulation and how the head-tail interaction is affected by Ca2+. Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca2+ regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca2+ regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca2+ induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function. PMID:22437832

The structural effects of mutations can aid in differential phenotype prediction of beta-myosin heavy chain (Myosin-7) missense variants.

PubMed

Al-Numair, Nouf S; Lopes, Luis; Syrris, Petros; Monserrat, Lorenzo; Elliott, Perry; Martin, Andrew C R

2016-10-01

High-throughput sequencing platforms are increasingly used to screen patients with genetic disease for pathogenic mutations, but prediction of the effects of mutations remains challenging. Previously we developed SAAPdap (Single Amino Acid Polymorphism Data Analysis Pipeline) and SAAPpred (Single Amino Acid Polymorphism Predictor) that use a combination of rule-based structural measures to predict whether a missense genetic variant is pathogenic. Here we investigate whether the same methodology can be used to develop a differential phenotype predictor, which, once a mutation has been predicted as pathogenic, is able to distinguish between phenotypes-in this case the two major clinical phenotypes (hypertrophic cardiomyopathy, HCM and dilated cardiomyopathy, DCM) associated with mutations in the beta-myosin heavy chain (MYH7) gene product (Myosin-7). A random forest predictor trained on rule-based structural analyses together with structural clustering data gave a Matthews' correlation coefficient (MCC) of 0.53 (accuracy, 75%). A post hoc removal of machine learning models that performed particularly badly, increased the performance (MCC = 0.61, Acc = 79%). This proof of concept suggests that methods used for pathogenicity prediction can be extended for use in differential phenotype prediction. Analyses were implemented in Perl and C and used the Java-based Weka machine learning environment. Please contact the authors for availability. andrew@bioinf.org.uk or andrew.martin@ucl.ac.uk Supplementary data are available at Bioinformatics online. © The Authors 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular mechanisms underlying deoxy‐ADP.Pi activation of pre‐powerstroke myosin

PubMed Central

Nowakowski, Sarah G.

2017-01-01

Abstract Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2‐deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross‐bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross‐bridge formation and reveal a potential mechanism that may underlie dATP‐induced improvements in cardiac function. PMID:28097776

Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

PubMed

Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

2010-08-06

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

Mathematical modeling of the relation between myosin phosphorylation and stress development in smooth muscles.

PubMed

Fajmut, Ales; Dobovisek, Andrej; Brumen, Milan

2005-01-01

In this paper the 4-state latch bridge model proposed by Rembold and Murphy is expanded; first by incorporation of the analytical expression of Ca2+ dependent MLCK activation from the work of Kato et al. and second, by inclusion of the myosin dephosphorylation based on the Michaelis-Menten kinetics. The analysis of the proposed model and the comparison with the original model results as well as with the experimental data is presented. The model is able to predict the steady-state isometric stress and the myosin phosphorylation in dependence on steady cytosolic [Ca2+] as well as the temporal evolution of the system in dependence on the input Ca2+ signal in the form of biphasic transient, whereby our model results are in several aspects in better agreement with experimental observations.

Mesenchymal chemotaxis requires selective inactivation of Myosin II at the leading edge via a non-canonical PLCγ/PKCα pathway

PubMed Central

Asokan, Sreeja B.; Johnson, Heath E.; Rahman, Anisur; King, Samantha J.; Rotty, Jeremy D.; Lebedeva, Irina P.; Haugh, Jason M.; Bear, James E.

2014-01-01

Summary Chemotaxis, migration towards soluble chemical cues, is critical for processes such as wound healing and immune surveillance, and is exhibited by various cell types from rapidly-migrating leukocytes to slow-moving mesenchymal cells. To interrogate the mechanisms involved in mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of the chemoattractant PDGF. Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mTOR signaling, are dispensable for chemotaxis to PDGF. Instead, we find that local inactivation of Myosin IIA, through a non-canonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of TIRF imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge is required for mesenchymal chemotaxis. PMID:25482883

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

PubMed Central

Kekenes-Huskey, Peter M.; Liao, Tao; Gillette, Andrew K.; Hake, Johan E.; Zhang, Yongjie; Michailova, Anushka P.; McCulloch, Andrew D.; McCammon, J. Andrew

2013-01-01

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions. PMID:24209858

Thick Filament Protein Network, Functions, and Disease Association.

PubMed

Wang, Li; Geist, Janelle; Grogan, Alyssa; Hu, Li-Yen R; Kontrogianni-Konstantopoulos, Aikaterini

2018-03-13

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

Watching single molecules dance

NASA Astrophysics Data System (ADS)

Mehta, Amit Dinesh

Molecular motors convert chemical energy, from ATP hydrolysis or ion flow, into mechanical motion. A variety of increasingly precise mechanical probes have been developed to monitor and perturb these motors at the single molecule level. Several outstanding questions can be best approached at the single molecule level. These include: how far does a motor progress per energy quanta consumed? how does its reaction cycle respond to load? how many productive catalytic cycles can it undergo per diffusional encounter with its track? and what is the mechanical stiffness of a single molecule connection? A dual beam optical trap, in conjunction with in vitro ensemble motility assays, has been used to characterize two members of the myosin superfamily: muscle myosin II and chick brain myosin V. Both move the helical polymer actin, but myosin II acts in large ensembles to drive muscle contraction or cytokinesis, while myosin V acts in small numbers to transport vesicles. An optical trapping apparatus was rendered sufficiently precise to identify a myosin working stroke with 1nm or so, barring systematic errors such as those perhaps due to random protein orientations. This and other light microscopic motility assays were used to characterize myosin V: unlike myosin II this vesicle transport protein moves through many increments of travel while remaining strongly bound to a single actin filament. The step size, stall force, and travel distance of myosin V reveal a remarkably efficient motor capable of moving along a helical track for over a micrometer without significantly spiraling around it. Such properties are fully consistent with the putative role of an organelle transport motor, present in small numbers to maintain movement over long ranges relative to cellular size scales. The contrast between myosin II and myosin V resembles that between a human running on the moon and one walking on earth, where the former allows for faster motion when in larger ensembles but for less travel distance when in smaller ones.

Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

PubMed Central

Winkelmann, D. A.; Bourdieu, L.; Kinose, F.; Libchaber, A.

1995-01-01

We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

PubMed

Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

2016-03-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. Copyright © 2016 the American Physiological Society.

Myosin content of individual human muscle fibers isolated by laser capture microdissection

PubMed Central

Stone, William L.; Howell, Mary E. A.; Brannon, Marianne F.; Hall, H. Kenton; Gibson, Andrew L.; Stone, Michael H.

2015-01-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. PMID:26676053

Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schewkunow, Vitali; Sharma, Karan P.; Diez, Gerold

2008-02-08

A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles mademore » of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 deg. C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.« less

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

PubMed Central

Noguchi, Taro Q. P.; Morimatsu, Masatoshi; Iwane, Atsuko H.; Yanagida, Toshio; Uyeda, Taro Q. P.

2015-01-01

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin. PMID:25945499

Fetal myosin immunoreactivity in human dystrophic muscle.

PubMed

Schiaffino, S; Gorza, L; Dones, I; Cornelio, F; Sartore, S

1986-01-01

We report immunofluorescence observations on normal and dystrophic human muscle using an antibody (anti-bF) raised against bovine fetal myosin and specific for fetal myosin heavy chains. In rat skeletal muscle, anti-bF was previously found to react selectively with myosin isoforms expressed during fetal and early postnatal development and in regenerating muscles. Anti-bF stained most fibers in human fetal and neonatal muscle, whereas only nuclear chain fibers of muscle spindles were labeled in normal adult muscle. In muscle biopsies from patients with Duchenne's muscular dystrophy, numerous extrafusal fibers were stained: some were small regenerating fibers, others were larger fibers presumably resulting from previous regenerative events. Fetal myosin immunoreactivity in Duchenne's dystrophy appears to reflect the reexpression of fetal-specific myosin isoforms and provides a new valuable tool for identifying regenerating fibers and following their destiny in dystrophic muscle.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization

PubMed Central

Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel H.; Jedlicka, Sabrina S.; Vavylonis, Dimitrios

2015-01-01

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. PMID:25641802

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.

Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosinmore » IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.« less

Novel roles for actin in mitochondrial fission

PubMed Central

Hatch, Anna L.; Gurel, Pinar S.; Higgs, Henry N.

2014-01-01

ABSTRACT Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER–mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals. PMID:25217628

No Difference in Myosin Kinetics and Spatial Distribution of the Lever Arm in the Left and Right Ventricles of Human Hearts

PubMed Central

Duggal, Divya; Requena, S.; Nagwekar, Janhavi; Raut, Sangram; Rich, Ryan; Das, Hriday; Patel, Vipul; Gryczynski, Ignacy; Fudala, Rafal; Gryczynski, Zygmunt; Blair, Cheavar; Campbell, Kenneth S.; Borejdo, Julian

2017-01-01

The systemic circulation offers larger resistance to the blood flow than the pulmonary system. Consequently, the left ventricle (LV) must pump blood with more force than the right ventricle (RV). The question arises whether the stronger pumping action of the LV is due to a more efficient action of left ventricular myosin, or whether it is due to the morphological differences between ventricles. Such a question cannot be answered by studying the entire ventricles or myocytes because any observed differences would be wiped out by averaging the information obtained from trillions of myosin molecules present in a ventricle or myocyte. We therefore searched for the differences between single myosin molecules of the LV and RV of failing hearts In-situ. We show that the parameters that define the mechanical characteristics of working myosin (kinetic rates and the distribution of spatial orientation of myosin lever arm) were the same in both ventricles. These results suggest that there is no difference in the way myosin interacts with thin filaments in myocytes of failing hearts, and suggests that the difference in pumping efficiencies are caused by interactions between muscle proteins other than myosin or that they are purely morphological. PMID:29081749

Stochastic dynamics and mechanosensitivity of myosin II minifilaments

NASA Astrophysics Data System (ADS)

Albert, Philipp J.; Erdmann, Thorsten; Schwarz, Ulrich S.

2014-09-01

Tissue cells are in a state of permanent mechanical tension that is maintained mainly by myosin II minifilaments, which are bipolar assemblies of tens of myosin II molecular motors contracting actin networks and bundles. Here we introduce a stochastic model for myosin II minifilaments as two small myosin II motor ensembles engaging in a stochastic tug-of-war. Each of the two ensembles is described by the parallel cluster model that allows us to use exact stochastic simulations and at the same time to keep important molecular details of the myosin II cross-bridge cycle. Our simulation and analytical results reveal a strong dependence of myosin II minifilament dynamics on environmental stiffness that is reminiscent of the cellular response to substrate stiffness. For small stiffness, minifilaments form transient crosslinks exerting short spikes of force with negligible mean. For large stiffness, minifilaments form near permanent crosslinks exerting a mean force which hardly depends on environmental elasticity. This functional switch arises because dissociation after the power stroke is suppressed by force (catch bonding) and because ensembles can no longer perform the power stroke at large forces. Symmetric myosin II minifilaments perform a random walk with an effective diffusion constant which decreases with increasing ensemble size, as demonstrated for rigid substrates with an analytical treatment.

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp; Hosaka, Toshio; Harada, Nagakatsu

2010-01-01

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4more » to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.« less

Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dey, Sumit K.; Saha, Shekhar; Das, Provas

2014-08-01

3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reducedmore » in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.« less

Myosin II–interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane

PubMed Central

Jiao, Meng; Wu, Di; Wei, Qize

2018-01-01

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II–interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction. PMID:29321250

Actin-myosin network is required for proper assembly of influenza virus particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregatedmore » on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.« less

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

PubMed

Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

2018-02-01

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

The myosin converter domain modulates muscle performance.

PubMed

Swank, Douglas M; Knowles, Aileen F; Suggs, Jennifer A; Sarsoza, Floyd; Lee, Annie; Maughan, David W; Bernstein, Sanford I

2002-04-01

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.

Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

NASA Astrophysics Data System (ADS)

Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

2013-12-01

Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

Effect of nitrofen in the final stages of development of the diaphragm muscle in rats.

PubMed

Gonçalves, Frances Lilian Lanhellas; Oliveira, Fábio Santana de; Schmidt, Augusto Frederico; Pereira, Luís Antônio Violin Dias; Gallindo, Rodrigo Melo; Figueira, Rebeca Lopes; Sbragia, Lourenço

2013-01-01

To evaluate the expression of myosin in muscle fibers of the diaphragm in experimental congenital diaphragmatic hernia (CDH). Fetuses of pregnant rats were divided into four groups: External Control (EC), composed of non-manipulated rats; Nitrofen, composed of pregnant rats that received 100 mg of nitrofen (2,4-dichloro-4'nitrodiphenyl ether) diluted in olive oil on gestational day (GD) 9.5, whose fetuses developed CDH (N+) or not (N-), and Olive Oil Placebo (OO), composed of pregnant rats that received the oil on the same GD. The fetuses were collected on GD 18.5, 19.5, 20.5 and 21.5 (term = 22 days). We obtained body weight (BW) and photographed the diaphragm area (DA), hernia area (HA) and subsequent calculated the HA/DA ratio in N+ group. Samples of Diaphragm muscle were processed for histological staining with H/E and immunohistochemistry (IHQ) for myosin. The fetuses of N- and N+ groups had decreased BW and DA compared to EC and OO groups (p < 0.001). HA was decreased on GD 18.5 compared to 21.5 (p < 0.001) and the HA/DA ratio showed no difference. IHQ showed decreased expression of myosin in nitrofen groups. CDH induced by nitrofen model contributes to the understanding of muscularization in the formation of the diaphragm where the myosin expression is decreased.

Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

PubMed Central

Cobley, James N.; Ab. Malik, Zulezwan; Morton, James P.; Close, Graeme L.; Edwards, Ben J.; Burniston, Jatin G.

2016-01-01

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping. PMID:28248225

Flexibility of myosin attachment to surfaces influences F-actin motion.

PubMed Central

Winkelmann, D A; Bourdieu, L; Ott, A; Kinose, F; Libchaber, A

1995-01-01

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. Images FIGURE 1 FIGURE 4 FIGURE 8 PMID:7544167

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle.

PubMed

Cooke, Roger

2011-03-01

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.

Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

PubMed Central

Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

2010-01-01

SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

Topology of interaction between titin and myosin thick filaments.

PubMed

Kellermayer, Miklós; Sziklai, Dominik; Papp, Zsombor; Decker, Brennan; Lakatos, Eszter; Mártonfalvi, Zsolt

2018-05-05

Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the A-band titin is associated with the myosin thick filament. It has been speculated that titin may serve as a blueprint for thick-filament formation due to the super-repeat structure of its A-band domains. Accordingly, titin might provide a template that determines the length and structural periodicity of the thick filament. Here we tested the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ratios (300 myosins per 12 titins) in buffers of different ionic strength (KCl concentration range 100-300 mM). The topology of the filamentous complexes was investigated with atomic force microscopy. We found that the samples contained distinct, segregated populations of titin molecules and myosin thick filaments. We were unable to identify complexes in which myosin molecules were regularly associated to either mono- or oligomeric titin in either relaxed or stretched states of the titin filaments. Thus, the electrostatically driven self-association is stronger in both myosin and titin than their binding to each other, and it is unlikely that titin functions as a geometrical template for thick-filament formation. However, when allowed to equilibrate configurationally, long myosin thick filaments appeared with titin oligomers attached to their surface. The titin meshwork formed on the thick-filament surface may play a role in controlling thick-filament length by regulating the structural dynamics of myosin molecules and placing a mechanical limit on the filament length. Copyright © 2018 Elsevier Inc. All rights reserved.

[Cleavage time for a hydrogen bond under a load].

PubMed

Bespalov, S V; Tolpygo, K B

1993-01-01

Statistics of the hydrogen bond formation and break in a bundle of actin and myosin filaments realizing the attractive force in the sarcomere of a muscle is studied. Purely mechanical problem of the attractive-force formation and motion of myosin heads and action globules under their action is supplemented by accounting for the irreversible processes: 1. Thermal de-excitation of the latter in the chain of hydrogen bond during the elementary act of the ATP energy use resulting in fixing the extended actin filament. 2. Break of the hydrogen bonds, realizing this fixing, due to thermal fluctuations for the time tau. The average life-time turns out to be the order of time necessary for the movement of z-membrane sarcomere for the value of action filament extension delta 1, which is necessary for the process of muscle contraction to be continued.

Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes

PubMed Central

White, Jennifer; Barro, Marietta V.; Makarenkova, Helen P.; Sanger, Joseph W.; Sanger, Jean M.

2014-01-01

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, non-muscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that non-muscle myosins II A and B are components of the Z-Bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-Bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. PMID:25125171

Unconstrained steps of myosin VI appear longest among known molecular motors.

PubMed

Ali, M Yusuf; Homma, Kazuaki; Iwane, Atsuko Hikikoshi; Adachi, Kengo; Itoh, Hiroyasu; Kinosita, Kazuhiko; Yanagida, Toshio; Ikebe, Mitsuo

2004-06-01

Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V.

Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

PubMed Central

Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

2010-01-01

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift

PubMed Central

Sun, Xiaojing; Wang, Yihua; Ajtai, Katalin

2017-01-01

Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC) N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function. A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N-terminus actin binding. Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin. PMID:28423017

Three Hierarchies in Skeletal Muscle Fibre Classification Allotype, Isotype and Phenotype

NASA Technical Reports Server (NTRS)

Hoh, Joseph F. Y.; Hughes, Suzanne; Hugh, Gregory; Pozgaj, Irene

1991-01-01

Immunocytochemical analyses using specific anti-myosin antibodies of mammalian muscle fibers during regeneration, development, and after denervation have revealed two distinct myogenic components determining fiber phenotype. The jaw-closing muscles of the cat contain superfast fibers which express a unique myosin not found in limb muscles. When superfast muscle is transplanted into a limb muscle bed, regenerating myotubes synthesize superfast myosin independent of innervation. Reinnervation by the nerve to a fast muscle leads to the expression of superfast and not fast myosin, while reinnervation by the nerve to a slow muscle leads to the expression of a slow myosin. When limb muscle is transplanted into the jaw muscle bed, only limb myosins are synthesized. Thus jaw and limb muscles belong to distinct allotypes, each with a unique range of phenotype options, the expressions of which may be modulated by the nerve. Primary and secondary myotubes in developing jaw and limb muscles are observed to belong to different categories characterized by different patterns of myosin gene expression. By taking into consideration the pattern of myosins synthesized and the changes in fiber size after denervation, 3 types of primary (fast, slow, and intermediate) fibers can be distinguished in rat fast limb muscles. All primaries synthesize slow myosin soon after their formation, but this is withdrawn in fast and intermediate primaries at different times. After neonatal denervation, slow and intermediate primaries express slow primaries hypertrophy with other fibers atrophy. In the mature rat, the number of slow fibers in the EDL is less than the number of slow primaries. Upon denervation, hypertrophic slow fibers matching the number and topographic distribution of slow primaries appear, suggesting that a subpopulation of the slow primaries acquire the fast phenotype during adult life, but reveal their original identity as slow primaries in response to denervation by hypertrophying and synthesizing slow myosin. It is proposed that within each muscle allotype, the various isotypes of primary and secondary fibers are myogenically determined, and are derived from different lineage of myoblasts.

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

PubMed Central

Kayser-Herold, Oliver; Stojanovic, Boban; Nedic, Djordje; Irving, Thomas C.; Geeves, Michael A.

2016-01-01

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to correctly describe the biochemical reactions of tethered molecules and their interaction energetics. PMID:27864330

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mijailovich, Srboljub M.; Kayser-Herold, Oliver; Stojanovic, Boban

2016-11-18

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulatemore » state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to correctly describe the biochemical reactions of tethered molecules and their interaction energetics.« less

Ultraslow myosin molecular motors of placental contractile stem villi in humans.

PubMed

Lecarpentier, Yves; Claes, Victor; Lecarpentier, Edouard; Guerin, Catherine; Hébert, Jean-Louis; Arsalane, Abdelilah; Moumen, Abdelouahab; Krokidis, Xénophon; Michel, Francine; Timbely, Oumar

2014-01-01

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

PubMed Central

Lelli, Andrea; Michel, Vincent; Boutet de Monvel, Jacques; Cortese, Matteo; Bosch-Grau, Montserrat; Aghaie, Asadollah; Perfettini, Isabelle; Dupont, Typhaine; Avan, Paul

2016-01-01

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a−/−Myo3b−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a−/−Myo3b−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a−/−Myo3b−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping. PMID:26754646

[Myosin storage myopathy: a rare subtype of protein aggregate myopathies].

PubMed

Kiphuth, I C; Neuen-Jacob, E; Struffert, T; Wehner, M; Wallefeld, W; Laing, N; Schröder, R

2010-04-01

Myopathies with pathological protein aggregates comprise a numerically significant group of sporadic and hereditary muscle disorders. A rare disease entity within the group of protein aggregate myopathies is the myosin storage myopathy, which is caused by heterozygous mutations in the MYH7 gene which encodes the slow/beta-myosin heavy chain. We report the clinical, myopathological and MRI findings in the first German patient suffering from a myosin storage myopathy due to a heterozygous R 1845W missense mutation.

Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms

PubMed Central

Shaffer, Justin F.; Kier, William M.

2015-01-01

The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities. PMID:26997860

Probing muscle myosin motor action: x-ray (m3 and m6) interference measurements report motor domain not lever arm movement.

PubMed

Knupp, Carlo; Offer, Gerald; Ranatunga, K W; Squire, John M

2009-07-10

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.

The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy

PubMed Central

Spudich, James A.

2015-01-01

No matter how many times one explores the structure of the myosin molecule, there is always something new to discover. Here, I describe the myosin mesa, a structural feature of the motor domain that has the characteristics of a binding domain for another protein, possibly myosin-binding protein C (MyBP-C). Interestingly, many well-known hypertrophic cardiomyopathy (HCM) mutations lie along this surface and may affect the putative interactions proposed here. A potential unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy is discussed here. It involves increased power output of the cardiac muscle as a result of HCM mutations causing the release of inhibition by myosin binding protein C. PMID:25619247

Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

PubMed Central

Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Lidofsky, Steven D.

2011-01-01

 The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress. PMID:21209319

Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

PubMed Central

Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P.

2012-01-01

Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections. PMID:22792440

Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

PubMed

Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P

2012-01-01

Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

Myosin-II controls cellular branching morphogenesis and migration in 3D by minimizing cell surface curvature

PubMed Central

Elliott, Hunter; Fischer, Robert A.; Myers, Kenneth A.; Desai, Ravi A.; Gao, Lin; Chen, Christopher S.; Adelstein, Robert; Waterman, Clare M.; Danuser, Gaudenz

2014-01-01

In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration. PMID:25621949

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy.

PubMed

Ortolano, Saida; Tarrío, Rosa; Blanco-Arias, Patricia; Teijeira, Susana; Rodríguez-Trelles, Francisco; García-Murias, María; Delague, Valerie; Lévy, Nicolas; Fernández, José M; Quintáns, Beatriz; Millán, Beatriz San; Carracedo, Angel; Navarro, Carmen; Sobrido, María-Jesús

2011-04-01

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy. Copyright © 2011 Elsevier B.V. All rights reserved.

Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution.

PubMed

Shimada, Masato; Takai, Eisuke; Ejima, Daisuke; Arakawa, Tsutomu; Shiraki, Kentaro

2015-02-01

Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 °C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution. Copyright © 2014 Elsevier B.V. All rights reserved.

Stabilization of Helivcal Order in the Thick Filaments by Blebbistatin: Further Evidence of Coexisting Multiple Conformations of Myosin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, S.; White, H; Offer, G

2009-01-01

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction undermore » the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder?order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.« less

Identifying the architecture of a supracellular actomyosin network that induces tissue folding

NASA Astrophysics Data System (ADS)

Yevick, Hannah; Stoop, Norbert; Dunkel, Jorn; Martin, Adam

During embryonic development, the establishment of correct tissue form ensures proper tissue function. Yet, how the thousands of cells within a tissue coordinate force production to sculpt tissue shape is poorly understood. One important tissue shape change is tissue folding where a cell sheet bends to form a closed tube. Drosophila (fruit fly) embryos undergo such a folding event, called ventral furrow formation. The ventral furrow is associated with a supracellular network of actin and myosin, where actin-myosin fibers assemble and connect between cells. It is not known how this tissue-wide network grows and connects over time, how reproducible it is between embryos, and what determines its architecture. Here, we used topological feature analysis to quantitatively and dynamically map the connections and architecture of this supracellular network across hundreds of cells in the folding tissue. We identified the importance of the cell unit in setting up the tissue-scale architecture of the network. Our mathematical framework allows us to explore stereotypic properties of the myosin network such that we can investigate the reproducibility of mechanical connections for a morphogenetic process. NIH F32.

Rab7b at the intersection of intracellular trafficking and cell migration.

PubMed

Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

2015-01-01

Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

Development and regeneration of the electric organ.

PubMed

Zakon, H H; Unguez, G A

1999-05-01

The electric organ has evolved independently from muscle in at least six lineages of fish. How does a differentiated muscle cell change its fate to become an electrocyte? Is the process by which this occurs similar in different lineages? We have begun to answer these questions by studying the formation and maintenance of electrocytes in the genus Sternopygus, a weakly electric teleost. Electrocytes arise from the fusion of fully differentiated muscle fibers, mainly those expressing fast isoforms of myosin. Electrocytes briefly co-express sarcomeric proteins, such as myosin and tropomyosin, and keratin, a protein not found in mature muscle. The sarcomeric proteins are subsequently down-regulated, but keratin expression persists. We investigated whether the maintenance of the electrocyte phenotype depends on innervation. We found that, after spinal cord transection, which silences the electromotor neurons that innervate the electrocytes, or destruction of the spinal cord, which denervates the electrocytes, mature electrocytes re-express sarcomeric myosin and tropomyosin, although keratin expression persists. Ultrastructural examination of denervated electrocytes revealed nascent sarcomeres. Thus, the maintenance of the electrocyte phenotype depends on neural activity.

Poorly Understood Aspects of Striated Muscle Contraction

PubMed Central

Månsson, Alf

2015-01-01

Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP). Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs. PMID:25961006

Poorly understood aspects of striated muscle contraction.

PubMed

Månsson, Alf; Rassier, Dilson; Tsiavaliaris, Georgios

2015-01-01

Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP). Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.

Force dependence of phagosome trafficking in retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Daniel, Rebekah; Koll, Andrew T.; Altman, David

2014-09-01

Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

Boundaries steer the contraction of active gels

NASA Astrophysics Data System (ADS)

Schuppler, Matthias; Keber, Felix C.; Kröger, Martin; Bausch, Andreas R.

2016-10-01

Cells set up contractile actin arrays to drive various shape changes and to exert forces to their environment. To understand their assembly process, we present here a reconstituted contractile system, comprising F-actin and myosin II filaments, where we can control the local activation of myosin by light. By stimulating different symmetries, we show that the force balancing at the boundaries determine the shape changes as well as the dynamics of the global contraction. Spatially anisotropic attachment of initially isotropic networks leads to a self-organization of highly aligned contractile fibres, being reminiscent of the order formation in muscles or stress fibres. The observed shape changes and dynamics are fully recovered by a minimal physical model.

Theoretical studies of the ATP hydrolysis mechanism of myosin.

PubMed

Okimoto, N; Yamanaka, K; Ueno, J; Hata, M; Hoshino, T; Tsuda, M

2001-11-01

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.

Resting myosin cross-bridge configuration in frog muscle thick filaments.

PubMed

Cantino, M; Squire, J

1986-02-01

Clear images of myosin filaments have been seen in shadowed freeze-fracture replicas of single fibers of relaxed frog semitendinosus muscles rapidly frozen using a dual propane jet freezing device. These images have been analyzed by optical diffraction and computer averaging and have been modelled to reveal details of the myosin head configuration on the right-handed, three-stranded helix of cross-bridges. Both the characteristic 430-A and 140-150-A repeats of the myosin cross-bridge array could be seen. The measured filament backbone diameter was 140-160 A, and the outer diameter of the cross-bridge array was 300 A. Evidence is presented that suggests that the observed images are consistent with a model in which both of the heads of one myosin molecule tilt in the same direction at an angle of approximately 50-70 degrees to the normal to the filament long axis and are slewed so that they lie alongside each other and their radially projected density lies along the three right-handed helical tracks. Any perturbation of the myosin heads away from their ideal lattice sites needed to account for x-ray reflections not predicted for a perfect helix must be essentially along the three helical tracks of cross-bridges. Little trace of the presence of non-myosin proteins could be seen.

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

PubMed Central

Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B

2014-01-01

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss. PMID:24105371

Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2.

PubMed

Zhang, Hai-Man; Ji, Huan-Hong; Ni, Tong; Ma, Rong-Na; Wang, Aibing; Li, Xiang-Dong

2017-08-15

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC 50 values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI.

PubMed

Marcotti, Walter; Corns, Laura F; Goodyear, Richard J; Rzadzinska, Agnieszka K; Avraham, Karen B; Steel, Karen P; Richardson, Guy P; Kros, Corné J

2016-07-01

The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells' apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca(2+) -dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca(2+) -dependent adaptation. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Mutating the converter-relay interface of Drosophila myosin perturbs ATPase activity, actin motility, myofibril stability and flight ability.

PubMed

Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

2010-05-21

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V(max)) by approximately 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K(m)). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by approximately 15% or approximately 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by approximately 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional "cracking" of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle. (c) 2010 Elsevier Ltd. All rights reserved.

Anisotropy of Crumbs and aPKC Drives Myosin Cable Assembly during Tube Formation

PubMed Central

Röper, Katja

2012-01-01

Summary The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized. PMID:23153493

Anisotropy of Crumbs and aPKC drives myosin cable assembly during tube formation.

PubMed

Röper, Katja

2012-11-13

The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized. Copyright © 2012 Elsevier Inc. All rights reserved.

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

PubMed Central

Kim, Seungil; Lewis, Ace E.; Singh, Vivek; Ma, Xuefei; Adelstein, Robert; Bush, Jeffrey O.

2015-01-01

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development. PMID:25848986

Structural Basis for the Regulation of Muscle Contraction by Troponin and Tropomyosin

PubMed Central

Galińska-Rakoczy, Agnieszka; Engel, Patti; Xu, Chen; Jung, HyunSuk; Craig, Roger; Tobacman, Larry S.; Lehman, William

2008-01-01

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca2+-binding to TnC presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI with tropomyosin and actin that cause tropomyosin movement are unknown and thus the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI and then 3D-EM was used to resolve where troponin is anchored on actin-tropomyosin. EM-reconstruction showed how TnI-binding to both actin and tropomyosin at low-Ca2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric-interference of actin-myosin interaction that regulates muscle contraction. PMID:18514658

Homozygous deletion in MYL9 expands the molecular basis of megacystis-microcolon-intestinal hypoperistalsis syndrome.

PubMed

Moreno, Carolina Araujo; Sobreira, Nara; Pugh, Elizabeth; Zhang, Peng; Steel, Gary; Torres, Fábio Rossi; Cavalcanti, Denise Pontes

2018-05-01

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a severe disease characterized by functional obstruction in the urinary and gastrointestinal tract. The molecular basis of this condition started to be defined recently, and the genes related to the syndrome (ACTG2-heterozygous variant in sporadic cases; and MYH11 (myosin heavy chain 11), LMOD1 (leiomodin 1) and MYLK (myosin light chain (MLC) kinase)-autosomal recessive inheritance), encode proteins involved in the smooth muscle contraction, supporting a myopathic basis for the disease. In the present article, we described a family with two affected siblings with MMIHS born to consanguineous parents and the molecular investigation performed to define the genetic etiology. Previous whole exome sequencing of the affected child and parents did not identify a candidate gene for the disease in this family, but now we present a reanalysis of the data that led to the identification of a homozygous deletion encompassing the last exon of MYL9 (myosin regulatory light chain 9) in the affected individual. MYL9 gene encodes a regulatory myosin MLC and the phosphorylation of this protein is a crucial step in the contraction process of smooth muscle cell. Despite the absence of human or animal phenotype related to MYL9, a cause-effect relationship between MYL9 and the MMIHS seems biologically plausible. The present study reveals a strong candidate gene for autosomal recessive forms of MMIHS, expanding the molecular basis of this disease and reinforces the myopathic basis of this condition.

Myocardial short-range force responses increase with age in F344 rats

PubMed Central

Mitov, Mihail I.; Holbrook, Anastasia M.; Campbell, Kenneth S.

2009-01-01

The mechanical properties of triton-permeabilized ventricular preparations isolated from 4, 18 and 24-month-old F344 rats were analyzed to provide information about the molecular mechanisms that lead to age-related increases in diastolic myocardial stiffness in these animals. Passive stiffness (measured in solutions with minimal free Ca2+) did not change with age. This implies that the aging-associated dysfunction is not due to changes in titin or collagen molecules. Ca2+-activated preparations exhibited a characteristic short-range force response: force rose rapidly until the muscle reached its elastic limit and less rapidly thereafter. The elastic limit increased from 0.43 ± 0.01 % l0 (where l0 is the initial muscle length) in preparations from 4-month-old animals to 0.49 ± 0.01 % l0 in preparations from 24-month-old rats (p<0.001, ANOVA). Relative short-range force was defined as the maximum force produced during the short-range response normalized to the prevailing tension. This parameter increased from 0.110 ± 0.002 to 0.142 ± 0.002 over the same age-span (p<0.001, ANOVA). Analytical gel electrophoresis showed that the maximum stiffness of the preparations during the short-range response and the relative short-range force increased (p=0.031 and p=0.005 respectively) with the relative content of slow β myosin heavy chain molecules. Elastic limit values did not correlate with myosin isoform content. Simulations based on these results suggest that attached β myosin heavy chain cross-bridges are stiffer than links formed by α myosin heads. In conclusion, elevated content of stiffer β myosin heavy chain molecules may contribute to aging-associated increases in myocardial stiffness. PMID:19007786

Sequential activation of apical and basolateral contractility drives ascidian endoderm invagination

PubMed Central

Sherrard, Kristin; Robin, François; Lemaire, Patrick; Munro1, Edwin

2014-01-01

SUMMARY Background Epithelial invagination is a fundamental morphogenetic behavior that transforms a flat cell sheet into a pit or groove. Previous studies of invagination have focused on the role of actomyosin-dependent apical contraction; other mechanisms remain largely unexplored. Results We combined experimental and computational approaches to identify a two-step mechanism for endoderm invagination during ascidian gastrulation. During Step 1, which immediately precedes invagination, endoderm cells constrict their apices due to Rho/Rhokinase-dependent apical enrichment of 1P–myosin. Our data suggest that endoderm invagination itself occurs during Step 2, without further apical shrinkage, via a novel mechanism we call collared rounding: Rho/Rho-kinase-independent lateral enrichment of 1P–myosin drives apico-basal shortening, while Rho/Rho-kinase-dependent enrichment of 1P and 2P myosin in circumapical collars is required to prevent apical expansion and for deep invagination. Simulations show that boundary-specific tension values consistent with these distributions of active myosin can explain the cell shape changes observed during invagination both in normal embryos and in embryos treated with pharmacological inhibitors of either Rho-kinase or Myosin II ATPase. Indeed, we find that the balance of strong circumapical and basolateral tension is the only mechanism based on differential cortical tension that can explain ascidian endoderm invagination. Finally, simulations suggest that mesectoderm cells resist endoderm shape changes during both steps and we confirm this prediction experimentally. Conclusions Our findings suggest that early ascidian gastrulation is driven by the coordinated apposition of circumapical and lateral endoderm contraction, working against a resisting mesectoderm. We propose that similar mechanisms may operate during other invaginations. PMID:20691592

UNC-45/CRO1/She4p (UCS) Protein Forms Elongated Dimer and Joins Two Myosin Heads Near Their Actin Binding Region

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Shi; G Blobel

2011-12-31

UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 {angstrom} resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-{angstrom}-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal regionmore » as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 {angstrom} thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments.« less

On the mechanism of energy transduction in myosin subfragment 1.

PubMed Central

Botts, J; Takashi, R; Torgerson, P; Hozumi, T; Muhlrad, A; Mornet, D; Morales, M F

1984-01-01

It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Förster equation and that the existence of an intersite communication system can be deduced from piece-wise detection of interacting pairs of points. PMID:6585786

Myosin-II-Mediated Directional Migration of Dictyostelium Cells in Response to Cyclic Stretching of Substratum

PubMed Central

Iwadate, Yoshiaki; Okimura, Chika; Sato, Katsuya; Nakashima, Yuta; Tsujioka, Masatsune; Minami, Kazuyuki

2013-01-01

Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin. PMID:23442953

Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

PubMed

Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

1989-03-01

To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass microneedle (tip diameter, approximately 7 microns; elastic coefficient, approximately 40 pN/microns), which was coated with skeletal muscle myosin at the tip and extended from a micromanipulator at right angles with the actin cables. When the myosin-coated tip of the microneedle was brought into contact with the exposed surface of the actin cables, it exhibited ATP-dependent movement along the actin cables over a distance of 20-150 microns in 20-200 s (20-23 degrees C) and eventually stopped due to a balance between forces generated by the actin-myosin interaction (800-6000 pN) and by the bent microneedle. Since the load on the force-generating myosin molecules increased with the bending displacement of the microneedle (auxotonic condition), the relation between the load and the sliding velocity of the myosin heads past the actin cables was determined from the time course of the microneedle movement recorded with a video system. The shape of the force-velocity curve thus obtained was convex upwards, similar to that of the force-velocity curve of intact frog muscle fibers obtained under the auxotonic condition.

Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

PubMed Central

Armel, Thomas Z.; Leinwand, Leslie A.

2009-01-01

Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, β-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the α-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule. PMID:19336582

Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

PubMed

Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

2016-05-24

Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation

PubMed Central

Mortal, Simone; Iseppon, Federico; Perissinotto, Andrea; D'Este, Elisa; Cojoc, Dan; Napolitano, Luisa M. R.; Torre, Vincent

2017-01-01

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration. PMID:29326552

Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

NASA Technical Reports Server (NTRS)

Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

1992-01-01

This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

Changes in the myosin secondary structure and shrimp surimi gel strength induced by dense phase carbon dioxide.

PubMed

Guo, Minghui; Liu, Shucheng; Ismail, Marliya; Farid, Mohammed M; Ji, Hongwu; Mao, Weijie; Gao, Jing; Li, Chengyong

2017-07-15

Dense phase carbon dioxide (DPCD) could induce protein conformation changes. Myosin and shrimp surimi from Litopenaeus vannamei were treated with DPCD at 5-25MPa and 40-60°C for 20min. Myosin secondary structure was investigated by circular dichroism and shrimp surimi gel strength was determined using textural analysis to develop correlations between them. DPCD had a greater effect on secondary structure and gel strength than heating. With increasing pressure and temperature, the α-helix content of DPCD-treated myosin decreased, while the β-sheet, β-turn and random coil contents increased, and the shrimp surimi gel strength increased. The α-helix content was negatively correlated with gel strength, while the β-sheet, β-turn and random coil contents were positively correlated with gel strength. Therefore, when DPCD induced myosin to form a gel, the α-helix of myosin was unfolded and gradually converted to a β-sheet. Such transformations led to protein-protein interactions and cross-linking, which formed a three-dimensional network to enhance the gel strength. Copyright © 2017 Elsevier Ltd. All rights reserved.

Blebbistatin, a myosin II inhibitor, suppresses Ca(2+)-induced and "sensitized"-contraction of skinned tracheal muscles from guinea pig.

PubMed

Yumoto, Masatoshi; Watanabe, Masaru

2013-01-01

Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

PubMed Central

Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.

2012-01-01

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility. PMID:22711700

Roles of an Unconventional Protein Kinase and Myosin II in Amoeba Osmotic Shock Responses

PubMed Central

Betapudi, Venkaiah; Egelhoff, Thomas T.

2009-01-01

The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. GFP-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical, and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa. PMID:19843280

Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes

PubMed Central

Alamo, Lorenzo; Ware, James S; Pinto, Antonio; Gillilan, Richard E; Seidman, Jonathan G; Seidman, Christine E; Padrón, Raúl

2017-01-01

Cardiac β-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures predict contractility effects but not the prominent relaxation and energetic deficits that characterize HCM. During relaxation, pairs of myosins form interacting-heads motif (IHM) structures that with other sarcomere proteins establish an energy-saving, super-relaxed (SRX) state. Using a human β-cardiac myosin IHM quasi-atomic model, we defined interactions sites between adjacent myosin heads and associated protein partners, and then analyzed rare variants from 6112 HCM and 1315 DCM patients and 33,370 ExAC controls. HCM variants, 72% that changed electrostatic charges, disproportionately altered IHM interaction residues (expected 23%; HCM 54%, p=2.6×10−19; DCM 26%, p=0.66; controls 20%, p=0.23). HCM variant locations predict impaired IHM formation and stability, and attenuation of the SRX state - accounting for altered contractility, reduced diastolic relaxation, and increased energy consumption, that fully characterizes HCM pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.24634.001 PMID:28606303

Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

PubMed Central

Boëda, Batiste; El-Amraoui, Aziz; Bahloul, Amel; Goodyear, Richard; Daviet, Laurent; Blanchard, Stéphane; Perfettini, Isabelle; Fath, Karl R.; Shorte, Spencer; Reiners, Jan; Houdusse, Anne; Legrain, Pierre; Wolfrum, Uwe; Richardson, Guy; Petit, Christine

2002-01-01

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia. PMID:12485990

Identification of a novel mutation in the myosin VIIA motor domain in a family with autosomal dominant hearing loss (DFNA11).

PubMed

Di Leva, Francesca; D'Adamo, Pio; Cubellis, Maria Vittoria; D'Eustacchio, Angela; Errichiello, Monica; Saulino, Claudio; Auletta, Gennaro; Giannini, Pasquale; Donaudy, Francesca; Ciccodicola, Alfredo; Gasparini, Paolo; Franzè, Annamaria; Marciano, Elio

2006-01-01

We ascertained a large Italian family with an autosomal dominant form of non-syndromic sensorineural hearing loss with vestibular involvement. A genome-wide scan found linkage to locus DFNA11. Sequencing of the MYO7A gene in the linked region identified a new missense mutation resulting in an Ala230Val change in the motor domain of the myosin VIIA. Myosin VIIA has already been implicated in several forms of deafness, but this is the third mutation causing a dominant form of deafness, located in the myosin VIIA motor domain in a region never involved in hearing loss until now. A modelled protein structure of myosin VII motor domain provides evidence for a significant functional effect of this missense mutation. Copyright (c) 2006 S. Karger AG, Basel.

Effect of inorganic phosphate on the force and number of myosin cross-bridges during the isometric contraction of permeabilized muscle fibers from rabbit psoas.

PubMed

Caremani, Marco; Dantzig, Jody; Goldman, Yale E; Lombardi, Vincenzo; Linari, Marco

2008-12-15

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12 degrees C, sarcomere length 2.5 mum). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 +/- 1.1 kPa nm(-1). Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 +/- 3.7 kPa nm(-1). An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r(F)) is 23.5 +/- 1.0 s(-1) in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 +/- 0.9 s(-1) at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex.

PubMed

Fabritius, Amy S; Flynn, Jonathan R; McNally, Francis J

2011-11-01

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole. 2011 Elsevier Inc. All rights reserved.

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

PubMed

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Mouse Nuclear Myosin I Knock-Out Shows Interchangeability and Redundancy of Myosin Isoforms in the Cell Nucleus

PubMed Central

Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

2013-01-01

Background Nuclear myosin I (NM1) is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes. PMID:23593477

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

PubMed

Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

2013-01-01

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions*

PubMed Central

Quintero, Omar A.; Weck, Meredith L.; Unrath, William C.; Gallagher, James W.; Cui, Runjia; Kachar, Bechara; Tyska, Matthew J.; Yengo, Christopher M.

2016-01-01

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30–34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance. PMID:27582493

The Role of the UNC-82 Protein Kinase in Organizing Myosin Filaments in Striated Muscle of Caenorhabditis elegans

PubMed Central

Schiller, NaTasha R.; Duchesneau, Christopher D.; Lane, Latrisha S.; Reedy, April R.; Manzon, Emily R.; Hoppe, Pamela E.

2017-01-01

We study the mechanisms that guide the formation and maintenance of the highly ordered actin-myosin cytoskeleton in striated muscle. The UNC-82 kinase of Caenorhabditis elegans is orthologous to mammalian kinases ARK5/NUAK1 and SNARK/NUAK2. UNC-82 localizes to the M-line, and is required for proper organization of thick filaments, but its substrate and mechanism of action are unknown. Antibody staining of three mutants with missense mutations in the UNC-82 catalytic domain revealed muscle structure that is less disorganized than in the null unc-82(0), but contained distinctive ectopic accumulations not found in unc-82(0). These accumulations contain paramyosin and myosin B, but lack myosin A and myosin A-associated proteins, as well as proteins of the integrin-associated complex. Fluorescently tagged missense mutant protein UNC-82 E424K localized normally in wild type; however, in unc-82(0), the tagged protein was found in the ectopic accumulations, which we also show to label with recently synthesized paramyosin. Recruitment of wild-type UNC-82::GFP to aggregates of differing protein composition in five muscle-affecting mutants revealed that colocalization of UNC-82 and paramyosin does not require UNC-96, UNC-98/ZnF, UNC-89/obscurin, CSN-5, myosin A, or myosin B individually. Dosage effects in paramyosin mutants suggest that UNC-82 acts as part of a complex, in which its stoichiometric relationship with paramyosin is critical. UNC-82 dosage affects muscle organization in the absence of paramyosin, perhaps through myosin B. We present evidence that the interaction of UNC-98/ZnF with myosin A is independent of UNC-82, and that UNC-82 acts upstream of UNC-98/ZnF in a pathway that organizes paramyosin during thick filament assembly. PMID:28040740

The Role of the UNC-82 Protein Kinase in Organizing Myosin Filaments in Striated Muscle of Caenorhabditis elegans.

PubMed

Schiller, NaTasha R; Duchesneau, Christopher D; Lane, Latrisha S; Reedy, April R; Manzon, Emily R; Hoppe, Pamela E

2017-03-01

We study the mechanisms that guide the formation and maintenance of the highly ordered actin-myosin cytoskeleton in striated muscle. The UNC-82 kinase of Caenorhabditis elegans is orthologous to mammalian kinases ARK5/NUAK1 and SNARK/NUAK2. UNC-82 localizes to the M-line, and is required for proper organization of thick filaments, but its substrate and mechanism of action are unknown. Antibody staining of three mutants with missense mutations in the UNC-82 catalytic domain revealed muscle structure that is less disorganized than in the null unc-82(0) , but contained distinctive ectopic accumulations not found in unc-82(0) These accumulations contain paramyosin and myosin B, but lack myosin A and myosin A-associated proteins, as well as proteins of the integrin-associated complex. Fluorescently tagged missense mutant protein UNC-82 E424K localized normally in wild type; however, in unc-82(0) , the tagged protein was found in the ectopic accumulations, which we also show to label with recently synthesized paramyosin. Recruitment of wild-type UNC-82::GFP to aggregates of differing protein composition in five muscle-affecting mutants revealed that colocalization of UNC-82 and paramyosin does not require UNC-96, UNC-98/ZnF, UNC-89/obscurin, CSN-5, myosin A, or myosin B individually. Dosage effects in paramyosin mutants suggest that UNC-82 acts as part of a complex, in which its stoichiometric relationship with paramyosin is critical. UNC-82 dosage affects muscle organization in the absence of paramyosin, perhaps through myosin B. We present evidence that the interaction of UNC-98/ZnF with myosin A is independent of UNC-82, and that UNC-82 acts upstream of UNC-98/ZnF in a pathway that organizes paramyosin during thick filament assembly. Copyright © 2017 by the Genetics Society of America.

Fast and slow myosins as markers of muscle injury.

PubMed

Guerrero, M; Guiu-Comadevall, M; Cadefau, J A; Parra, J; Balius, R; Estruch, A; Rodas, G; Bedini, J L; Cussó, R

2008-07-01

The diagnosis of muscular lesions suffered by athletes is usually made by clinical criteria combined with imaging of the lesion (ultrasonography and/or magnetic resonance) and blood tests to detect the presence of non-specific muscle markers. This study was undertaken to evaluate injury to fast and slow-twitch fibres using specific muscle markers for these fibres. Blood samples were obtained from 51 non-sports people and 38 sportsmen with skeletal muscle injury. Western blood analysis was performed to determine fast and slow myosin and creatine kinase (CK) levels. Skeletal muscle damage was diagnosed by physical examination, ultrasonography and magnetic resonance and biochemical markers. The imaging tests were found to be excellent for detecting and confirming grade II and III lesions. However, grade I lesions were often unconfirmed by these techniques. Grade I lesions have higher levels of fast myosin than slow myosin with a very small increase in CK levels. Grade II and III lesions have high values of both fast and slow myosin. The evaluation of fast and slow myosin in the blood 48 h after the lesion occurs is a useful aid for the detection of type I lesions in particular, since fast myosin is an exclusive skeletal muscle marker. The correct diagnosis of grade I lesions can prevent progression of the injury in athletes undergoing continual training sessions and competitions, thus aiding sports physicians in their decision making.

Biochemical and bioinformatic analysis of the MYO19 motor domain

PubMed Central

Adikes, Rebecca C.; Unrath, William C.; Yengo, Christopher M.; Quintero, Omar A.

2014-01-01

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-cosedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment. PMID:23568824

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II*

PubMed Central

Bloemink, Marieke J.; Melkani, Girish C.; Bernstein, Sanford I.; Geeves, Michael A.

2016-01-01

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis. PMID:26586917

Tropomyosin movement on F-actin during muscle activation explained by energy landscapes

PubMed Central

Orzechowski, Marek; Moore, Jeffrey R.; Fischer, Stefan; Lehman, William

2014-01-01

Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced “open-state” position. This indicates that spontaneous movement of tropomyosin away from its energetic “ground-state” to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations. PMID:24412204

Tropomyosin movement on F-actin during muscle activation explained by energy landscapes.

PubMed

Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan; Lehman, William

2014-03-01

Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced "open-state" position. This indicates that spontaneous movement of tropomyosin away from its energetic "ground-state" to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations. Copyright © 2014 Elsevier Inc. All rights reserved.

CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

PubMed

Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

2015-01-01

Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss.

PubMed

Kwon, Tae-Jun; Oh, Se-Kyung; Park, Hong-Joon; Sato, Osamu; Venselaar, Hanka; Choi, Soo Young; Kim, SungHee; Lee, Kyu-Yup; Bok, Jinwoong; Lee, Sang-Heun; Vriend, Gert; Ikebe, Mitsuo; Kim, Un-Kyung; Choi, Jae Young

2014-07-01

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

PubMed

Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

2008-09-09

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss

PubMed Central

Kwon, Tae-Jun; Oh, Se-Kyung; Park, Hong-Joon; Sato, Osamu; Venselaar, Hanka; Choi, Soo Young; Kim, SungHee; Lee, Kyu-Yup; Bok, Jinwoong; Lee, Sang-Heun; Vriend, Gert; Ikebe, Mitsuo; Kim, Un-Kyung; Choi, Jae Young

2014-01-01

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene. PMID:25080041

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

PubMed Central

Coffman, Valerie C.; Nile, Aaron H.; Lee, I-Ju; Liu, Huayang

2009-01-01

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis. PMID:19864459

Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a.

PubMed

Yengo, Christopher M; Ananthanarayanan, Shobana K; Brosey, Chris A; Mao, Suli; Tyska, Matthew J

2008-01-15

Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V(MAX) was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.

Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle.

PubMed

Kampourakis, Thomas; Ponnam, Saraswathi; Irving, Malcolm

2018-04-01

Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants

PubMed Central

Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

2016-01-01

The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function. PMID:27378946

A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

PubMed Central

Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

2014-01-01

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

PubMed Central

Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

1989-01-01

To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass microneedle (tip diameter, approximately 7 microns; elastic coefficient, approximately 40 pN/microns), which was coated with skeletal muscle myosin at the tip and extended from a micromanipulator at right angles with the actin cables. When the myosin-coated tip of the microneedle was brought into contact with the exposed surface of the actin cables, it exhibited ATP-dependent movement along the actin cables over a distance of 20-150 microns in 20-200 s (20-23 degrees C) and eventually stopped due to a balance between forces generated by the actin-myosin interaction (800-6000 pN) and by the bent microneedle. Since the load on the force-generating myosin molecules increased with the bending displacement of the microneedle (auxotonic condition), the relation between the load and the sliding velocity of the myosin heads past the actin cables was determined from the time course of the microneedle movement recorded with a video system. The shape of the force-velocity curve thus obtained was convex upwards, similar to that of the force-velocity curve of intact frog muscle fibers obtained under the auxotonic condition. Images PMID:2922395

Qdot Labeled Actin Super Resolution Motility Assay Measures Low Duty Cycle Muscle Myosin Step-Size

PubMed Central

Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

2013-01-01

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot’s were imaged in time with total internal reflection fluorescence microscopy then spatially localized to 1-3 nanometers using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full length β-cardiac myosin (MYH7). Average Qdot-actin velocity matches measurements with rhodamine-phalloidin labeled actin. The sHMM Qdot-actin velocity histogram contains low velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm, and, larger compliance than sHMM depending on MYH7 surface concentration. Low duty cycle skeletal and cardiac myosin present challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach provides quick, quantitative, and inexpensive step-size measurement for low duty cycle muscle myosin. PMID:23383646

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-05-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

Myosin XI-Dependent Formation of Tubular Structures from Endoplasmic Reticulum Isolated from Tobacco Cultured BY-2 Cells1[W][OA

PubMed Central

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-01-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER. PMID:21427277

A Failure to Communicate

PubMed Central

Kronert, William A.; Melkani, Girish C.; Melkani, Anju; Bernstein, Sanford I.

2015-01-01

Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (∼80%) as well as in basal (∼67%) and actin-activated (∼84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ∼73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease. PMID:26446785

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.

PubMed

Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella

2014-03-01

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

PubMed Central

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog

PubMed Central

Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella

2014-01-01

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6–3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments. PMID:24344169

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Kinesins and Myosins: Molecular Motors that Coordinate Cellular Functions in Plants.

PubMed

Nebenführ, Andreas; Dixit, Ram

2018-04-29

Kinesins and myosins are motor proteins that can move actively along microtubules and actin filaments, respectively. Plants have evolved a unique set of motors that function as regulators and organizers of the cytoskeleton and as drivers of long-distance transport of various cellular components. Recent progress has established the full complement of motors encoded in plant genomes and has revealed valuable insights into the cellular functions of many kinesin and myosin isoforms. Interestingly, several of the motors were found to functionally connect the two cytoskeletal systems and thereby to coordinate their activities. In this review, we discuss the available genetic, cell biological, and biochemical data for each of the plant kinesin and myosin families from the context of their subcellular mechanism of action as well as their physiological function in the whole plant. We particularly emphasize work that illustrates mechanisms by which kinesins and myosins coordinate the activities of the cytoskeletal system.

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

PubMed

Duque, Julia; Gorfinkiel, Nicole

2016-12-15

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Calcium-responsive contractility during fertilization in sea urchin eggs.

PubMed

Stack, Christianna; Lucero, Amy J; Shuster, Charles B

2006-04-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. (c) 2006 Wiley-Liss, Inc.

Calcium-Responsive Contractility During Fertilization in Sea Urchin Eggs

PubMed Central

Stack, Christianna; Lucero, Amy J.; Shuster, Charles B.

2008-01-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both prior to- and following fertilization, and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed- and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs, but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. PMID:16470603

Sliding movement of single actin filaments on one-headed myosin filaments

NASA Astrophysics Data System (ADS)

Harada, Yoshie; Noguchi, Akira; Kishino, Akiyoshi; Yanagida, Toshio

1987-04-01

The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread1, superprecipitation2-4 and chemical modification of muscle fibres5. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly6,7. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis

PubMed Central

Granato, Michael

2016-01-01

During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator. PMID:27855159

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus.

PubMed

Sobczak, Magdalena; Wasik, Anna; Kłopocka, Wanda; Redowicz, Maria Jolanta

2008-12-01

Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart

PubMed Central

1986-01-01

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development. PMID:3514633

Axial rotation of sliding actin filaments revealed by single-fluorophore imaging

PubMed Central

Sase, Ichiro; Miyata, Hidetake; Ishiwata, Shin’ichi; Kinosita, Kazuhiko

1997-01-01

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of ≈72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of ≈1 μm, which is much greater than the 72 nm pitch. Thus, myosin does not “walk” on the helical array of actin protomers; rather it “runs,” skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each “kick” of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions. PMID:9159126

­Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage

PubMed Central

2016-01-01

Contraction of actomyosin rings during cytokinesis is typically attributed to actin filaments sliding toward each other via Myosin-2 motor activity. However, rings constrict in some cells in the absence of Myosin-2 activity. Thus, ring closure uses Myosin-2–dependent and –independent mechanisms. But what the Myosin-2–independent mechanisms are, and to what extent they are sufficient to drive closure, remains unclear. During cleavage in Drosophila melanogaster embryos, actomyosin rings constrict in two sequential and mechanistically distinct phases. We show that these phases differ in constriction speed and are genetically and pharmacologically separable. Further, Myosin-2 activity is required for slow constriction in “phase 1” but is largely dispensable for fast constriction in “phase 2,” and F-actin disassembly is only required for fast constriction in phase 2. Switching from phase 1 to phase 2 seemingly relies on the spatial organization of F-actin as controlled by Cofilin, Anillin, and Septin. Our work shows that fly embryos present a singular opportunity to compare separable ring constriction mechanisms, with varying Myosin-2 dependencies, in one cell type and in vivo. PMID:27799369

Axial rotation of sliding actin filaments revealed by single-fluorophore imaging.

PubMed

Sase, I; Miyata, H; Ishiwata, S; Kinosita, K

1997-05-27

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of approximately 72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of approximately 1 microm, which is much greater than the 72 nm pitch. Thus, myosin does not "walk" on the helical array of actin protomers; rather it "runs," skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each "kick" of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

Neonatal myosin in bovine and pig tensor tympani muscle fibres.

PubMed Central

Scapolo, P A; Rowlerson, A; Mascarello, F; Veggetti, A

1991-01-01

In previous studies of middle ear muscles, the classification of fibre types by histochemical methods was particularly difficult in the bovine and porcine tensor tympani muscle, suggesting the presence of immature fibres. We therefore reexamined the tensor tympani from pigs and cattle of various ages immunohistochemically, using a panel of antimyosin antibodies, including one (anti-NE) specific for neonatal and embryonic myosins. Fibres positive to anti-NE were found in tensor tympani in both species in all ages examined; only a few of these fibres reacted exclusively with this antibody; some also contained slow myosin and the majority also contained adult fast (type IIA) myosin. Furthermore, although the remaining fibres included some of the classical types I and IIA, the majority of them showed a mismatch between their histochemical and immunohistochemical profiles. The morphological appearance of the muscle, the widespread presence of neonatal myosin (often together with another myosin in the same fibre) and the persistence of this composition from birth to adulthood, could be explained by an incomplete development of the muscle fibres, resulting in a 'muscle' much better suited to the role of a ligament. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:1810932

CRYO-EM STRUCTURES OF THE ACTIN:TROPOMYOSIN FILAMENT REVEAL THE MECHANISM FOR THE TRANSITION FROM C- TO M-STATE

PubMed Central

Sousa, Duncan R.; Stagg, Scott M.; Stroupe, M. Elizabeth

2013-01-01

Tropomyosin is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of tropomyosin along the actin:tropomyosin:troponin thin filament to block or expose myosin binding sites on actin. In striated muscle, including involuntary cardiac muscle, tropomyosin regulates muscle contraction by coupling Ca2+ binding to troponin with myosin binding to the thin filament. In smooth muscle, the switch is the post-translational modification of the myosin. Depending on the activation state of troponin and the binding state of myosin, tropomyosin can occupy the blocked, closed, or open position on actin. Using native cryogenic 3DEM, we have directly resolved and visualized cardiac and gizzard muscle tropomyosin on filamentous actin in the position that corresponds to the closed state. From the 8-Å resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed state position. PMID:24021812

Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

PubMed

Lan, Bo; Deng, Linhong; Donovan, Graham M; Chin, Leslie Y M; Syyong, Harley T; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D; Norris, Brandon A; Liu, Jeffrey C-Y; Swyngedouw, Nicholas E; Banaem, Saleha M; Paré, Peter D; Seow, Chun Y

2015-01-01

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. Copyright © 2015 the American Physiological Society.

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

PubMed

Bunda, Jordan; Gittings, William; Vandenboom, Rene

2018-01-30

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK -/- ) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK -/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK -/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK -/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK -/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK -/- muscles without RLC phosphorylation. © 2018. Published by The Company of Biologists Ltd.

Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle

PubMed Central

Lan, Bo; Deng, Linhong; Donovan, Graham M.; Chin, Leslie Y. M.; Syyong, Harley T.; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Swyngedouw, Nicholas E.; Banaem, Saleha M.; Paré, Peter D.

2014-01-01

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. PMID:25305246

Self-polarization and directional motility of cytoplasm.

PubMed

Verkhovsky, A B; Svitkina, T M; Borisy, G G

1999-01-14

Directional cell motility implies the presence of a steering mechanism and a functional asymmetry between the front and rear of the cell. How this functional asymmetry arises and is maintained during cell locomotion is, however, unclear. Lamellar fragments of fish epidermal keratocytes, which lack nuclei, microtubules and most organelles, present a simplified, perhaps minimal, system for analyzing this problem because they consist of little other than the motile machinery enclosed by a membrane and yet can move with remarkable speed and persistence. We have produced two types of cellular fragments: discoid stationary fragments and polarized fragments undergoing locomotion. The organization and dynamics of the actin-myosin II system were isotropic in stationary fragments and anisotropic in the moving fragments. To investigate whether the creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin-myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to undergo locomotion and the locomotion and associated anisotropic organization of the actin-myosin II system were sustained after withdrawal of the stimulus. We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.

Structural basis for the regulation of muscle contraction by troponin and tropomyosin.

PubMed

Galińska-Rakoczy, Agnieszka; Engel, Patti; Xu, Chen; Jung, Hyunsuk; Craig, Roger; Tobacman, Larry S; Lehman, William

2008-06-20

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca2+ binding to TnC (the Ca2+ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the "inhibitory" subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.

Early stages of the recovery stroke in myosin II studied by molecular dynamics simulations

PubMed Central

Baumketner, Andrij; Nesmelov, Yuri

2011-01-01

The recovery stroke is a key step in the functional cycle of muscle motor protein myosin, during which pre-recovery conformation of the protein is changed into the active post-recovery conformation, ready to exersice force. We study the microscopic details of this transition using molecular dynamics simulations of atomistic models in implicit and explicit solvent. In more than 2 μs of aggregate simulation time, we uncover evidence that the recovery stroke is a two-step process consisting of two stages separated by a time delay. In our simulations, we directly observe the first stage at which switch II loop closes in the presence of adenosine triphosphate at the nucleotide binding site. The resulting configuration of the nucleotide binding site is identical to that detected experimentally. Distribution of inter-residue distances measured in the force generating region of myosin is in good agreement with the experimental data. The second stage of the recovery stroke structural transition, rotation of the converter domain, was not observed in our simulations. Apparently it occurs on a longer time scale. We suggest that the two parts of the recovery stroke need to be studied using separate computational models. PMID:21922589

Model of myosin node aggregation into a contractile ring: the effect of local alignment

NASA Astrophysics Data System (ADS)

Ojkic, Nikola; Wu, Jian-Qiu; Vavylonis, Dimitrios

2011-09-01

Actomyosin bundles frequently form through aggregation of membrane-bound myosin clusters. One such example is the formation of the contractile ring in fission yeast from a broad band of cortical nodes. Nodes are macromolecular complexes containing several dozens of myosin-II molecules and a few formin dimers. The condensation of a broad band of nodes into the contractile ring has been previously described by a search, capture, pull and release (SCPR) model. In SCPR, a random search process mediated by actin filaments nucleated by formins leads to transient actomyosin connections among nodes that pull one another into a ring. The SCPR model reproduces the transport of nodes over long distances and predicts observed clump-formation instabilities in mutants. However, the model does not generate transient linear elements and meshwork structures as observed in some wild-type and mutant cells during ring assembly. As a minimal model of node alignment, we added short-range aligning forces to the SCPR model representing currently unresolved mechanisms that may involve structural components, cross-linking and bundling proteins. We studied the effect of the local node alignment mechanism on ring formation numerically. We varied the new parameters and found viable rings for a realistic range of values. Morphologically, transient structures that form during ring assembly resemble those observed in experiments with wild-type and cdc25-22 cells. Our work supports a hierarchical process of ring self-organization involving components drawn together from distant parts of the cell followed by progressive stabilization.

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

PubMed

Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I; Geeves, Michael A

2016-01-22

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Myosin filament activation in the heart is tuned to the mechanical task

PubMed Central

Reconditi, Massimo; Caremani, Marco; Pinzauti, Francesca; Powers, Joseph D.; Narayanan, Theyencheri; Stienen, Ger J. M.; Linari, Marco; Lombardi, Vincenzo

2017-01-01

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank–Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank–Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer–nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole–systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank–Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors. PMID:28265101

An overview and online registry of microvillus inclusion disease patients and their MYO5B mutations.

PubMed

van der Velde, K Joeri; Dhekne, Herschel S; Swertz, Morris A; Sirigu, Serena; Ropars, Virginie; Vinke, Petra C; Rengaw, Trebor; van den Akker, Peter C; Rings, Edmond H H M; Houdusse, Anne; van Ijzendoorn, Sven C D

2013-12-01

Microvillus inclusion disease (MVID) is one of the most severe congenital intestinal disorders and is characterized by neonatal secretory diarrhea and the inability to absorb nutrients from the intestinal lumen. MVID is associated with patient-, family-, and ancestry-unique mutations in the MYO5B gene, encoding the actin-based motor protein myosin Vb. Here, we review the MYO5B gene and all currently known MYO5B mutations and for the first time methodologically categorize these with regard to functional protein domains and recurrence in MYO7A associated with Usher syndrome and other myosins. We also review animal models for MVID and the latest data on functional studies related to the myosin Vb protein. To congregate existing and future information on MVID geno-/phenotypes and facilitate its quick and easy sharing among clinicians and researchers, we have constructed an online MOLGENIS-based international patient registry (www.MVID-central.org). This easily accessible database currently contains detailed information of 137 MVID patients together with reported clinical/phenotypic details and 41 unique MYO5B mutations, of which several unpublished. The future expansion and prospective nature of this registry is expected to improve disease diagnosis, prognosis, and genetic counseling. © 2013 WILEY PERIODICALS, INC.

Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays

PubMed Central

Scheid, Lisa-Mareike; Weber, Cornelia; Bopp, Nasrin; Mosqueira, Matias; Fink, Rainer H. A.

2017-01-01

The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish—or other origin—and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models. PMID:28620318

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

PubMed

Kampourakis, Thomas; Zhang, Xuemeng; Sun, Yin-Biao; Irving, Malcolm

2018-01-01

Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament

PubMed Central

Kampourakis, Thomas; Zhang, Xuemeng; Sun, Yin‐Biao

2017-01-01

Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.Thick filament regulation is a promising target for novel therapeutics in heart disease. Abstract Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. PMID:29052230

Toluene Dose-Response and Preliminary Study of Proteomics for Neuronal Cell Lines

DTIC Science & Technology

2015-07-01

related to oxidative stress such as energy reserve metabolism, cell -death signaling, reactive oxygen species (ROS) defense, cytoskeletal rearrangement...protein nodes related to oxidative stress as characterized by gene ontologies for energy reserve metabolism, cell -death signaling, reactive oxygen ...process Myosin I complex myofibril assembly Cytoskeletal matrix assembly DNA methyltransferase Activity Cellular ketone Metabolic process Mesenchymal stem

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells.

PubMed

Janson, L W; Taylor, D L

1993-10-01

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.

Sarcomeric Pattern Formation by Actin Cluster Coalescence

PubMed Central

Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

2012-01-01

Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals

PubMed Central

Milberg, Oleg; Shitara, Akiko; Ebrahim, Seham; Tora, Muhibullah; Tran, Duy T.; Chen, Yun; Conti, Mary Anne; Ten Hagen, Kelly G.

2017-01-01

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules. PMID:28600434

Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

PubMed

Lee, Eunhee; Stafford, Walter F

2015-01-01

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

PubMed

Okubo, Hiroyuki; Iwai, Masanori; Iwai, Sosuke; Chaen, Shigeru

2010-05-28

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Betaine protects urea-induced denaturation of myosin subfragment-1.

PubMed

Ortiz-Costa, Susana; Sorenson, Martha M; Sola-Penna, Mauro

2008-07-01

We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous portion. The inhibition of catalytic activity was counteracted by methylamines. With the aim of comprehending the effects of urea on the catalytic (globular) portion of myosin, this study examines the effects of urea and the countereffects of betaine on the catalytic activity and structure of myosin subfragment-1. It is shown that urea inactivates subfragment-1 in parallel with its ability to induce exposure of the enzyme hydrophobic domains, as assessed using intrinsic and extrinsic fluorescence. Both effects are counteracted by betaine, which alone does not significantly affect subfragment-1. Urea also enhances the accessibility of thiol groups, promotes aggregation and decreases the alpha-helix content of S1, effects that are also counteracted by betaine. We conclude that urea-induced inactivation of the enzyme is caused by partial unfolding of the myosin catalytic domain.

The juxtamembrane domain of the E-cadherin cytoplasmic tail contributes to its interaction with Myosin VI

PubMed Central

Mangold, Sabine; Norwood, Suzanne J.; Yap, Alpha S.; Collins, Brett M.

2012-01-01

We recently identified the atypical myosin, Myosin VI, as a component of epithelial cell-cell junctions that interacts with E-cadherin. Recombinant proteins bearing the cargo-binding domain of Myosin VI (Myo VI-CBD) or the cytoplasmic tail of E-cadherin can interact directly with one another. In this report we further investigate the molecular requirements of the interaction between Myo VI-CBD and E-cadherin combining truncation mutation analysis with in vitro binding assays. We report that a short (28 amino acid) juxtamembrane region of the cadherin cytoplasmic tail is sufficient to bind Myo VI-CBD. However, central regions of the cadherin tail adjacent to the juxtamembrane sequence also display binding activity for Myo VI-CBD. It is therefore possible that the cadherin tail bears two binding sites for Myosin VI, or an extended binding site that includes the juxtamembrane region. Nevertheless, our biochemical data highlight the capacity for the juxtamembrane region to interact with functionally-significant cytoplasmic proteins. PMID:23007415

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor.

PubMed

Takano, Mitsunori; Terada, Tomoki P; Sasai, Masaki

2010-04-27

The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor.

Using electrical and optical tweezers to facilitate studies of molecular motors.

PubMed

Arsenault, Mark E; Sun, Yujie; Bau, Haim H; Goldman, Yale E

2009-06-28

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.

Using electrical and optical tweezers to facilitate studies of molecular motors†

PubMed Central

Arsenault, Mark E.; Sun, Yujie; Bau, Haim H.; Goldman, Yale E.

2013-01-01

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells. PMID:19506758

Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects.

PubMed

Patterson, B; Ruppel, K M; Wu, Y; Spudich, J A

1997-10-31

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

Optical trapping studies of acto-myosin motor proteins

NASA Astrophysics Data System (ADS)

Farrow, Rachel E.; Rosenthal, Peter B.; Mashanov, Gregory I.; Holder, Anthony A.; Molloy, Justin E.

2007-09-01

Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical work. This paper focuses on studies of the acto-myosin motor system that is responsible for muscle contraction and a host of other cellular motilities. Myosin works by binding to filamentous actin, pulling and then releasing. Each cycle of interaction produces a few nanometres movement and a few piconewtons force. Individual interactions can be observed directly by holding an individual actin filament between two optically trapped microspheres and positioning it in the immediate vicinity of a single myosin motor. When the chemical fuel (adenosine triphosphate or ATP) is present the myosin undergoes repeated cycles of interaction with the actin filament producing square-wave like displacements and forces. Analysis of optical trapping data sets enables the size and timing of the molecular motions to be deduced.

Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

PubMed Central

Weng, Mo

2016-01-01

Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT. PMID:26754645

The UNC-45 Myosin Chaperone: From Worms to Flies to Vertebrates

PubMed Central

Lee, Chi F.; Melkani, Girish C.; Bernstein, Sanford I.

2014-01-01

UNC-45 is a UCS domain protein that is critical for myosin stability and function. It likely aides in folding myosin during cellular differentiation and maintenance and protects myosin from denaturation during stress. Invertebrates have a single unc-45 gene that is expressed in both muscle and non-muscle tissues. Vertebrates possess one gene expressed in striated muscle (unc-45b) and one that is more generally expressed (unc-45a). Structurally, UNC-45 is composed of a series of alpha-helices connected by loops. It has an N-terminal TPR domain that binds to Hsp90 and a central domain composed of armadillo repeats. Its C-terminal UCS domain, which is also comprised of helical armadillo repeats, interacts with myosin. In this review, we present biochemical, structural and genetic analyses of UNC-45 in Caenorhabditis elegans, Drosophila melanogaster and various vertebrates. Further, we provide insights into UNC-45 functions, its potential mechanism of action and its roles in human disease. PMID:25376491

Structure of MyTH4-FERM domains in myosin VIIa tail bound to cargo.

PubMed

Wu, Lin; Pan, Lifeng; Wei, Zhiyi; Zhang, Mingjie

2011-02-11

The unconventional myosin VIIa (MYO7A) is one of the five proteins that form a network of complexes involved in formation of stereocilia. Defects in these proteins cause syndromic deaf-blindness in humans [Usher syndrome I (USH1)]. Many disease-causing mutations occur in myosin tail homology 4-protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) domains in the myosin tail that binds to another USH1 protein, Sans. We report the crystal structure of MYO7A MyTH4-FERM domains in complex with the central domain (CEN) of Sans at 2.8 angstrom resolution. The MyTH4 and FERM domains form an integral structural and functional supramodule binding to two highly conserved segments (CEN1 and 2) of Sans. The MyTH4-FERM/CEN complex structure provides mechanistic explanations for known deafness-causing mutations in MYO7A MyTH4-FERM. The structure will also facilitate mechanistic and functional studies of MyTH4-FERM domains in other myosins.

Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony

2010-05-28

UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for themore » first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.« less

Intramuscular variations of proteome and muscle fiber type distribution in semimembranosus and semitendinosus muscles associated with pork quality.

PubMed

Kim, Gap-Don; Yang, Han-Sul; Jeong, Jin-Yeon

2018-04-01

Proteome analysis was performed to understand intramuscular variations in muscle fiber distribution in semimembranosus (SM) and semitendinosus (ST) muscles associated with pork quality. Fifteen SM and ST muscles were separated into dark and light portions. The relative area of oxidative fiber was higher (P < .0001) in dark portion than that in light portion, while glycolytic fiber types were distributed primarily (P < .01) in light portions regardless of muscle types. Myosin-1, myosin-4, troponin complex (fast), myosin light chains, and metabolic enzymes responsible for fast-twitch glycolytic types were overexpressed in light portions (P < .05). However, myosin-2, myosin-7, myoglobin, and mitochondrial oxidative metabolic enzymes were closely related to slow-twitch oxidative fibers. These resulted in high pH, redness, and tenderness but low lightness and drip loss of pork quality. In conclusion, differentially expressed muscle proteins are associated with fiber type (oxidative vs. glycolytic) distribution, resulting in intramuscular variations of pork quality. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antenna Mechanism of Length Control of Actin Cables

PubMed Central

Mohapatra, Lishibanya; Goode, Bruce L.; Kondev, Jane

2015-01-01

Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This “antenna mechanism” involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control. PMID:26107518

Antenna Mechanism of Length Control of Actin Cables.

PubMed

Mohapatra, Lishibanya; Goode, Bruce L; Kondev, Jane

2015-06-01

Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This "antenna mechanism" involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

PubMed

O'Loughlin, Thomas; Masters, Thomas A; Buss, Folma

2018-04-01

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine-tune motor activity in time and space. These motor-adaptor-cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling-based proteomics strategy to map the interactome of the unique minus end-directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6-adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG-Induced F-actin for Tethering) complex that controls endosome positioning and motility through RHO-driven actin polymerisation; and the DISP (DOCK7-Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Force generation within tissues during development

NASA Astrophysics Data System (ADS)

Kasza, Karen

During embryonic development, multicellular tissues physically change shape, move, and grow. Changes in epithelial tissue organization are often accomplished by local movements of cells that are driven largely by forces generated by the motor protein myosin II. These forces are patterned to orient cell movements, resulting in changes in tissue shape and organization to build functional tissues and organs. To investigate the mechanisms of force generation in vivo, we use the fruit fly embryo as a model system. Spatial patterns of forces orient cell movements to drive rapid tissue elongation along the head-to-tail axis of the embryo. I will describe how studying embryos generated with engineered myosin variants provides insight into where, when, and how forces are generated to efficiently reorganize tissues. We found that a myosin variant that is locked-in to the active or ``on'' state accelerates cell movements, while two mutant myosin variants associated with human disease produce slowed cell movement. These myosin variants all disrupt tissue elongation, but live imaging and biophysical measurements reveal distinct effects on myosin organization and dynamics within cells and uncover mechanisms that control the spatial and temporal patterns of force generation. These studies shed light not only on how defects in force generation contribute to disease but also on physical principles at work in active, living materials.

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation.

PubMed

Miyake, Yuka; Inoue, Naoko; Nishimura, Koji; Kinoshita, Nagatoki; Hosoya, Hiroshi; Yonemura, Shigenobu

2006-05-15

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.

In the Thick of It: HCM-Causing Mutations in Myosin Binding Proteins of the Thick Filament

PubMed Central

Harris, Samantha P.; Lyons, Ross G.; Bezold, Kristina L.

2010-01-01

In the 20 yrs since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM) an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins including the myosin essential and regulatory light chains and cardiac myosin binding protein-C (cMyBP-C). However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes. PMID:21415409

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres.

PubMed

Avrova, Stanislava V; Rysev, Nikita A; Matusovsky, Oleg S; Shelud'ko, Nikolay S; Borovikov, Yurii S

2012-05-01

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin. Copyright © 2012 Elsevier Inc. All rights reserved.

A free-boundary model of a motile cell explains turning behavior.

PubMed

Nickaeen, Masoud; Novak, Igor L; Pulford, Stephanie; Rumack, Aaron; Brandon, Jamie; Slepchenko, Boris M; Mogilner, Alex

2017-11-01

To understand shapes and movements of cells undergoing lamellipodial motility, we systematically explore minimal free-boundary models of actin-myosin contractility consisting of the force-balance and myosin transport equations. The models account for isotropic contraction proportional to myosin density, viscous stresses in the actin network, and constant-strength viscous-like adhesion. The contraction generates a spatially graded centripetal actin flow, which in turn reinforces the contraction via myosin redistribution and causes retraction of the lamellipodial boundary. Actin protrusion at the boundary counters the retraction, and the balance of the protrusion and retraction shapes the lamellipodium. The model analysis shows that initiation of motility critically depends on three dimensionless parameter combinations, which represent myosin-dependent contractility, a characteristic viscosity-adhesion length, and a rate of actin protrusion. When the contractility is sufficiently strong, cells break symmetry and move steadily along either straight or circular trajectories, and the motile behavior is sensitive to conditions at the cell boundary. Scanning of a model parameter space shows that the contractile mechanism of motility supports robust cell turning in conditions where short viscosity-adhesion lengths and fast protrusion cause an accumulation of myosin in a small region at the cell rear, destabilizing the axial symmetry of a moving cell.

Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

PubMed Central

Samant, Sadhana A.; Pillai, Vinodkumar B.; Sundaresan, Nagalingam R.; Shroff, Sanjeev G.; Gupta, Mahesh P.

2015-01-01

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:25911107

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

PubMed

He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

2015-01-01

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization

PubMed Central

He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

2015-01-01

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation. PMID:26562659

Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration

PubMed Central

Croft, Daniel R.; Coleman, Mathew L.; Li, Shuixing; Robertson, David; Sullivan, Teresa; Stewart, Colin L.; Olson, Michael F.

2005-01-01

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization. PMID:15657395

Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin, myosin, desmin, alpha-actinin, troponin and titin.

PubMed

Costa, Manoel L; Escaleira, Roberta C; Rodrigues, Viviane B; Manasfi, Muhamed; Mermelstein, Claudia S

2002-08-01

The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.

Structural Characterization of the Binding of Myosin*ADP*Pi to Actin in Permeabilized Rabbit Psoas Muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu,S.; Gu, J.; Belknap, B.

2006-01-01

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A{center_dot}M{center_dot}ADP and A{center_dot}M) and the weakly bound A{center_dot}M{center_dot}ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ('stereospecific' attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A{center_dot}M{center_dot}ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A{center_dot}M{center_dot}ADP{center_dot}P{sub i}, however, is poorly understood. Thismore » state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M{center_dot}ATP, M{center_dot}ADP{center_dot}P{sub i} states and the weakly attached A{center_dot}M{center_dot}ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A{center_dot}M{center_dot}ADP{center_dot}P{sub i}. The series of experiments presented in this article were carried out under relaxing conditions at 25{sup o}C, where {approx}95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M{center_dot}ADP{center_dot}P{sub i} with strongly coupled domains may contribute to the unique attachment configuration: the 'primed' myosin heads may function as 'transient struts' when attached to the thin filaments.« less

Assembly and mechanosensory function of focal adhesions: experiments and models.

PubMed

Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

2006-04-01

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

The unit event of sliding of the chemo-mechanical enzyme composed of myosin and actin with regulatory proteins.

PubMed

Oosawa, Fumio

2008-04-25

Various myosin-actin systems do not always show the same sliding behaviors. To make the situation clear, discussions are concentrated on the unit event of sliding of the chemo-mechanical enzyme composed of a single myosin head and a single actin filament with regulatory proteins. The popular idea of the one-to-one correspondence between the chemical state and the physical state or between the chemical reaction step and the physical conformational change is reexamined. It is likely that the sites and the modes of interaction between myosin head and actin filament during the ATP hydrolysis are more multiple and variable, and the input-output coupling in the chemo-mechanical enzyme is loose.

Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

PubMed Central

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Background Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the “cytoplasmic” myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. Methodology/Principal Findings We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. Conclusions/Significance We have shown that the novel specific NLS brings to the cell nucleus not only the “nuclear” isoform of myosin I (NM1 protein) but also its “cytoplasmic” isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus. PMID:22295092

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age.

PubMed

Gannon, Joan; Doran, Philip; Kirwan, Anne; Ohlendieck, Kay

2009-11-01

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

Loss of zebrafish Smyd1a interferes with myofibrillar integrity without triggering the misfolded myosin response.

PubMed

Paone, Christoph; Rudeck, Steven; Etard, Christelle; Strähle, Uwe; Rottbauer, Wolfgang; Just, Steffen

2018-02-05

Sarcomeric protein turnover needs to be tightly balanced to assure proper assembly and renewal of sarcomeric units within muscle tissues. The mechanisms regulating these fundamental processes are only poorly understood, but of great clinical importance since many cardiac and skeletal muscle diseases are associated with defective sarcomeric organization. The SET- and MYND domain containing protein 1b (Smyd1b) is known to play a crucial role in myofibrillogenesis by functionally interacting with the myosin chaperones Unc45b and Hsp90α1. In zebrafish, Smyd1b, Unc45b and Hsp90α1 are part of the misfolded myosin response (MMR), a regulatory transcriptional response that is activated by disturbed myosin homeostasis. Genome duplication in zebrafish led to a second smyd1 gene, termed smyd1a. Morpholino- and CRISPR/Cas9-mediated knockdown of smyd1a led to significant perturbations in sarcomere structure resulting in decreased cardiac as well as skeletal muscle function. Similar to Smyd1b, we found Smyd1a to localize to the sarcomeric M-band in skeletal and cardiac muscles. Overexpression of smyd1a efficiently compensated for the loss of Smyd1b in flatline (fla) mutant zebrafish embryos, rescued the myopathic phenotype and suppressed the MMR in Smyd1b-deficient embryos, suggesting overlapping functions of both Smyd1 paralogs. Interestingly, Smyd1a is not transcriptionally activated in Smyd1b-deficient fla mutants, demonstrating lack of genetic compensation despite the functional redundancy of both zebrafish Smyd1 paralogs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Wavelet Imaging on Multiple Scales (WIMS) reveals focal adhesion distributions, dynamics and coupling between actomyosin bundle stability

PubMed Central

Toplak, Tim; Palmieri, Benoit; Juanes-García, Alba; Vicente-Manzanares, Miguel; Grant, Martin; Wiseman, Paul W.

2017-01-01

We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales. PMID:29049414

Myocardial infarction triggers chronic cardiac autoimmunity in type 1 diabetes.

PubMed

Gottumukkala, Raju V S R K; Lv, HuiJuan; Cornivelli, Lizbeth; Wagers, Amy J; Kwong, Raymond Y; Bronson, Roderick; Stewart, Garrick C; Schulze, P Christian; Chutkow, William; Wolpert, Howard A; Lee, Richard T; Lipes, Myra A

2012-06-13

Patients with type 1 diabetes (T1D) suffer excessive morbidity and mortality after myocardial infarction (MI) that is not fully explained by the metabolic effects of diabetes. Acute MI is known to trigger a profound innate inflammatory response with influx of mononuclear cells and production of proinflammatory cytokines that are crucial for cardiac repair. We hypothesized that these same pathways might exert "adjuvant effects" and induce pathological responses in autoimmune-prone T1D hosts. Here, we show that experimental MI in nonobese diabetic mice, but not in control C57BL/6 mice, results in a severe post-infarction autoimmune (PIA) syndrome characterized by destructive lymphocytic infiltrates in the myocardium, infarct expansion, sustained cardiac autoantibody production, and T helper type 1 effector cell responses against cardiac (α-)myosin. PIA was prevented by inducing tolerance to α-myosin, demonstrating that immune responses to cardiac myosin are essential for this disease process. Extending these findings to humans, we developed a panel of immunoassays for cardiac autoantibody detection and found autoantibody positivity in 83% post-MI T1D patients. We further identified shared cardiac myosin autoantibody signatures between post-MI T1D patients and nondiabetic patients with myocarditis, which were absent in post-MI type 2 diabetic patients, and confirmed the presence of myocarditis in T1D by cardiac magnetic resonance imaging techniques. These data provide experimental and clinical evidence for a distinct post-MI autoimmune syndrome in T1D. Our findings suggest that PIA may contribute to worsened post-MI outcomes in T1D and highlight a role for antigen-specific immunointervention to selectively block this pathway.

Second harmonic generation imaging of skeletal muscle tissue and myofibrils

NASA Astrophysics Data System (ADS)

Campagnola, Paul J.; Mohler, William H.; Plotnikov, Sergey; Millard, Andrew C.

2006-02-01

Second Harmonic Generation (SHG) imaging microscopy is used to examine the morphology and structural properties of intact muscle tissue. Using biochemical and optical analysis, we characterize the molecular structure underlying SHG from the complex muscle sarcomere. We find that SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. We thus determined that the SHG emission arises from domains of the sarcomere containing thick filaments. By fitting the SHG polarization anisotropy to theoretical response curves, we find an orientation for the harmonophore that corresponds well to the pitch angle of the myosin rod α-helix with respect to the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving rise to SHG from striated muscle. Using SHG imaging microscopy, we have also examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5 fold increase in achievable SHG imaging depth. Fast Fourier Transform (FFT) analysis shows quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process. Also, comparison of the SHG angular polarization dependence shows no change in the supramolecular organization of acto-myosin complexes. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The pronounced lack of dependence of glycerol concentration on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle.

A novel quantitative morphometry approach to assess regeneration in dystrophic skeletal muscle.

PubMed

Buttgereit, Andreas; Weber, Cornelia; Friedrich, Oliver

2014-07-01

Duchenne muscular dystrophy is an inherited degenerative muscle disease with progressive weakness of skeletal and cardiac muscle. Disturbed calcium homeostasis and signalling pathways result in degeneration/regeneration cycles with fibrotic remodelling of muscle tissue, sustained by chronic inflammation. In addition to altered microarchitecture, regeneration in dystrophic muscle fibres is often only classified by centrally located nuclei but correlation of the regeneration process to nuclear volumes, myosin amounts, architecture and functional quality are missing, in particular in old muscles where the regenerative capacity is exhausted. Such information could yield novel regeneration-to-function biomarkers. Here we used second harmonic generation and multi photon fluorescence microscopy in intact single muscle fibres from wild-type, dystrophic mdx and transgenic mdx mice expressing an Δex 17-48 mini-dystrophin to determine the percentage of centronucleated fibres and nucleus-to-myosin volume ratio as a function of age. Based on this ratio we define a 'biomotoric efficiency' as an optical measure for fibre maturation, which is close to unity in adult wild-type and mini-dystrophin fibres, but smaller in very young and old mdx mice as a result of ongoing cell maturation (young) and regeneration (aged). With these parameters it is possible to provide a quantitative measure about muscle fibre regeneration. Copyright © 2014 Elsevier B.V. All rights reserved.

Thermal gelation profile changes in reconstituted actomyosin due to storage under a high salt concentration and low temperature.

PubMed

Tanji, H; Ikeuchi, Y; Yoshizawa, M; Suzuki, A

1997-05-01

Changes in the heat-induced gelation properties of reconstituted rabbit skeletal actomyosin stored under a high salt concentration at pH 6.0 and 0 degree C were investigated at different weight ratios of actin to myosin by using dynamic rheological and biochemical measurements. The addition of actin resulted in a pronounced peak maximum at about 50 degrees C and an accompanying temporary reduction in the range at about 50 degrees C to 60 degrees C. The more the initial actin concentration was increased, the greater was the area of the peak/shoulder. However, this area was markedly diminished with increasing storage time. As a result, the dynamic rheological pattern was transformed from an actomyosin type into a myosin type. The relationship between the G' value at 80 degrees C and the actin/myosin weight ratio was curvilinear, with a peak at the ratio of 0.05, immediately after storage was started. This profile changed during storage, depending on the extent to denaturation of actin and myosin in the reconstituted actomyosin (RAM). The G' value of actomyosin in 0.5 M KCl with a small actin/myosin ratio of 0.05 decreased to one-half of its initial value after 7 days of storage, whereas the G' value with a large actin/myosin ratio of 0.225 increased by about 1.6 times. In 1.5 M KCl, all the G' values declined to the level with myosin alone after 7 days of storage. The time-course plots of the remaining actin concentration in RAM at different weight ratios of actin to myosin after being treated with 0.5 M or 1.5 M KCl showed a decrease in the actin content with increasing storage time, and an increase in the KCl concentration to 1.5 M KCl promoted the denaturation of actin in RAM faster than with 0.5 M KCl. The surface hydrophobicity of each RAM sample progressively increased with increasing storage time, while little significant increase in the sulfhydryl (SH) content during storage was observed. It is concluded that changes in the heat-induced gelation properties of actomyosin during storage are largely attributable to the denaturation of actin rather than to the denaturation of myosin or to quantitative changes in the SH content and hydrophobicity.

Myosin II-interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane.

PubMed

Jiao, Meng; Wu, Di; Wei, Qize

2018-03-01

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II-interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction. © 2018 Jiao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

PubMed Central

Karatzaferi, Christina; Adamek, Nancy

2017-01-01

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue. PMID:28931538

A new model for the surface arrangement of myosin molecules in tarantula thick filaments.

PubMed

Offer, G; Knight, P J; Burgess, S A; Alamo, L; Padrón, R

2000-04-28

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head. We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing. Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail. Copyright 2000 Academic Press.

Mutation of the myosin converter domain alters cross-bridge elasticity

PubMed Central

Köhler, Jan; Winkler, Gerhard; Schulte, Imke; Scholz, Tim; McKenna, William; Brenner, Bernhard; Kraft, Theresia

2002-01-01

Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the β-myosin heavy chain. We found that the Arg-719 → Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48–59%. Under rigor and relaxing conditions, fiber stiffness was 45–47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself. PMID:11904418

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

PubMed

Hong, Feng; Brizendine, Richard K; Carter, Michael S; Alcala, Diego B; Brown, Avery E; Chattin, Amy M; Haldeman, Brian D; Walsh, Michael P; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

2015-10-01

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. © 2015 Hong et al.

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

PubMed

Duggal, D; Nagwekar, J; Rich, R; Huang, W; Midde, K; Fudala, R; Das, H; Gryczynski, I; Szczesna-Cordary, D; Borejdo, J

2015-05-15

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC. Copyright © 2015 the American Physiological Society.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

DOE PAGES

Taylor, Keenan C.; Buvoli, Massimo; Korkmaz, Elif Nihal; ...

2015-07-06

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report in this paper that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility.more » Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. Finally, in addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.« less

Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function.

PubMed

Schmitt, Joachim P; Debold, Edward P; Ahmad, Ferhaan; Armstrong, Amy; Frederico, Andrea; Conner, David A; Mende, Ulrike; Lohse, Martin J; Warshaw, David; Seidman, Christine E; Seidman, J G

2006-09-26

Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the alpha-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin (V(actin)) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower V(actin) in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated.

The R403Q Myosin Mutation Implicated in Familial Hypertrophic Cardiomyopathy Causes Disorder at the Actomyosin Interface

PubMed Central

Volkmann, Niels; Lui, HongJun; Hazelwood, Larnele; Trybus, Kathleen M.; Lowey, Susan; Hanein, Dorit

2007-01-01

Background Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the β-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype. Principal Findings Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow β-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface. Significance These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state. PMID:17987111

Eccentric contraction-induced injury to type I, IIa, and IIa/IIx muscle fibers of elderly adults

USDA-ARS?s Scientific Manuscript database

Muscles of old laboratory rodents experience exaggerated force losses after eccentric contractile activity. We extended this line of inquiry to humans and investigated the influence of fiber myosin heavy chain (MHC) isoform content on the injury process. Skinned muscle fiber segments, prepared from ...

Mitosis-Specific Mechanosensing and Contractile Protein Redistribution Control Cell Shape

PubMed Central

Effler, Janet C.; Kee, Yee-Seir; Berk, Jason M.; Tran, Minhchau N.; Iglesias, Pablo A.; Robinson, Douglas N.

2008-01-01

Summary Because cell division failure is deleterious, promoting tumorigenesis in mammals [1], cells utilize numerous mechanisms to control their cell-cycle progression [2–4]. Though cell division is considered a well-ordered sequence of biochemical events [5], cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Since cells respond to their mechanical environment [6, 7], we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin-II are vital to cell division [8, 9], we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins, myosin-II and cortexillin-I, redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, is independent of spindle orientation, but is dependent on myosin-II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis. PMID:17027494

Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?

NASA Astrophysics Data System (ADS)

Shi, Yunfei; Varner, Victor D.; Taber, Larry A.

2015-02-01

Cytoskeletal contraction is crucial to numerous morphogenetic processes, but its role in early heart development is poorly understood. Studies in chick embryos have shown that inhibiting myosin-II-based contraction prior to Hamburger-Hamilton (HH) stage 10 (33 h incubation) impedes fusion of the mesodermal heart fields that create the primitive heart tube (HT), as well as the ensuing process of cardiac looping. If contraction is inhibited at or after looping begins at HH10, however, fusion and looping proceed relatively normally. To explore the mechanisms behind this seemingly fundamental change in behavior, we measured spatiotemporal distributions of tissue stiffness, stress, and strain around the anterior intestinal portal (AIP), the opening to the foregut where contraction and cardiac fusion occur. The results indicate that stiffness and tangential tension decreased bilaterally along the AIP with distance from the embryonic midline. The gradients in stiffness and tension, as well as strain rate, increased to peaks at HH9 (30 h) and decreased afterward. Exposure to the myosin II inhibitor blebbistatin reduced these effects, suggesting that they are mainly generated by active cytoskeletal contraction, and finite-element modeling indicates that the measured mechanical gradients are consistent with a relatively uniform contraction of the endodermal layer in conjunction with constraints imposed by the attached mesoderm. Taken together, our results suggest that, before HH10, endodermal contraction pulls the bilateral heart fields toward the midline where they fuse to create the HT. By HH10, however, the fusion process is far enough along to enable apposing cardiac progenitor cells to keep ‘zipping’ together during looping without the need for continued high contractile forces. These findings should shed new light on a perplexing question in early heart development.

Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

PubMed Central

Gabriele, Sylvain; Benoliel, Anne-Marie; Bongrand, Pierre; Théodoly, Olivier

2009-01-01

Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments. PMID:19450501

Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

PubMed

Chopra, Anant; Kutys, Matthew L; Zhang, Kehan; Polacheck, William J; Sheng, Calvin C; Luu, Rebeccah J; Eyckmans, Jeroen; Hinson, J Travis; Seidman, Jonathan G; Seidman, Christine E; Chen, Christopher S

2018-01-08

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Force Generation by Membrane-Associated Myosin-I

PubMed Central

Pyrpassopoulos, Serapion; Arpağ, Göker; Feeser, Elizabeth A.; Shuman, Henry; Tüzel, Erkan; Ostap, E. Michael

2016-01-01

Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions. PMID:27156719

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

PubMed Central

Takano, Mitsunori; Terada, Tomoki P.; Sasai, Masaki

2010-01-01

The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor. PMID:20385833

Model of myosin recruitment to the cell equator for cytokinesis: feedback mechanisms and dynamical regimes

NASA Astrophysics Data System (ADS)

Veksler, Alexander; Vavylonis, Dimitrios

2011-03-01

The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During animal cell cytokinesis, cortical myosin filaments (MF) disassemble at the flanking regions and concentrate in the equator. This recruitment depends on myosin motor activity and the Rho proteins that regulate MF assembly and disassembly. Central spindle and astral microtubules help establish a spatial pattern of differential Rho activity. We propose a reaction-diffusion model for the dynamics of MF recruitment to the equatorial region. In the model, the central spindle and mechanical stress promote self-reinforcing MF assembly. Negative feedback is introduced by MF-induced recruitment of inhibitor myosin phosphatase. Our model yields various dynamical regimes and explains both the recruitment of MF to the cleavage furrow and the observed damped MF oscillations in the flanking regions, as well as steady MF assembly. Space and time parameters of MF oscillations are calculated. We predict oscillatory relaxation of cortical MF upon removal of locally-applied external stress.

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

PubMed

Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

2015-10-16

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture

PubMed Central

Kubis, Hans-Peter; Haller, Ernst-August; Wetzel, Petra; Gros, Gerolf

1997-01-01

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. PMID:9108130

Leveraging the membrane-cytoskeleton interface with myosin-1

PubMed Central

McConnell, Russell E.; Tyska, Matthew J.

2010-01-01

Class 1 myosins are small motor proteins with the ability to simultaneously bind to actin filaments and cellular membranes. Given their ability to generate mechanical force, and their high prevalence in many cell types, these molecules are well positioned to carry out a number of important biological functions at the interface of membrane and the actin cytoskeleton. Indeed, recent studies implicate these motors in endocytosis, exocytosis, release of extracellular vesicles, and the regulation of tension between membrane and the cytoskeleton. Many class 1 myosins also exhibit a load-dependent mechano-chemical cycle that enables them to maintain tension for long periods of time without hydrolyzing ATP. These properties put myosins-1 in a unique position to regulate dynamic membrane-cytoskeleton interactions and respond to physical forces during these events. PMID:20471271

Thick-Filament Strain and Interfilament Spacing in Passive Muscle: Effect of Titin-Based Passive Tension

PubMed Central

Irving, Thomas; Wu, Yiming; Bekyarova, Tanya; Farman, Gerrie P.; Fukuda, Norio; Granzier, Henk

2011-01-01

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d1,0) and Z-disk (dZ) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I1,1/I1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I1,1/I1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed. PMID:21402032

Cortical Actomyosin Breakage Triggers Shape Oscillations in Cells and Cell Fragments

PubMed Central

Paluch, Ewa; Piel, Matthieu; Prost, Jacques; Bornens, Michel; Sykes, Cécile

2005-01-01

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization. PMID:15879479

Increased myocardial short-range forces in a rodent model of diabetes reflect elevated content of β myosin heavy chain.

PubMed

Chung, Charles S; Mitov, Mihail I; Callahan, Leigh Ann; Campbell, Kenneth S

2014-06-15

Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12week old rats that had been injected with STZ or vehicle 4weeks earlier, and activated in solutions with pCa (=-log10[Ca(2+)]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ∼67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of β myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if β myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes. Copyright © 2013 Elsevier Inc. All rights reserved.

An intermediate along the recovery stroke of myosin VI revealed by X-ray crystallography and molecular dynamics.

PubMed

Blanc, Florian; Isabet, Tatiana; Benisty, Hannah; Sweeney, H Lee; Cecchini, Marco; Houdusse, Anne

2018-06-12

Myosins form a class of actin-based, ATPase motor proteins that mediate important cellular functions such as cargo transport and cell motility. Their functional cycle involves two large-scale swings of the lever arm: the force-generating powerstroke, which takes place on actin, and the recovery stroke during which the lever arm is reprimed into an armed configuration. Previous analyses of the prerecovery (postrigor) and postrecovery (prepowerstroke) states predicted that closure of switch II in the ATP binding site precedes the movement of the converter and the lever arm. Here, we report on a crystal structure of myosin VI, called pretransition state (PTS), which was solved at 2.2 Å resolution. Structural analysis and all-atom molecular dynamics simulations are consistent with PTS being an intermediate along the recovery stroke, where the Relay/SH1 elements adopt a postrecovery conformation, and switch II remains open. In this state, the converter appears to be largely uncoupled from the motor domain and explores an ensemble of partially reprimed configurations through extensive, reversible fluctuations. Moreover, we found that the free energy cost of hydrogen-bonding switch II to ATP is lowered by more than 10 kcal/mol compared with the prerecovery state. These results support the conclusion that closing of switch II does not initiate the recovery stroke transition in myosin VI. Rather, they suggest a mechanism in which lever arm repriming would be mostly driven by thermal fluctuations and eventually stabilized by the switch II interaction with the nucleotide in a ratchet-like fashion.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

PubMed

Alamo, Lorenzo; Koubassova, Natalia; Pinto, Antonio; Gillilan, Richard; Tsaturyan, Andrey; Padrón, Raúl

2017-10-01

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca 2+ -activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP

PubMed Central

Sugi, Haruo; Yamaguchi, Maki; Ohno, Tetsuo; Kobayashi, Takakazu; Chaen, Shigeru; Okuyama, Hiroshi

2016-01-01

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles. PMID:27583360

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP.

PubMed

Sugi, Haruo; Yamaguchi, Maki; Ohno, Tetsuo; Kobayashi, Takakazu; Chaen, Shigeru; Okuyama, Hiroshi

2016-01-01

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles.

Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex

PubMed Central

Sipos, Adrienn; Darula, Zsuzsanna; Bécsi, Bálint; Nagy, Dénes; Iván, Judit; Erdődi, Ferenc

2017-01-01

Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138. PMID:28486561

A calcium-driven mechanochemical model for prediction of force generation in smooth muscle.

PubMed

Murtada, Sae-Il; Kroon, Martin; Holzapfel, Gerhard A

2010-12-01

A new model for the mechanochemical response of smooth muscle is presented. The focus is on the response of the actin-myosin complex and on the related generation of force (or stress). The chemical (kinetic) model describes the cross-bridge interactions with the thin filament in which the calcium-dependent myosin phosphorylation is the only regulatory mechanism. The new mechanical model is based on Hill's three-component model and it includes one internal state variable that describes the contraction/relaxation of the contractile units. It is characterized by a strain-energy function and an evolution law incorporating only a few material parameters with clear physical meaning. The proposed model satisfies the second law of thermodynamics. The results of the combined coupled model are broadly consistent with isometric and isotonic experiments on smooth muscle tissue. The simulations suggest that the matrix in which the actin-myosin complex is embedded does have a viscous property. It is straightforward for implementation into a finite element program in order to solve more complex boundary-value problems such as the control of short-term changes in lumen diameter of arteries due to mechanochemical signals.

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

PubMed

Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

2006-04-01

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

PubMed Central

Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, John; Van Engelenburg, Schuyler B.; Ohi, Ryoma; Burnette, Dylan T.

2016-01-01

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled. PMID:26960797

Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.

2011-11-02

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This resultmore » provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.« less

Determination of the myosin step size from mechanical and kinetic data.

PubMed Central

Pate, E; White, H; Cooke, R

1993-01-01

During muscle contraction, work is generated when a myosin cross-bridge attaches to an actin filament and exerts a force on it through some power-stroke distance, h. At the end of this power stroke, attached myosin heads are carried into regions where they exert a negative force on the actin filament (the drag stroke) and where they are released rapidly from actin by ATP binding. Although the length of the power stroke remains controversial, average distance traversed in the drag-stroke region can be determined when one knows both rate of cross-bridge dissociation and filament-sliding velocity. At maximum contraction velocity, the average force exerted in the drag stroke must balance that exerted in the power stroke. We discuss here a simple model of cross-bridge interaction that allows one to calculate the force exerted in the drag stroke and to relate this to the power-stroke distance h traversed by cross-bridges in the positive-force region. Both the rate at which myosin can be dissociated from actin and the velocity at which an actin filament can be translated have been measured for a series of myosin isozymes and for different substrates, producing a wide range of values for each. Nonetheless, we show here that the rate of myosin dissociation from actin correlates well with the velocity of filament sliding, providing support for the simple model presented and suggesting that the power stroke is approximately 10 nm in length. PMID:8460156

Allosteric modulation of cardiac myosin dynamics by omecamtiv mecarbil

PubMed Central

Tiberti, Matteo

2017-01-01

New promising avenues for the pharmacological treatment of skeletal and heart muscle diseases rely on direct sarcomeric modulators, which are molecules that can directly bind to sarcomeric proteins and either inhibit or enhance their activity. A recent breakthrough has been the discovery of the myosin activator omecamtiv mecarbil (OM), which has been shown to increase the power output of the cardiac muscle and is currently in clinical trials for the treatment of heart failure. While the overall effect of OM on the mechano-chemical cycle of myosin is to increase the fraction of myosin molecules in the sarcomere that are strongly bound to actin, the molecular basis of its action is still not completely clear. We present here a Molecular Dynamics study of the motor domain of human cardiac myosin bound to OM, where the effects of the drug on the dynamical properties of the protein are investigated for the first time with atomistic resolution. We found that OM has a double effect on myosin dynamics, inducing a) an increased coupling of the motions of the converter and lever arm subdomains to the rest of the protein and b) a rewiring of the network of dynamic correlations, which produces preferential communication pathways between the OM binding site and distant functional regions. The location of the residues responsible for these effects suggests possible strategies for the future development of improved drugs and the targeting of specific cardiomyopathy-related mutations. PMID:29108014

Time-resolved microrheology of actively remodeling actomyosin networks

NASA Astrophysics Data System (ADS)

Silva, Marina Soares e.; Stuhrmann, Björn; Betz, Timo; Koenderink, Gijsje H.

2014-07-01

Living cells constitute an extraordinary state of matter since they are inherently out of thermal equilibrium due to internal metabolic processes. Indeed, measurements of particle motion in the cytoplasm of animal cells have revealed clear signatures of nonthermal fluctuations superposed on passive thermal motion. However, it has been difficult to pinpoint the exact molecular origin of this activity. Here, we employ time-resolved microrheology based on particle tracking to measure nonequilibrium fluctuations produced by myosin motor proteins in a minimal model system composed of purified actin filaments and myosin motors. We show that the motors generate spatially heterogeneous contractile fluctuations, which become less frequent with time as a consequence of motor-driven network remodeling. We analyze the particle tracking data on different length scales, combining particle image velocimetry, an ensemble analysis of the particle trajectories, and finally a kymograph analysis of individual particle trajectories to quantify the length and time scales associated with active particle displacements. All analyses show clear signatures of nonequilibrium activity: the particles exhibit random motion with an enhanced amplitude compared to passive samples, and they exhibit sporadic contractile fluctuations with ballistic motion over large (up to 30 μm) distances. This nonequilibrium activity diminishes with sample age, even though the adenosine triphosphate level is held constant. We propose that network coarsening concentrates motors in large clusters and depletes them from the network, thus reducing the occurrence of contractile fluctuations. Our data provide valuable insight into the physical processes underlying stress generation within motor-driven actin networks and the analysis framework may prove useful for future microrheology studies in cells and model organisms.

Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.

PubMed

Ripoll, Léa; Heiligenstein, Xavier; Hurbain, Ilse; Domingues, Lia; Figon, Florent; Petersen, Karl J; Dennis, Megan K; Houdusse, Anne; Marks, Michael S; Raposo, Graça; Delevoye, Cédric

2018-06-06

Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that myosin VI motor activity mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the myosin adaptor optineurin and the activity of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new myosin VI- and actin-dependent membrane fission mechanism required for organelle function. © 2018 Ripoll et al.

The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C

2006-12-01

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by {approx}60 degrees. This recovery stroke is coupled to the activation of myosin's ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a see-saw motionmore » of the relay helix, followed by a piston/seesaw motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery stroke by using Principal Component Analysis. This reveals that the only principal motions of these two helices that make a large amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions.« less

Ca2+-Induced Rigidity Change of the Myosin VIIa IQ Motif-Single α Helix Lever Arm Extension.

PubMed

Li, Jianchao; Chen, Yiyun; Deng, Yisong; Unarta, Ilona Christy; Lu, Qing; Huang, Xuhui; Zhang, Mingjie

2017-04-04

Several unconventional myosins contain a highly charged single α helix (SAH) immediately following the calmodulin (CaM) binding IQ motifs, functioning to extend lever arms of these myosins. How such SAH is connected to the IQ motifs and whether the conformation of the IQ motifs-SAH segments are regulated by Ca 2+ fluctuations are not known. Here, we demonstrate by solving its crystal structure that the predicted SAH of myosin VIIa (Myo7a) forms a stable SAH. The structure of Myo7a IQ5-SAH segment in complex with apo-CaM reveals that the SAH sequence can extend the length of the Myo7a lever arm. Although Ca 2+ -CaM remains bound to IQ5-SAH, the Ca 2+ -induced CaM binding mode change softens the conformation of the IQ5-SAH junction, revealing a Ca 2+ -induced lever arm flexibility change for Myo7a. We further demonstrate that the last IQ motif of several other myosins also binds to both apo- and Ca 2+ -CaM, suggesting a common Ca 2+ -induced conformational regulation mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

A simplified method for identification of human cardiac myosin heavy-chain isoforms.

PubMed

Piao, Shengfu; Yu, Fushun; Mihm, Michael J; Reiser, Peter J; McCarthy, Patrick M; Van Wagoner, David R; Bauer, John Anthony

2003-02-01

Cardiac myosin is a central participant in the cross-bridge cycling that mediates myocyte contraction and consists of multiple subunits that mediate both hydrolysis of ATP and mechanical production of contractile force Two isoforms of myosin heavy chain (MHC- alpha and MHC- beta ) are known to exist in mammalian cardiac tissue, and it is within this myosin subunit that ATPase activity resides. These isoforms differ by less than 0.2% in total molecular mass and amino acid sequence, but, strikingly, influence the rate and efficiency of energy utilization for generation of contractile force. Changes in the MHC- alpha /MHC- beta ratio has been classically viewed as an adaptation of a failing myocyte in both animal models and humans; however, their measurement has traditionally required specialized preparations and materials for sufficient resolution. Here we describe a greatly simplified method for routine assessments of myosin isoform composition in human cardiac tissues. The primary advantages of our approach include higher throughput and reduced supply costs with no apparent loss of statistical power, reproducibility or achieved results. Use of this more convenient method may provide enhanced access to an otherwise specialized technique and could provide additional opportunity for investigation of cardiac myocyte adaptive changes.

Identification and functional study of a new missense mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11).

PubMed

Sang, Qing; Yan, Xukun; Wang, Huan; Feng, Ruizhi; Fei, Xiang; Ma, Duan; Xing, Qinghe; Li, Qiaoli; Zhao, Xinzhi; Jin, Li; He, Lin; Li, Huawei; Wang, Lei

2013-01-01

The MYO7A encodes a protein classified as an unconventional myosin. Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles other previously published DFNA11 families. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. Genome-wide linkage analysis using Illumina Cyto-12 Chip mapped the disease locus to the DFNA11 interval in the family. A c.2003G→A (p.R668H) mutation of the MYO7A, is heterozygous in all affected family members and absent in 100 healthy individuals. Arg668His is located in a region of the myosin VIIA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural role in linking different lobes of motor domain together. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin VIIA, indicating that the ATPase activity in the p.R668H mutant myosin VIIA was significantly destroyed.

Invited Review: The Myosins: Exploration of the Development of Our Current Understanding of These Mutations in the Motor

PubMed Central

Moore, Jeffrey R.; Leinwand, Leslie; Warshaw, David M.

2013-01-01

Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are inherited diseases with a high incidence of death due to electrical abnormalities or outflow tract obstruction. In many of the families afflicted with either disease, causative mutations have been identified in various sarcomeric proteins. In this review, we focus on mutations in the cardiac muscle molecular motor, myosin and its associated light chains. Despite the >300 identified mutations there is still no clear understanding of how these mutations within the same myosin molecule can lead to the dramatically different clinical phenotypes associated with HCM and DCM. Localizing mutations within myosin’s molecular structure provides insight into the potential consequence of these perturbations to key functional domains of the motor. Review of biochemical and biophysical data that characterize the functional capacities of these mutant myosins suggests that mutant myosins with enhanced contractility lead to HCM while those displaying reduced contractility lead to DCM. With gain and loss of function potentially being the primary consequence of a specific mutation, how these functional changes trigger the hypertrophic response and lead to the distinct HCM and DCM phenotypes will be the future investigative challenge. PMID:22821910

Model for adhesion clutch explains biphasic relationship between actin flow and traction at the cell leading edge

PubMed Central

Craig, Erin M.; Stricker, Jonathan; Gardel, Margaret L.; Mogilner, Alex

2015-01-01

Cell motility relies on the continuous reorganization of a dynamic actin-myosin-adhesion network at the leading edge of the cell, in order to generate protrusion at the leading edge and traction between the cell and its external environment. We analyze experimentally measured spatial distributions of actin flow, traction force, myosin density, and adhesion density in control and pharmacologically perturbed epithelial cells in order to develop a mechanical model of the actin-adhesion-myosin self-organization at the leading edge. A model in which the F-actin network is treated as a viscous gel, and adhesion clutch engagement is strengthened by myosin but weakened by actin flow, can explain the measured molecular distributions and correctly predict the spatial distributions of the actin flow and traction stress. We test the model by comparing its predictions with measurements of the actin flow and traction stress in cells with fast and slow actin polymerization rates. The model predicts how the location of the lamellipodium-lamellum boundary depends on the actin viscosity and adhesion strength. The model further predicts that the location of the lamellipodium-lamellum boundary is not very sensitive to the level of myosin contraction. PMID:25969948

Yuji Tonomura: a pioneer in the field of energy transduction in muscle contraction.

PubMed

Onishi, Hirofumi

2009-07-01

Late Professor Yuji Tonomura has made a great contribution in the study of energy transduction in muscle contraction. He was the investigator who first proposed that a myosin-phosphate intermediate is produced subsequently to the Michaelis-Menten complex in the pre-steady state of the myosin ATPase reaction and that it is a key intermediate for muscle contraction. Here, his proposed intermediate will be viewed from the prospective of today's understanding of actomyosin ATPase kinetics and in the context of myosin motor domain crystal structures.

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

PubMed

Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

2016-07-01

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as a common pathway contributing to the activity-dependent regulation of vesicle endocytosis at synapses. © 2016 International Society for Neurochemistry.

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Shenping; Liu, Jun; Reedy, Mary C.

2010-10-22

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filamentmore » density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.« less

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle.

PubMed

Kang, J O; Ito, T; Fukazawa, T

1983-01-01

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week. Copyright © 1983. Published by Elsevier Ltd.

Direct real-time detection of the structural and biochemical events in the myosin power stroke.

PubMed

Muretta, Joseph M; Rohde, John A; Johnsrud, Daniel O; Cornea, Sinziana; Thomas, David D

2015-11-17

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)(2)FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure.

PubMed

Pasakarnis, Laurynas; Frei, Erich; Caussinus, Emmanuel; Affolter, Markus; Brunner, Damian

2016-11-01

Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.

Crystal structure of the motor domain of a class-I myosin

PubMed Central

Kollmar, Martin; Dürrwang, Ulrike; Kliche, Werner; Manstein, Dietmar J.; Kull, F.Jon

2002-01-01

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is ∼30° further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain. PMID:12032065

Long single [alpha]-helical tail domains bridge the gap between structure and function of myosin VI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spink, Benjamin J.; Sivaramakrishnan, Sivaraj; Lipfert, Jan

2008-09-29

Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take {approx}36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single {alpha}-helix of {approx}10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the {approx}36-nm step, rather than dimerizing as previously proposed, we show that themore » cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.« less

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.

PubMed

Fusi, L; Brunello, E; Yan, Z; Irving, M

2016-10-31

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

PubMed Central

Fusi, L.; Brunello, E.; Yan, Z.; Irving, M.

2016-01-01

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. PMID:27796302

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application

PubMed Central

Sirigu, Serena; Hartman, James J.; Ropars, Virginie; Clancy, Sheila; Wang, Xi; Chuang, Grace; Qian, Xiangping; Lu, Pu-Ping; Barrett, Edward; Rudolph, Karin; Royer, Christopher; Morgan, Bradley P.; Stura, Enrico A.; Malik, Fady I.; Houdusse, Anne M.

2016-01-01

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the “recovery stroke” transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents. PMID:27815532

Myo1g is an active player in maintaining cell stiffness in B-lymphocytes.

PubMed

López-Ortega, O; Ovalle-García, E; Ortega-Blake, I; Antillón, A; Chávez-Munguía, B; Patiño-López, G; Fragoso-Soriano, R; Santos-Argumedo, L

2016-05-01

B-lymphocytes are migrating cells that specialize in antigen presentation, antibody secretion, and endocytosis; these processes implicate the modulation of plasma membrane elasticity. Cell stiffness is a force generated by the interaction between the actin-cytoskeleton and the plasma membrane, which requires the participation of several proteins. These proteins include class I myosins, which are now considered to play a role in controlling membrane-cytoskeleton interactions. In this study, we identified the motor protein Myosin 1g (Myo1g) as a mediator of this phenomenon. The absence of Myo1g decreased the cell stiffness, affecting cell adhesion, cell spreading, phagocytosis, and endocytosis in B-lymphocytes. The results described here reveal a novel molecular mechanism by which Myo1g mediates and regulates cell stiffness in B-lymphocytes. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

Cofilin, But Not Profilin, Is Required for Myosin-I-Induced Actin Polymerization and the Endocytic Uptake in Yeast

PubMed Central

Idrissi, Fatima-Zahra; Wolf, Bianka L.; Geli, M. Isabel

2002-01-01

Mutations in the budding yeast myosins-I (MYO3 and MYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable. PMID:12429847

Ontogenetic, gravity-dependent development of rat soleus muscle

NASA Technical Reports Server (NTRS)

Ohira, Y.; Tanaka, T.; Yoshinaga, T.; Kawano, F.; Nomura, T.; Nonaka, I.; Allen, D. L.; Roy, R. R.; Edgerton, V. R.

2001-01-01

We tested the hypothesis that rat soleus muscle fiber growth and changes in myosin phenotype during the postnatal, preweaning period would be largely independent of weight bearing. The hindlimbs of one group of pups were unloaded intermittently from postnatal day 4 to day 21: the pups were isolated from the dam for 5 h during unloading and returned for nursing for 1 h. Control pups were either maintained with the dam as normal or put on an alternating feeding schedule as described above. The enlargement of mass (approximately 3 times), increase in myonuclear number (approximately 1.6 times) and myonuclear domain (approximately 2.6 times), and transformation toward a slow fiber phenotype (from 56 to 70% fibers expressing type I myosin heavy chain) observed in controls were inhibited by hindlimb unloading. These properties were normalized to control levels or higher within 1 mo of reambulation beginning immediately after the unloading period. Therefore, chronic unloading essentially stopped the ontogenetic developmental processes of 1) net increase in DNA available for transcription, 2) increase in amount of cytoplasm sustained by that DNA pool, and 3) normal transition of myosin isoforms that occur in some fibers from birth to weaning. It is concluded that normal ontogenetic development of a postural muscle is highly dependent on the gravitational environment even during the early postnatal period, when full weight-bearing activity is not routine.

Sarcomere protein gene mutations and inherited heart disease: a beta-cardiac myosin heavy chain mutation causing endocardial fibroelastosis and heart failure.

PubMed

Kamisago, Mitsuhiro; Schmitt, Joachim P; McNamara, Dennis; Seidman, Christine; Seidman, J G

2006-01-01

Inherited human cardiomyopathies often lead to heart failure. A common feature of these conditions is that affected individuals can express the disease causing mutations for many years without showing clinical signs of the disease. Previous studies have demonstrated that sarcomere protein gene mutations can cause either dilated cardiomyopathy or hypertrophic cardiomyopathy. Here we demonstrate that the Arg442His missense mutation in beta-cardiac myosin heavy chain (betaMHC) causes dilated cardiomyopathy, endocardial fibroelastosis and heart failure at a very early age. Using standard genetic engineering tools we and others have made murine models by introducing human disease causing mutations into mice. The central hypothesis of these studies has been that by identifying the pathophysiological pathways activated by these mutations we can define enzymatic activities that are modified during the disease process and which may be involved in pathways that involve more common forms of cardiac disease. Murine models bearing different mutant myosins are being used to address whether each disease causing mutant betaMHC activates the same or different cellular pathways. Dissecting the molecular pathways modulated by mutations in sarcomere protein genes as well as other genes has already demonstrated that there are multiple pathways leading to cardiac remodelling and heart failure. Defining the mechanisms by which mutations in the same genes activate different cellular pathways remains an important question.

Different Head Environments in Tarantula Thick Filaments Support a Cooperative Activation Process

PubMed Central

Sulbarán, Guidenn; Biasutto, Antonio; Alamo, Lorenzo; Riggs, Claire; Pinto, Antonio; Méndez, Franklin; Craig, Roger; Padrón, Raúl

2013-01-01

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism. PMID:24209856