Science.gov

Sample records for inhibits myosin motor

  1. Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a

    PubMed Central

    Yao, Lin-Lin; Cao, Qing-Juan; Zhang, Hai-Man; Zhang, Jie; Cao, Yang; Li, Xiang-dong

    2015-01-01

    The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca2+ or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca2+, little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function. PMID:26039755

  2. A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

    PubMed Central

    Heaslip, Aoife T.; Leung, Jacqueline M.; Carey, Kimberly L.; Catti, Federica; Warshaw, David M.; Westwood, Nicholas J.; Ballif, Bryan A.; Ward, Gary E.

    2010-01-01

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains. PMID:20084115

  3. Myosin VI: cellular functions and motor properties.

    PubMed Central

    Roberts, Rhys; Lister, Ida; Schmitz, Stephan; Walker, Matthew; Veigel, Claudia; Trinick, John; Buss, Folma; Kendrick-Jones, John

    2004-01-01

    Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed. PMID:15647169

  4. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew

    2015-01-01

    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting non-processive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs. PMID:25712463

  5. Arabidopsis myosin XI: a motor rules the tracks.

    PubMed

    Cai, Chao; Henty-Ridilla, Jessica L; Szymanski, Daniel B; Staiger, Christopher J

    2014-11-01

    Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change. PMID:25237128

  6. Phosphate and ADP differently inhibit coordinated smooth muscle myosin groups.

    PubMed

    Hilbert, Lennart; Balassy, Zsombor; Zitouni, Nedjma B; Mackey, Michael C; Lauzon, Anne-Marie

    2015-02-01

    Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state. PMID:25650929

  7. Collective Dynamics of Elastically Coupled Myosin V Motors*

    PubMed Central

    Lu, Hailong; Efremov, Artem K.; Bookwalter, Carol S.; Krementsova, Elena B.; Driver, Jonathan W.; Trybus, Kathleen M.; Diehl, Michael R.

    2012-01-01

    Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain. PMID:22718762

  8. Force generation by kinesin and myosin cytoskeletal motor proteins

    PubMed Central

    Kull, F. Jon; Endow, Sharyn A.

    2013-01-01

    Summary Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central β-sheet – proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins – is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease. PMID:23487037

  9. Force generation by kinesin and myosin cytoskeletal motor proteins.

    PubMed

    Kull, F Jon; Endow, Sharyn A

    2013-01-01

    Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central β-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease. PMID:23487037

  10. 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

  11. 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

  12. Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

    SciTech Connect

    Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen; Kerekes, Ryan A; Gleason, Shaun Scott; Hatten, Mary E

    2009-01-01

    Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.

  13. Myosin-I molecular motors at a glance.

    PubMed

    McIntosh, Betsy B; Ostap, E Michael

    2016-07-15

    Myosin-I molecular motors are proposed to play various cellular roles related to membrane dynamics and trafficking. In this Cell Science at a Glance article and the accompanying poster, we review and illustrate the proposed cellular functions of metazoan myosin-I molecular motors by examining the structural, biochemical, mechanical and cell biological evidence for their proposed molecular roles. We highlight evidence for the roles of myosin-I isoforms in regulating membrane tension and actin architecture, powering plasma membrane and organelle deformation, participating in membrane trafficking, and functioning as a tension-sensitive dock or tether. Collectively, myosin-I motors have been implicated in increasingly complex cellular phenomena, yet how a single isoform accomplishes multiple types of molecular functions is still an active area of investigation. To fully understand the underlying physiology, it is now essential to piece together different approaches of biological investigation. This article will appeal to investigators who study immunology, metabolic diseases, endosomal trafficking, cell motility, cancer and kidney disease, and to those who are interested in how cellular membranes are coupled to the underlying actin cytoskeleton in a variety of different applications. PMID:27401928

  14. Myosin VI is a processive motor with a large step size

    PubMed Central

    Rock, Ronald S.; Rice, Sarah E.; Wells, Amber L.; Purcell, Thomas J.; Spudich, James A.; Sweeney, H. Lee

    2001-01-01

    Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes. PMID:11707568

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

    PubMed

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

    2014-08-26

    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 (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 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

  16. Myosin VI as a transporter and an anchor: A model for kinetics of the motor under load

    NASA Astrophysics Data System (ADS)

    Chuan, Peiying; Spudich, James; Dunn, Alexander

    2010-03-01

    Myosin VI is an actin-based motor that is thought to function both as a transporter and an anchor in vivo. In an earlier study (Altman et al, Cell 2004), inhibition of myosin VI stepping kinetics by load applied using an optical trap was observed at saturating ATP and low ADP concentrations (< 2.5 μM). A simple mechanism whereby the rate of ADP binding increases exponentially with load was proposed. This model predicts that myosin VI functions primarily as an anchor at loads greater than ˜0.5 pN under physiological nucleotide conditions, which is potentially inconsistent with its roles in vivo. Here we present myosin VI stepping data taken at a variety of applied loads and ADP concentrations, and show that the Altman model only holds at low ADP concentrations. At higher, physiologically relevant ADP concentrations under load we observe dwell times that are an order of magnitude smaller than predicted by the Altman model. We present a modified model in which applied load alters the equilibrium between two myosin VI states with different nucleotide affinities. This new kinetic scheme accurately describes myosin VI behavior at various nucleotide conditions under a large range of loads, and explains how the motor is able to carry out its roles in vivo, both as a force-generating transporter and as an anchor.

  17. Life without double-headed non-muscle myosin II motor proteins

    NASA Astrophysics Data System (ADS)

    Betapudi, Venkaiah

    2014-07-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  18. Life without double-headed non-muscle myosin II motor proteins

    PubMed Central

    Betapudi, Venkaiah

    2014-01-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients' life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life. PMID:25072053

  19. The myosin X motor is optimized for movement on actin bundles.

    PubMed

    Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana; Blanc, Florian; Zhou, Kaifeng; Lin, Tianming; Liu, Xiaoyan; Hissier, Pascale; Samazan, Frédéric; Amigues, Béatrice; Yang, Eric D; Park, Hyokeun; Pylypenko, Olena; Cecchini, Marco; Sindelar, Charles V; Sweeney, H Lee; Houdusse, Anne

    2016-01-01

    Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles. PMID:27580874

  20. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments.

    PubMed

    Hariadi, R F; Sommese, R F; Adhikari, A S; Taylor, R E; Sutton, S; Spudich, J A; Sivaramakrishnan, S

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes. PMID:26149240

  1. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

    NASA Astrophysics Data System (ADS)

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.; Sivaramakrishnan, S.

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  2. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    PubMed

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia

    2012-08-10

    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  3. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity

    PubMed Central

    Jana, Biman; Onuchic, José N.

    2016-01-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

  4. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity.

    PubMed

    Jana, Biman; Onuchic, José N

    2016-08-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

  5. Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

    PubMed Central

    Nelson, Shane R.; Trybus, Kathleen M.; Warshaw, David M.

    2014-01-01

    Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm2), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track. PMID:25201964

  6. Molecular genetics of myosin motors in Arabidopsis. Progress report, [July 1, 1992--February 28, 1994

    SciTech Connect

    Not Available

    1994-06-01

    We have evidence for at least nine myosin-like genes in Arbidopsis, six of which have been cloned by a PCR-based method from genomic DNA, two have been isolated by genomic DNA cloning, and four have been identified by cDNA cloning. Most of our attention has been focused on the four myosin genes for which we have cDNA clones, and these cDNAs have now been sequenced to completion. Each of these myosins is similar in overall structure, with each containing the characteristic myosin head (motor) domain, which possesses ATP- and actin-binding motifs, a series of IQ repeats, which may be involved in calmodulin binding, a domain with a high probability of forming an alpha-helical coiled-coil secondary structure, which may allow the polypeptides to form dimers, and a variable tail domain, which may serve to define the specific cellular component that each myosin interacts with. One of these myosin genes, called MYA1, displays structural similarity to class of myosins that includes the yeast MYO2, mouse Dilute, and chicken p190 proteins, and this group of myosins is thought to play a role in intracellular trafficking of organelles. Because MYA1 is similar to this interesting class of myosins, we have chosen to conduct detailed studies of MYA1.

  7. Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    PubMed Central

    Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

    2011-01-01

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. PMID:22022566

  8. 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. PMID:27129208

  9. A small molecule species specifically inhibits Fusarium myosin I.

    PubMed

    Zhang, Chengqi; Chen, Yun; Yin, Yanni; Ji, Huan-Hong; Shim, Won-Bo; Hou, Yiping; Zhou, Mingguo; Li, Xiang-Dong; Ma, Zhonghua

    2015-08-01

    Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of cereal crops worldwide. Recently, a novel fungicide JS399-19 has been launched into the marketplace to manage FHB. It is compelling that JS399-19 shows highly inhibitory activity towards some Fusarium species, but not to other fungi, indicating that it is an environmentally compatible fungicide. To explore the mode of action of this species-specific compound, we conducted a whole-genome transcript profiling together with genetic and biochemical assays, and discovered that JS399-19 targets the myosin I of F. graminearum (FgMyo1). FgMyo1 is essential for F. graminearum growth. A point mutation S217L or E420K in FgMyo1 is responsible for F. graminearum resistance to JS399-19. In addition, transformation of F. graminearum with the myosin I gene of Magnaporthe grisea, the causal agent of rice blast, also led to JS399-19 resistance. JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1, but not the mutated FgMyo1(S217L/E420K) . These results provide us a new insight into the design of species-specific antifungal compounds. Furthermore, our strategy can be applied to identify novel drug targets in various pathogenic organisms. PMID:25404531

  10. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

    PubMed

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas; Houdusse, Anne; Kerkhoff, Eugen

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. PMID:27623148

  11. New insight into role of myosin motors for activation of RNA polymerases.

    PubMed

    Sarshad, Aishe A; Percipalle, Piergiorgio

    2014-01-01

    In the eukaryotic cell nucleus, actin and myosin are emerging as essential regulators of nuclear function. At gene level, they regulate chromatin and modulate RNA polymerase transcription, and at the RNA level, they are involved in the metabolism of ribonucleoprotein complexes. Furthermore, actin and myosin are involved in maintaining the structure of cell nucleus by mediating chromatin movement and by interacting with components of the nuclear lamina. This plethora of functions is now supported by evidence that nuclear actin polymerizes just like the cytoplasmic actin fraction. Based on these considerations, we now hypothesize that the nuclear myosin forms function as actin-based motors. In this chapter, our goal is to start from the knowledge acquired in the cytoplasmic field to explore how nuclear myosin functions in gene transcription. One of the pressing issues discussed here is whether nuclear myosin produces local tension or functions as transporters. Based on two current models reported in the literature, we discuss the topology of the actin-based nuclear myosin 1 motor and how it is believed to facilitate propulsion of the RNA polymerase machinery while maintaining chromatin that is compatible with transcription. These mechanisms will be placed in the context of cell cycle progression. PMID:24952918

  12. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26945064

  14. 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

    2016-05-01

    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. PMID:26945064

  15. Insights into the Chemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism

    SciTech Connect

    Schwarzl, S.M.; Smith, Jeremy C; Fischer, S.

    2006-03-01

    The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP{circle_dot}Pi state of nucleotide-bound myosin.

  16. The myofilament elasticity and its effect on kinetics of force generation by the myosin motor.

    PubMed

    Piazzesi, Gabriella; Dolfi, Mario; Brunello, Elisabetta; Fusi, Luca; Reconditi, Massimo; Bianco, Pasquale; Linari, Marco; Lombardi, Vincenzo

    2014-06-15

    The half-sarcomere is the functional unit of striated muscle, in which, according to a "linear" mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance Cf. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (Chs-T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a Cf of 12.6nm/MPa. With this value of Cf, the fit of Chs-T relation during the isometric force rise gives s=1.8-1.9nm, similar to the values estimated using the linear model. PMID:24631572

  17. Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

    ERIC Educational Resources Information Center

    Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin

    2012-01-01

    Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

  18. Molecular genetics of myosin motors in Arabidopsis. Final report, July 1, 1992--June 30, 1996

    SciTech Connect

    Schiefelbein, J.

    1997-02-01

    The normal growth and development of plant cells depends on the precise organization and distribution of the cellular contents. The basic goal of this investigation was to define a group of the molecules that are involved in organizing and transporting plant cell components. Based largely on studies of animal and fungal cells, one of the molecules thought to be involved in intracellular trafficking in plants is the actin-based motor protein myosin. Therefore, the major aim of this study was to isolate and analyze plant genes encoding myosin proteins. The plant of choice for these experiments was Arabidopsis thaliana, which offers numerous advantages for molecular genetics research.

  19. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    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 motion 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.

  20. Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

    PubMed Central

    Logvinova, Daria S.; Markov, Denis I.; Nikolaeva, Olga P.; Sluchanko, Nikolai N.; Ushakov, Dmitry S.; Levitsky, Dmitrii I.

    2015-01-01

    Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle. PMID:26356744

  1. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    NASA Astrophysics Data System (ADS)

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-08-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  2. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    PubMed Central

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-01-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin. PMID:26246073

  3. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor.

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-03-01

    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 60{sup o}. This recovery stroke is coupled to the activation of myosin 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 seesaw motion of the relay helix, followed by a piston-like 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. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.

  4. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway.

    PubMed

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang; McCrae, Keith R

    2013-11-28

    The antiphospholipid syndrome is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLAs). Most pathogenic APLAs are directed against β2-glycoprotein I (β2GPI), a plasma phospholipid binding protein. One mechanism by which circulating antiphospholipid/anti-β2GPI antibodies may promote thrombosis is by inducing the release of procoagulant microparticles from endothelial cells. However, there is no information available concerning the mechanisms by which anti-β2GPI antibodies induce microparticle release. In seeking to identify proteins phosphorylated during anti-β2GPI antibody-induced endothelial activation, we observed phosphorylation of nonmuscle myosin II regulatory light chain (RLC), which regulates cytoskeletal assembly. In parallel, we observed a dramatic increase in the formation of filamentous actin, a two- to fivefold increase in the release of endothelial cell microparticles, and a 10- to 15-fold increase in the expression of E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and tissue factor messenger RNA. Microparticle release, but not endothelial cell surface E-selectin expression, was blocked by inhibiting RLC phosphorylation or nonmuscle myosin II motor activity. These results suggest that distinct pathways, some of which mediate cytoskeletal assembly, regulate the endothelial cell response to anti-β2GPI antibodies. Inhibition of nonmuscle myosin II activation may provide a novel approach for inhibiting microparticle release by endothelial cells in response to anti-β2GPI antibodies. PMID:23954892

  5. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway

    PubMed Central

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang

    2013-01-01

    The antiphospholipid syndrome is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLAs). Most pathogenic APLAs are directed against β2-glycoprotein I (β2GPI), a plasma phospholipid binding protein. One mechanism by which circulating antiphospholipid/anti-β2GPI antibodies may promote thrombosis is by inducing the release of procoagulant microparticles from endothelial cells. However, there is no information available concerning the mechanisms by which anti-β2GPI antibodies induce microparticle release. In seeking to identify proteins phosphorylated during anti-β2GPI antibody-induced endothelial activation, we observed phosphorylation of nonmuscle myosin II regulatory light chain (RLC), which regulates cytoskeletal assembly. In parallel, we observed a dramatic increase in the formation of filamentous actin, a two- to fivefold increase in the release of endothelial cell microparticles, and a 10- to 15-fold increase in the expression of E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and tissue factor messenger RNA. Microparticle release, but not endothelial cell surface E-selectin expression, was blocked by inhibiting RLC phosphorylation or nonmuscle myosin II motor activity. These results suggest that distinct pathways, some of which mediate cytoskeletal assembly, regulate the endothelial cell response to anti-β2GPI antibodies. Inhibition of nonmuscle myosin II activation may provide a novel approach for inhibiting microparticle release by endothelial cells in response to anti-β2GPI antibodies. PMID:23954892

  6. The structural coupling between ATPase activation and recovery stroke in the myosin II motor

    SciTech Connect

    Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-07-01

    Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

  7. Myosin Vc Is a Molecular Motor That Functions in Secretory Granule Trafficking

    PubMed Central

    Jacobs, Damon T.; Weigert, Roberto; Grode, Kyle D.; Donaldson, Julie G.

    2009-01-01

    Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (∼30 nm/s) and microtubule independently, whereas tubules move rapidly (∼440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking. PMID:19741097

  8. Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

    PubMed Central

    Caremani, Marco; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo; Linari, Marco

    2015-01-01

    The chemo-mechanical cycle of the myosin II–actin reaction in situ has been investigated in Ca2+-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi), shortening at V ≤ 350 nm s–1 per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mm, which reduces T0 and the number of motors by 40–50%, does not influence the dependence on V of number and strain. A model simulation of the myosin–actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. Key points Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin–actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to

  9. Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes.

    PubMed

    Shin, Jae-Won; Swift, Joe; Spinler, Kyle R; Discher, Dennis E

    2011-07-12

    Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbβ3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation. PMID:21709232

  10. Harmonic force spectroscopy reveals a force-velocity curve from a single human beta cardiac myosin motor

    NASA Astrophysics Data System (ADS)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian; Mortensen, Kim; Flyvbjerg, Henrik; Spudich, James

    2014-03-01

    A muscle contracts rapidly under low load, but slowly under high load. Its molecular mechanisms remain to be elucidated, however. During contraction, myosins in thick filaments interact with actin in thin filaments in the sarcomere, cycling between a strongly bound (force producing) state and a weakly bound (relaxed) state. Huxley et al. have previously proposed that the transition from the strong to the weak interaction can be modulated by a load. We use a new method we call ``harmonic force spectroscopy'' to extract a load-velocity curve from a single human beta cardiac myosin II motor. With a dual-beam optical trap, we hold an actin dumbbell over a myosin molecule anchored to the microscope stage that oscillates sinusoidally. Upon binding, the motor experiences an oscillatory load with a mean that is directed forward or backward, depending on binding location We find that the bound time at saturating [ATP] is exponentially correlated with the mean load, which is explained by Arrhenius transition theory. With a stroke size measurement, we obtained a load-velocity curve from a single myosin. We compare the curves for wild-type motors with mutants that cause hypertrophic cardiomyopathies, to understand the effects on the contractile cycle

  11. 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

  12. Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis

    PubMed Central

    Spudich, James A.; Sivaramakrishnan, Sivaraj

    2010-01-01

    The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual ~ 180° lever arm swing and achieves its large step size using special structural features in its tail domain. PMID:20094053

  13. Response Inhibition in Motor Conversion Disorder

    PubMed Central

    Voon, Valerie; Ekanayake, Vindhya; Wiggs, Edythe; Kranick, Sarah; Ameli, Rezvan; Harrison, Neil A.; Hallett, Mark

    2014-01-01

    Conversion disorders (CDs) are unexplained neurological symptoms presumed to be related to a psychological issue. Studies focusing on conversion paralysis have suggested potential impairments in motor initiation or execution. Here we studied CD patients with aberrant or excessive motor movements and focused on motor response inhibition. We also assessed cognitive measures in multiple domains. We compared 30 CD patients and 30 age-, sex-, and education-matched healthy volunteers on a motor response inhibition task (go/no go), along with verbal motor response inhibition (color-word interference) and measures of attention, sustained attention, processing speed, language, memory, visuospatial processing, and executive function including planning and verbal fluency. CD patients had greater impairments in commission errors on the go/no go task (P <.001) compared with healthy volunteers, which remained significant after Bonferroni correction for multiple comparisons and after controlling for attention, sustained attention, depression, and anxiety. There were no significant differences in other cognitive measures. We highlight a specific deficit in motor response inhibition that may play a role in impaired inhibition of unwanted movement such as the excessive and aberrant movements seen in motor conversion. Patients with nonepileptic seizures, a different form of conversion disorder, are commonly reported to have lower IQ and multiple cognitive deficits. Our results point toward potential differences between conversion disorder subgroups. PMID:23554084

  14. Analysis of flexural rigidity of actin filaments propelled by surface adsorbed myosin motors.

    PubMed

    Bengtsson, Elina; Persson, Malin; Månsson, Alf

    2013-11-01

    Actin filaments are central components of the cytoskeleton and the contractile machinery of muscle. The filaments are known to exist in a range of conformational states presumably with different flexural rigidity and thereby different persistence lengths. Our results analyze the approaches proposed previously to measure the persistence length from the statistics of the winding paths of actin filaments that are propelled by surface-adsorbed myosin motor fragments in the in vitro motility assay. Our results suggest that the persistence length of heavy meromyosin propelled actin filaments can be estimated with high accuracy and reproducibility using this approach provided that: (1) the in vitro motility assay experiments are designed to prevent bias in filament sliding directions, (2) at least 200 independent filament paths are studied, (3) the ratio between the sliding distance between measurements and the camera pixel-size is between 4 and 12, (4) the sliding distances between measurements is less than 50% of the expected persistence length, and (5) an appropriate cut-off value is chosen to exclude abrupt large angular changes in sliding direction that are complications, e.g., due to the presence of rigor heads. If the above precautions are taken the described method should be a useful routine part of in vitro motility assays thus expanding the amount of information to be gained from these. PMID:24039103

  15. 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. PMID:21767493

  16. Dasatinib affects focal adhesion and myosin regulation to inhibit matrix contraction by Müller cells.

    PubMed

    Tsukahara, Rintaro; Umazume, Kazuhiko; Yamakawa, Naoyuki; McDonald, Kevin; Kaplan, Henry J; Tamiya, Shigeo

    2015-10-01

    Epiretinal membrane (ERM) contraction is associated with a variety of ocular diseases that cause macular dysfunction. Trans-differentiated Müller cells have been identified in ERMs, and have been implicated to be involved in the contractile process. In this study, we tested the effect of dasatinib, an FDA-approved tyrosine kinase inhibitor, on matrix contraction caused by Müller cells, and examined molecular mechanism of action. Type I collagen matrix contraction assays were used to examine the effect of drugs on matrix contraction by trans-differentiated Müller cells. Fluophore-conjugated phalloidin was used for the detection of actin cytoskeleton, and Western-blot analyses were carried out to examine protein expression and phosphorylation status. Dasatinib inhibited collagen matrix contraction by trans-differentiated Müller cells that was associated with decreased cell spreading and reduction of actomyosin stress fibers. Concomitantly, dasatinib-treated Müller cells had reduced phosphorylation of Src family kinase, paxillin, as well as myosin II light chain. Specific inhibitors of Rho/ROCK and myosin II confirmed the critical role played by this pathway in Müller cell contraction. Our data demonstrate that dasatinib significantly reduced matrix contraction by Müller cells via inhibition of focal adhesion, as well as actomyosin contraction. PMID:26240967

  17. Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin

    PubMed Central

    Wendt, Thomas; Taylor, Dianne; Messier, Terri; Trybus, Kathleen M.; Taylor, Kenneth A.

    1999-01-01

    The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited. PMID:10613897

  18. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

    PubMed Central

    Ebrahim, Seham; Avenarius, Matthew R.; Grati, M'hamed; Krey, Jocelyn F.; Windsor, Alanna M.; Sousa, Aurea D.; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A.; Salles, Felipe T.; Baird, Michelle A.; Davidson, Michael W.; Jones, Sherri M.; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H.; Yengo, Christopher M.; Barr-Gillespie, Peter G.; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

  19. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

    PubMed

    Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed; Krey, Jocelyn F; Windsor, Alanna M; Sousa, Aurea D; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A; Salles, Felipe T; Baird, Michelle A; Davidson, Michael W; Jones, Sherri M; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H; Yengo, Christopher M; Barr-Gillespie, Peter G; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

  20. The neck region of the myosin motor domain acts as a lever arm to generate movement.

    PubMed Central

    Uyeda, T Q; Abramson, P D; Spudich, J A

    1996-01-01

    The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8633089

  1. The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function.

    PubMed

    Iizumi, Yosuke; Sagara, Hiroshi; Kabe, Yasuaki; Azuma, Motoki; Kume, Kanako; Ogawa, Michinaga; Nagai, Takeshi; Gillespie, Peter G; Sasakawa, Chihiro; Handa, Hiroshi

    2007-12-13

    Enteropathogenic Escherichia coli (EPEC) destroys intestinal microvilli and suppresses phagocytosis by injecting effectors into infected cells through a type III secretion system (TTSS). EspB, a component of the TTSS, is also injected into the cytoplasm of host cells. However, the physiological functions of EspB within the host cell cytoplasm remain unclear. We show that EspB binds to myosins, which are a superfamily of proteins that interact with actin filaments and mediate essential cellular processes, including microvillus formation and phagocytosis. EspB inhibits the interaction of myosins with actin, and an EspB mutant that lacks the myosin-binding region maintained its TTSS function but could not induce microvillus effacing or suppress phagocytosis. Moreover, the myosin-binding region of EspB is essential for Citrobacter rodentium, an EPEC-related murine pathogen, to efficiently infect mice. These results suggest that EspB inhibits myosin functions and thereby facilitates efficient infection by EPEC. PMID:18078690

  2. Lentivirus-Mediated Knockdown of Myosin VI Inhibits Cell Proliferation of Breast Cancer Cell.

    PubMed

    Wang, Hong; Wang, Biyun; Zhu, Wei; Yang, Ziang

    2015-10-01

    Myosin VI (MYO6) is a unique member of the myosin superfamily, and almost no experimental studies link MYO6 to tumorigenesis of breast cancer. However, previous microarray data demonstrated that MYO6 was frequently overexpressed in breast cancer tissues. In this study, to further develop its role in breast cancer, endogenous expression of MYO6 was significantly inhibited in breast cancer ZR-75-30 and MDA-MB-231 cells using lentivirus-mediated RNA interference. Quantitative polymerase chain reaction and western blot were applied to detect the expression level of MYO6. Cell viability of both cell lines was measured by methylthiazol tetrazolium and colony formation assays. Besides, cell cycle assay was utilized to acquire the distribution information of cell phase. The results demonstrated that knockdown of MYO6 markedly reduced cell viability and colony formation, as well as suppressed cell cycle progression in breast cancer cells. The results suggested that MYO6 played a vital role in breast cancer cells and might provide useful information for diagnosis and therapy of human breast cancer in future. PMID:26407123

  3. Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

    PubMed Central

    Malmqvist, Ulf; Trybus, Kathleen M.; Yagi, Shinobu; Carmichael, Jeff; Fay, Fredric S.

    1997-01-01

    A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle. PMID:9207148

  4. Switching of actin-myosin motors by voltage-induced pH bias in vitro.

    PubMed

    Hatori, Kuniyuki; Iwase, Takahiro; Wada, Reito

    2016-08-01

    ATP-driven motor proteins, which function in cell motility and organelle transport, have potential applications as bio-inspired micro-devices; however, their control remains unsatisfactory. Here, we show rapid-velocity control of actin filaments interacting with myosin motors using voltage applied to Pt electrodes in an in vitro motility system, by which immediate increases and decreases in velocity were induced beside the cathode and anode, respectively. Indicator dye revealed pH changes after voltage application, and alternate voltage switching allowed actin filaments to cyclically alter their velocity in response to these changes. This principle provides a basis for on-demand control of not only motor proteins but also pH-sensitive events at a microscopic level. PMID:27210738

  5. Unconventional myosins acting unconventionally

    PubMed Central

    Woolner, Sarah; Bement, William M.

    2016-01-01

    Unconventional myosins are proteins that bind actin filaments in an ATP-regulated manner. Because of their association with membranes, they have traditionally been viewed as motors that function primarily to transport membranous organelles along actin filaments. Recently, however, a wealth of roles for myosins that are not obviously related to organelle transport have been uncovered, including organization of F-actin, mitotic spindle regulation and gene transcription. Furthermore, it has also become apparent that the motor domains of different myosins vary strikingly in their biophysical attributes. We suggest that the assumption that most unconventional myosins function primarily as organelle transporters might be misguided. PMID:19406643

  6. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

    PubMed Central

    Nag, Suman; Sommese, Ruth F.; Ujfalusi, Zoltan; Combs, Ariana; Langer, Stephen; Sutton, Shirley; Leinwand, Leslie A.; Geeves, Michael A.; Ruppel, Kathleen M.; Spudich, James A.

    2015-01-01

    Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation. PMID:26601291

  7. The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site

    PubMed Central

    Caremani, Marco; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo; Linari, Marco

    2013-01-01

    Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere. PMID:23878374

  8. Myosin II Activity Softens Cells in Suspension

    PubMed Central

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

    2015-01-01

    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. PMID:25902426

  9. 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. PMID:25902426

  10. Olanzapine May Inhibit Colonic Motility Associated with the 5-HT Receptor and Myosin Light Chain Kinase

    PubMed Central

    Zhang, Jiarui; Qiao, Ying; Le, Jingjing

    2016-01-01

    Objective To study whether the effects of olanzapine on gastrointestinal motility is related to the serotonin antagonism and myosin light chain kinase. Methods Male Sprague-Dawley rats were randomly divided into four groups. Olanzapine gavage was performed for each treatment group during the course of 30 continuous days, while the same volume of saline was given to the rats in the control group. Defecation of the rats was observed on days 7 and 30 after olanzapine gavage. The effects of olanzapine on contraction of colonic smooth muscles were observed in ex vivo experiments. A Western blot was used to evaluate expression levels of the serotonin transporter (SERT) and MLCK in colon segments of the rats. Results ResultsaaCompared to the control group, 5-160 µ M of olanzapine could inhibit dose-dependently the contraction of colonic smooth muscle ex vivo experiments. The maximum smooth muscle contraction effects of 5-HT and acetylcholine significantly decreased after treatment with 40-160 µ M of olanzapine. Constipation was found in the olanzapine-treated rats on day 7 and have sustained day 30 after gavage. Expression of MLCK in olanzapine-treated rats was significantly decreased, whereas the expression of SERT significantly increased on the day 7, then significantly decreased on the day 30 after olanzapine gavage. Conclusion SERT and MLCK may involve in the inhibition of colonic contraction induced by olanzapine. PMID:27081386

  11. Simulations of the myosin II motor reveal a nucleotide-state sensing element that controls the recovery stroke.

    PubMed

    Koppole, Sampath; Smith, Jeremy C; Fischer, Stefan

    2006-08-18

    During the recovery stroke, the myosin motor is primed for the next power stroke by a 60 degree rotation of its lever arm. This reversible motion is coupled to the activation of the ATPase function of myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456 peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and gamma-phosphate that is essential for ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum myosin II in the two end conformations of the recovery stroke with different nucleotide states (ATP, ADP x Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the nucleotide state is achieved by a small displacement of the cleaved gamma-phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery stroke while the nucleotide is in the ADP x Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when ATP is added to the pre-recovery stroke conformer and remains stable after the recovery stroke, indicating that it has a role in shaping the ATP binding site by induced fit. PMID:16859703

  12. The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

    PubMed

    Tumbarello, David A; Manna, Paul T; Allen, Mark; Bycroft, Mark; Arden, Susan D; Kendrick-Jones, John; Buss, Folma

    2015-10-01

    Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity. PMID:26451915

  13. The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy

    PubMed Central

    Tumbarello, David A.; Manna, Paul T.; Allen, Mark; Bycroft, Mark; Arden, Susan D.; Kendrick-Jones, John; Buss, Folma

    2015-01-01

    Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity. PMID:26451915

  14. Surround inhibition in motor execution and motor imagery.

    PubMed

    Aoyama, Toshiyuki; Kaneko, Fuminari; Ohashi, Yukari; Nagata, Hiroshi

    2016-08-26

    Surround inhibition (SI) is a neural mechanism to focus neuronal activity and facilitate selective motor execution (ME). The aim of the present study was to investigate whether SI is also generated during motor imagery (MI). Furthermore, we investigated whether the extent of SI during MI depends on the strength of SI during ME and/or vividness of MI. The extent of SI was examined during MI and ME of index finger flexion. Transcranial magnetic stimulation was applied at rest, during initiation of the movement (phasic phase) and during tonic muscle contraction of the index finger flexors. Motor evoked potentials (MEPs) were recorded from a surround muscle, abductor digiti minimi (ADM) and a synergistic muscle, the first dorsal interosseous muscle. The amplitude of ADM MEP was reduced during the phasic phase, which indicates that SI occurred during ME. In seven of 14 subjects, SI was also observed during MI, although this effect was not significant. There was a moderate correlation between the extent of SI during ME and MI. Furthermore, good imagers who experienced vivid MI during the MI task showed stronger SI than poor imagers. These results indicate that common neural substrates involved in SI during ME are at least in part recruited during MI. In clinical situations, the therapeutic use of MI to generate vivid MI may be one of effective tool to develop the strength of SI, which facilitate selective execution of desired movements. PMID:27418120

  15. Characterization of F-Actin Tryptophan Phosphorescence in the Presence and Absence of Tryptophan-Free Myosin Motor Domain

    PubMed Central

    Bódis, Emöke; Strambini, Giovanni B.; Gonnelli, Margherita; Málnási-Csizmadia, András; Somogyi, Béla

    2004-01-01

    The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140–293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (τ1 and τ2) and increasing by ∼20% the longest component (τ3). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (τ4, twice as long as τ3) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique. PMID:15298917

  16. Engineered kinesin motor proteins amenable to small-molecule inhibition

    PubMed Central

    Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  17. Engineered kinesin motor proteins amenable to small-molecule inhibition.

    PubMed

    Engelke, Martin F; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T Lynne; Soppina, Pushpanjali; Hancock, William O; Gelfand, Vladimir I; Verhey, Kristen J

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  18. 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

  19. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

    PubMed

    Samant, Sadhana A; Pillai, Vinodkumar B; Sundaresan, Nagalingam R; Shroff, Sanjeev G; Gupta, Mahesh P

    2015-06-19

    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

  20. Remote control of myosin and kinesin motors using light-activated gearshifting.

    PubMed

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C; Schindler, Tony D; Nogales, Eva; Bryant, Zev

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

  1. Remote control of myosin and kinesin motors using light-activated gearshifting

    PubMed Central

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C.; Schindler, Tony D.; Nogales, Eva

    2015-01-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells1,2. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems3,4. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears—speed up, slow down or switch directions—when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

  2. Myosins, Actin and Autophagy.

    PubMed

    Kruppa, Antonina J; Kendrick-Jones, John; Buss, Folma

    2016-08-01

    Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome. PMID:27146966

  3. Maximum limit to the number of myosin II motors participating in processive sliding of actin.

    PubMed

    Rastogi, Khushboo; Puliyakodan, Mohammed Shabeel; Pandey, Vikas; Nath, Sunil; Elangovan, Ravikrishnan

    2016-01-01

    In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads. PMID:27554800

  4. Maximum limit to the number of myosin II motors participating in processive sliding of actin

    PubMed Central

    Rastogi, Khushboo; Puliyakodan, Mohammed Shabeel; Pandey, Vikas; Nath, Sunil; Elangovan, Ravikrishnan

    2016-01-01

    In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads. PMID:27554800

  5. Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum

    PubMed Central

    Swärd, Karl; Dreja, Karl; Susnjar, Marija; Hellstrand, Per; Hartshorne, David J; Walsh, Michael P

    2000-01-01

    Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK).The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum.Contraction of permeabilized muscle strips induced by GTPγS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known Ki values for inhibition of ROK. GTPγS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077.Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]i induced by carbachol.LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation.In the absence of Ca2+, GTPγS stimulated 35S incorporation from [35S]ATPγS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected.These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction. PMID:10618150

  6. Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

    SciTech Connect

    Gerthoffer, W.T.; Murphey, K.A.; Khoyi, M.A.

    1988-08-01

    Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased /sup 45/Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on /sup 45/Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.

  7. Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

    PubMed Central

    Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo

    2015-01-01

    Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii. PMID:26544049

  8. Contingent Involuntary Motoric Inhibition: The Involuntary Inhibition of a Motor Response Contingent on Top-Down Goals

    ERIC Educational Resources Information Center

    Anderson, Brian A.; Folk, Charles L.

    2012-01-01

    Effective motor control involves both the execution of appropriate responses and the inhibition of inappropriate responses that are evoked by response-associated stimuli. The inhibition of a motor response has traditionally been characterized as either a voluntary act of cognitive control or a low-level perceptual bias arising from processes such…

  9. Using voluntary motor commands to inhibit involuntary arm movements.

    PubMed

    Ghosh, Arko; Rothwell, John; Haggard, Patrick

    2014-11-01

    A hallmark of voluntary motor control is the ability to stop an ongoing movement. Is voluntary motor inhibition a general neural mechanism that can be focused on any movement, including involuntary movements, or is it mere termination of a positive voluntary motor command? The involuntary arm lift, or 'floating arm trick', is a distinctive long-lasting reflex of the deltoid muscle. We investigated how a voluntary motor network inhibits this form of involuntary motor control. Transcranial magnetic stimulation of the motor cortex during the floating arm trick produced a silent period in the reflexively contracting deltoid muscle, followed by a rebound of muscle activity. This pattern suggests a persistent generator of involuntary motor commands. Instructions to bring the arm down voluntarily reduced activity of deltoid muscle. When this voluntary effort was withdrawn, the involuntary arm lift resumed. Further, voluntary motor inhibition produced a strange illusion of physical resistance to bringing the arm down, as if ongoing involuntarily generated commands were located in a 'sensory blind-spot', inaccessible to conscious perception. Our results suggest that voluntary motor inhibition may be a specific neural function, distinct from absence of positive voluntary motor commands. PMID:25253453

  10. Actin age orchestrates myosin-5 and myosin-6 run lengths.

    PubMed

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

    2015-08-01

    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 in which 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 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 toward the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks toward the pointed end of F-actin [5], traveling toward 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, whereas 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

  11. Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V

    PubMed Central

    Rogers, Stephen L.; Karcher, Ryan L.; Roland, Joseph T.; Minin, Alexander A.; Steffen, Walter; Gelfand, Vladimir I.

    1999-01-01

    Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161–164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis. PMID:10491390

  12. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  13. Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: a genetic tool for the next generation

    PubMed Central

    Sparkes, Imogen A.; Teanby, Nicholas A.; Hawes, Chris

    2008-01-01

    Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed. PMID:18503043

  14. Porcine myosin-VI: characterization of a new mammalian unconventional myosin

    PubMed Central

    1994-01-01

    We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain. PMID:7929586

  15. Transport of ER vesicles on actin filaments in neurons by myosin V.

    PubMed

    Tabb, J S; Molyneaux, B J; Cohen, D L; Kuznetsov, S A; Langford, G M

    1998-11-01

    Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for

  16. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins

    PubMed Central

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan

    2016-01-01

    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes. PMID:27597808

  17. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins.

    PubMed

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan

    2016-01-01

    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes. PMID:27597808

  18. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  19. Roles for kinesin and myosin during cytokinesis.

    PubMed Central

    Hepler, Peter K; Valster, Aline; Molchan, Tasha; Vos, Jan W

    2002-01-01

    Cytokinesis in higher plants involves the phragmoplast, a complex cytoplasmic structure that consists of microtubules (MTs), microfilaments (MFs) and membrane elements. Both MTs and MFs are essential for cell plate formation, although it is not clear which motor proteins are involved. Some candidate processes for motor proteins include transport of Golgi vesicles to the plane of the cell plate and the spatiotemporal organization of the cytoskeletal elements in order to achieve proper deposition and alignment of the cell plate. We have focused on the kinesin-like calmodulin binding protein (KCBP) and, more broadly, on myosins. Using an antibody that constitutively activates KCBP, we find that this MT motor, which is minus-end directed, contributes to the organization of the spindle and phragmoplast MTs. It does not participate in vesicle transport; rather, because of the orientation of the phragmoplast MTs, it is supposed that plus-end kinesins fill this role. Myosins, on the other hand, based on their inhibition with 2,3-butanedione monoxime and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), are associated with the process of post-mitotic spindle/phragmoplast alignment and with late lateral expansion of the cell plate. They are also not the principal motors involved in vesicle transport. PMID:12079671

  20. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Day, I. S.

    2001-01-01

    BACKGROUND: Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. RESULTS: Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. CONCLUSIONS: Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

  1. Alpha oscillatory correlates of motor inhibition in the aged brain

    PubMed Central

    Bönstrup, Marlene; Hagemann, Julian; Gerloff, Christian; Sauseng, Paul; Hummel, Friedhelm C.

    2015-01-01

    Exerting inhibitory control is a cognitive ability mediated by functions known to decline with age. The goal of this study is to add to the mechanistic understanding of cortical inhibition during motor control in aged brains. Based on behavioral findings of impaired inhibitory control with age we hypothesized that elderly will show a reduced or a lack of EEG alpha-power increase during tasks that require motor inhibition. Since inhibitory control over movements has been shown to rely on prior motor memory formation, we investigated cortical inhibitory processes at two points in time—early after learning and after an overnight consolidation phase and hypothesized an overnight increase of inhibitory capacities. Young and elderly participants acquired a complex finger movement sequence and in each experimental session brain activity during execution and inhibition of the sequence was recorded with multi-channel EEG. We assessed cortical processes of sustained inhibition by means of task-induced changes of alpha oscillatory power. During inhibition of the learned movement, young participants showed a significant alpha power increase at the sensorimotor cortices whereas elderly did not. Interestingly, for both groups, the overnight consolidation phase improved up-regulation of alpha power during sustained inhibition. This points to deficits in the generation and enhancement of local inhibitory mechanisms at the sensorimotor cortices in aged brains. However, the alpha power increase in both groups implies neuroplastic changes that strengthen the network of alpha power generation over time in young as well as elderly brains. PMID:26528179

  2. Motor cortical excitability and inhibition in acquired mirror pain.

    PubMed

    Fitzgibbon, Bernadette M; Enticott, Peter G; Bradshaw, John L; Giummarra, Melita J; Georgiou-Karistianis, Nellie; Chou, Michael; Fitzgerald, Paul B

    2012-11-21

    'Mirror pain' describes when the observation of another's pain experience induces a personal experience of pain. It has been suggested that mirror pain could result from changes in neural excitability or inhibition. In this study we used transcranial magnetic stimulation (TMS) to investigate motor cortical excitability in lower-limb amputees who experience mirror pain. Using paired-pulse TMS to assess motor cortical inhibition (CI) and cortical facilitation (CF), recordings were taken from the right first dorsal interosseus in lower-limb amputees who experience mirror pain (MP+), lower-limb amputees who do not experience mirror pain (MP-), and non-amputee controls. No differences in CI or CF were observed between the MP+ and both control groups. Thus, when not paired with a pain-related stimulus, changes in motor cortical excitability do not appear to contribute to the experience of mirror pain in lower-limb amputees. PMID:23022471

  3. Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

    PubMed Central

    Root, D D; Reisler, E

    1992-01-01

    The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin. PMID:1420910

  4. Myosin light-chain phosphatase.

    PubMed Central

    Morgan, M; Perry, S V; Ottaway, J

    1976-01-01

    1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain. Images PLATE 1 PMID:186030

  5. Directional Mechanosensing in Myosin VI

    NASA Astrophysics Data System (ADS)

    Yang, Yubo; Tehver, Riina

    2013-03-01

    Myosin is a family of versatile motor proteins that perform various tasks, such as organelle transport, anchoring and cell deformation. Although the general mechanism of the motors has been fairly well established, details on dynamic aspects like force response of the motor, and force propagation are yet to be fully understood. In this poster, we present the response of the ATP binding region to force exerted on the tail domain in order to test the proposed tension-dependent gating mechanism of myosin VI processive motion. We employed the Self-Organized Polymer model in a computer simulation to explore the effect. Current results show that the ATP binding domain of myosin VI indeed exhibits tension dependence - both structurally and dynamically.

  6. The myosin inhibitor blebbistatin stabilizes the super-relaxed state in skeletal muscle.

    PubMed

    Wilson, Clyde; Naber, Nariman; Pate, Edward; Cooke, Roger

    2014-10-01

    The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX. PMID:25296316

  7. The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

    PubMed Central

    Wilson, Clyde; Naber, Nariman; Pate, Edward; Cooke, Roger

    2014-01-01

    The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX. PMID:25296316

  8. 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

  9. A role for myosin IXb, a motor-RhoGAP chimera, in epithelial wound healing and tight junction regulation.

    PubMed

    Chandhoke, Surjit K; Mooseker, Mark S

    2012-07-01

    Polymorphisms in the gene encoding the heavy chain of myosin IXb (Myo9b) have been linked to several forms of inflammatory bowel disease (IBD). Given that Myo9b contains a RhoGTPase-activating protein domain within its tail, it may play key roles in Rho-mediated actin cytoskeletal modifications critical to intestinal barrier function. In wounded monolayers of the intestinal epithelial cell line Caco2(BBe) (BBe), Myo9b localizes to the extreme leading edge of lamellipodia of migrating cells. BBe cells exhibiting loss of Myo9b expression with RNA interference or Myo9b C-terminal dominant-negative (DN) tail-tip expression lack lamellipodia, fail to migrate into the wound, and form stress fiber-like arrays of actin at the free edges of cells facing the wound. These cells also exhibit disruption of tight junction (TJ) protein localization, including ZO-1, occludin, and claudin-1. Torsional motility and junctional permeability to dextran are greatly increased in cells expressing DN-tail-tip. Of interest, this effect is propagated to neighboring cells. Consistent with a role for Myo9b in regulating levels of active Rho, localization of both RhoGTP and myosin light chain phosphorylation corresponds to Myo9b-knockdown regions of BBe monolayers. These data reveal critical roles for Myo9b during epithelial wound healing and maintenance of TJ integrity-key functions that may be altered in patients with Myo9b-linked IBD. PMID:22573889

  10. Dissociation of motor and sensory inhibition processes in normal aging

    PubMed Central

    Anguera, Joaquin A.; Gazzaley, Adam

    2011-01-01

    Objective Age-related cognitive impairments have been attributed to deficits in inhibitory processes that mediate both motor restraint and sensory filtering. However, behavioral studies have failed to show an association between tasks that measure these distinct types of inhibition. In the present study, we hypothesized neural markers reflecting each type of inhibition may reveal a relationship across inhibitory domains in older adults. Methods Electroencephalography (EEG) and behavioral measures were used to explore whether there was an across-participant correlation between sensory suppression and motor inhibition. Sixteen healthy older adult participants (65-80 years) engaged in two separate experimental paradigms: a selective attention, delayed-recognition task and a stop-signal task. Results Findings revealed no significant relationship existed between neural markers of sensory suppression (P1 amplitude; N170 latency) and markers of motor inhibition (N2 and P3 amplitude and latency) in older adults. Conclusions These distinct inhibitory domains are differentially impacted in normal aging, as evidenced by previous behavioral work and the current neural findings. Thus a generalized inhibitory deficit may not be a common impairment in cognitive aging. Significance Given that some theories of cognitive aging suggest age-related failure of inhibitory mechanisms may span different modalities, the present findings contribute to an alternative view where age-related declines within each inhibitory modality are unrelated. PMID:21963321

  11. The unique enzymatic and mechanistic properties of plant myosins.

    PubMed

    Henn, Arnon; Sadot, Einat

    2014-12-01

    Myosins are molecular motors that move along actin-filament tracks. Plants express two main classes of myosins, myosin VIII and myosin XI. Along with their relatively conserved sequence and functions, plant myosins have acquired some unique features. Myosin VIII has the enzymatic characteristics of a tension sensor and/or a tension generator, similar to functions found in other eukaryotes. Interestingly, class XI plant myosins have gained a novel function that consists of propelling the exceptionally rapid cytoplasmic streaming. This specific class includes the fastest known translocating molecular motors, which can reach an extremely high velocity of about 60μms(-1). However, the enzymatic properties and mechanistic basis for these remarkable manifestations are not yet fully understood. Here we review recent progress in understanding the uniqueness of plant myosins, while emphasizing the unanswered questions. PMID:25435181

  12. 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

  13. A mechanochemical model for myosin VI

    NASA Astrophysics Data System (ADS)

    Tehver, Riina; Jack, Amanda; Lowe, Ian

    Myosin VI is a motor protein that transports cellular cargo along actin filaments. This transport takes place as a result of a coordinated mechano-chemical cycle that is controlled by external variables including imposed force and nucleotide concentrations. We present a model that captures the different dynamic pathways that myosin VI can take in response to these variables. The results of our model for experimentally observable quantities, such as the motor velocity or run length, agree with available experimental data, and we can also make predictions beyond the tested regimes. Using the model, we study how myosin VI reacts to its environment and test its operational efficiency.

  14. Abnormal dorsal premotor-motor inhibition in writer's cramp.

    PubMed

    Pirio Richardson, Sarah; Beck, Sandra; Bliem, Barbara; Hallett, Mark

    2014-05-01

    The authors hypothesized that a deficient premotor-motor inhibitory network contributes to the unwanted involuntary movements in dystonia. The authors studied nine controls and nine patients with writer's cramp (WC). Dorsal premotor-motor cortical inhibition (dPMI) was tested by applying conditioning transcranial magnetic stimulation (TMS) to the dorsal premotor cortex and then a test pulse to the ipsilateral motor cortex at an interval of 6 ms. The authors used an H-reflex in flexor carpi radialis paired with TMS over the premotor cortex to assess for spinal cord excitability change. Finally, the authors interrupted a choice reaction time task with TMS over dorsal premotor cortex to assess performance in a nondystonic task. The results showed that WC patients exhibited dPMI at rest (88.5%, the ratio of conditioned to unconditioned test pulse), in contrast to controls, who did not show dPMI (109.6%) (P = 0.0198). This difference between patients and controls persisted during contraction (100% vs. 112%) and pen-holding (95.6% vs. 111%). The H-reflex in the arm was not modulated by the premotor cortex stimulation. The WC patients made more errors, and the error rate improved with TMS over the premotor cortex. These results suggest that abnormal premotor-motor interactions may play a role in the pathophysiology of focal dystonia. The dPMI was not modulated by task in either group, but was constantly greater in the patients. The significance of the increased inhibition is likely to be compensatory. It appears to be a robust finding and, in combination with other features, could be further explored as a biomarker. PMID:24710852

  15. Central inhibition regulates motor output during physical fatigue.

    PubMed

    Tanaka, Masaaki; Shigihara, Yoshihito; Watanabe, Yasuyoshi

    2011-09-15

    Central fatigue refers to a progressive decline in the ability to activate muscles voluntarily. Although the existence of central inhibition of the motor area via visual feedback during physical fatigue was noted in a behavioral study, neural evidence has not been presented. The central mechanism to regulate physical fatigue was examined using a magnetoencephalographic (MEG) system. The study group consisted of eight healthy participants. They were randomly assigned to two groups in a crossover fashion to perform fatigue-inducing physical task sessions, in which they performed repetitive grips of a dominant hand at maximal voluntary contraction levels every second without using Ramachandran's mirror box to see the dominant hand or with using the mirror box to see the mirror reflection of the non-dominant hand to perceive that the fatigued dominant hand was not fatigued. Before and after each session, imagery of maximum grips of the dominant hand was performed for the evaluation with MEG. Beta-band event-related desynchronization (ERD) level of motor readiness magnetic field in the sensorimotor area in the contralateral hemisphere to the handgrips was not altered after the fatigue-inducing session without or with the mirror box. In contrast, the ERD level of motor movement-evoked magnetic field in the contralateral sensorimotor area was reduced after the fatigue-inducing session without the mirror box, although it was not altered after the session with the mirror box. We identified neural evidence of the central inhibition and showed that the visual feedback system is involved in the central mechanism regulating motor output. PMID:21803341

  16. CsmA, a Class V Chitin Synthase with a Myosin Motor-like Domain, Is Localized through Direct Interaction with the Actin Cytoskeleton in Aspergillus nidulans

    PubMed Central

    Takeshita, Norio; Ohta, Akinori; Horiuchi, Hiroyuki

    2005-01-01

    One of the essential features of fungal morphogenesis is the polarized synthesis of cell wall components such as chitin. The actin cytoskeleton provides the structural basis for cell polarity in Aspergillus nidulans, as well as in most other eukaryotes. A class V chitin synthase, CsmA, which contains a myosin motor-like domain (MMD), is conserved among most filamentous fungi. The ΔcsmA null mutant showed remarkable abnormalities with respect to cell wall integrity and the establishment of polarity. In this study, we demonstrated that CsmA tagged with 9× HA epitopes localized near actin structures at the hyphal tips and septation sites and that its MMD was able to bind to actin. Characterization of mutants bearing a point mutation or deletion in the MMD suggests that the interaction between the MMD and actin is not only necessary for the proper localization of CsmA, but also for CsmA function. Thus, the finding of a direct interaction between the chitin synthase and the actin cytoskeleton provides new insight into the mechanisms of polarized cell wall synthesis and fungal morphogenesis. PMID:15703213

  17. PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

    PubMed Central

    MEDEIROS, NELSON A.; REESE, THOMAS S.; JAFFE, HOWARD; DEGIORGIS, JOSEPH A.; BEARER, ELAINE L.

    2013-01-01

    The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60–95%) and chick pectoralis muscle (31–83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50–60%, identities), and some also matched unconventional myosins (33–50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles. PMID:9878103

  18. 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.

  19. Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy.

    PubMed

    Tang, Hong-Wen; Wang, Yu-Bao; Wang, Shiu-Lan; Wu, Mei-Hsuan; Lin, Shu-Yu; Chen, Guang-Chao

    2011-02-16

    Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein. PMID:21169990

  20. A Model of Motor Inhibition for a Complex Skill: Baseball Batting

    ERIC Educational Resources Information Center

    Gray, Rob

    2009-01-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different…

  1. Perceptual and Motor Inhibition in Individuals With Vestibular Disorders

    PubMed Central

    Mohammad, Maha T.; Whitney, Susan L.; Sparto, Patrick J.; Jennings, J. Richard; Furman, Joseph M.

    2016-01-01

    Background and Purpose Vestibular dysfunction has been shown to be associated with altered cognitive function. The purpose of this study was to examine changes in cognitive function in participants with vestibular disease during the course of vestibular physical therapy. Methods Twenty-two participants (mean age = 52, standard deviation = 11) with previously diagnosed vestibular disorders were tested at the beginning and end of rehabilitation. The Motor and Perceptual Inhibition Test (MAPIT) was used to assess manual reaction times when responding to various stimuli presented on a computer screen. Additional physical performance measures and questionnaires related to dizziness, fear of falling, and activities of daily living were used to quantify change during the 6-week intervention period. The repeatable battery for the assessment of neuropsychological status (a measure of memory and executive function) was used to ensure that participants did not have memory or executive function deficits. Results Overall, there were no significant differences in MAPIT score before versus after physical therapy intervention, however there were some participants who demonstrated improvements in motor inhibition (MI) and perceptual inhibition (PI) scores. Interstingly, 8 of the 9 participants with abnormal caloric test findings had improvements on 2 of the PI scores. Overall 50% to 64% of the participants demonstrated improvement in the 4 different MAPIT scores. There were improvements in physical performance and self-report measures at the end of the 6-week physical therapy intervention program. Discussion/Conclusion Individuals with vestibular disorders may show improvement in MI and PI after a 6-week physical therapy intervention program; those with abnormalities on caloric and rotational chair tests appear especially likely to experience improvement in PI. Additional study is needed to determine whether individuals with vestibular disorders have remediable deficits in MI and

  2. Myosin VI contributes to malignant proliferation of human glioma cells

    PubMed Central

    Xu, Rong; Fang, Xu-hao

    2016-01-01

    Previously characterized as a backward motor, myosin VI (MYO6), which belongs to myosin family, moves toward the minus end of the actin track, a direction opposite to all other known myosin members. Recent researches have illuminated the role of MYO6 in human cancers, particularly in prostate cancer. However, the role of MYO6 in glioma has not yet been determined. In this study, to explore the role of MYO6 in human glioma, lentivirus-delivered short hairpin RNA (shRNA) targeting MYO6 was designed to stably down-regulate its endogenous expression in glioblastoma cells U251. Knockdown of MYO6 signifi cantly inhibited viability and proliferation of U251 cells in vitro. Moreover, the cell cycle of U251 cells was arrested at G0/G1 phase with the absence of MYO6, which could contribute to the suppression of cell proliferation. In conclusion, we firstly identified the crucial involvement of MYO6 in human glioma. The inhibition of MYO6 by shRNA might be a potential therapeutic method in human glioma. PMID:26937209

  3. Imagining is Not Doing but Involves Specific Motor Commands: A Review of Experimental Data Related to Motor Inhibition

    PubMed Central

    Guillot, Aymeric; Di Rienzo, Franck; MacIntyre, Tadhg; Moran, Aidan; Collet, Christian

    2012-01-01

    There is now compelling evidence that motor imagery (MI) and actual movement share common neural substrate. However, the question of how MI inhibits the transmission of motor commands into the efferent pathways in order to prevent any movement is largely unresolved. Similarly, little is known about the nature of the electromyographic activity that is apparent during MI. In addressing these gaps in the literature, the present paper argues that MI includes motor execution commands for muscle contractions which are blocked at some level of the motor system by inhibitory mechanisms. We first assemble data from neuroimaging studies that demonstrate that the neural networks mediating MI and motor performance are not totally overlapping, thereby highlighting potential differences between MI and actual motor execution. We then review MI data indicating the presence of subliminal muscular activity reflecting the intrinsic characteristics of the motor command as well as increased corticomotor excitability. The third section not only considers the inhibitory mechanisms involved during MI but also examines how the brain resolves the problem of issuing the motor command for action while supervising motor inhibition when people engage in voluntary movement during MI. The last part of the paper draws on imagery research in clinical contexts to suggest that some patients move while imagining an action, although they are not aware of such movements. In particular, experimental data from amputees as well as from patients with Parkinson’s disease are discussed. We also review recent studies based on comparing brain activity in tetraplegic patients with that from healthy matched controls that provide insights into inhibitory processes during MI. We conclude by arguing that based on available evidence, a multifactorial explanation of motor inhibition during MI is warranted. PMID:22973214

  4. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    PubMed Central

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  5. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  6. Modulation of motor activity by cutaneous input: inhibition of the magnetic motor evoked potential by digital electrical stimulation.

    PubMed

    Clouston, P D; Kiers, L; Menkes, D; Sander, H; Chiappa, K; Cros, D

    1995-04-01

    We examined the inhibitory effect of a brief train of digital (D2) electrical stimuli at 4 times perception threshold on transcranial magnetic motor evoked potentials (MEPs) recorded from abductor pollicis brevis (APB) and flexor carpi radialis (FCR) muscles ipsilateral to the side of D2 stimulation. We compared this to the inhibitory effect of ipsilateral D2 stimulation on averaged rectified EMG recorded at 10% maximum voluntary contraction and on F-responses and H-reflexes recorded from these same muscles. We also compared MEPs recorded following D2 stimulation just above perception threshold to MEPs following higher intensity D2 stimulation. As well, we assessed the effect of preceding D2 stimulation on MEPs recorded from a relaxed versus tonically contracted hand muscle. D2 stimulation elicited a triphasic response of modest MEP facilitation followed by inhibition and further facilitation. The duration and onset of MEP inhibition correlated with those of the initial period of rectified EMG inhibition, however, the magnitude of MEP inhibition was generally less than the magnitude of EMG inhibition, consistent with a greater inhibitory effect of digital afferents on smaller motor neurons. MEPs were not facilitated during the rebound of EMG activity (the E2 period) that usually followed the initial period of EMG inhibition (I1 period). The behavior of H-reflexes and F-responses following ipsilateral D2 stimulation suggested that inhibition of both EMG and MEPs is not mediated via presynaptic inhibition of Ia afferents, and that inhibition is augmented by descending rather than segmental input to spinal motor neurons. Tonic contraction of the target muscle during D2 stimulation decreased the inhibitory effect of the preceding digital stimulus possibly due to recruitment of larger spinal motor neurons less likely to be inhibited by cutaneous input. PMID:7537203

  7. Cognitive Control Reflects Context Monitoring, Not Motoric Stopping, in Response Inhibition

    PubMed Central

    Chatham, Christopher H.; Claus, Eric D.; Kim, Albert; Curran, Tim; Banich, Marie T.; Munakata, Yuko

    2012-01-01

    The inhibition of unwanted behaviors is considered an effortful and controlled ability. However, inhibition also requires the detection of contexts indicating that old behaviors may be inappropriate – in other words, inhibition requires the ability to monitor context in the service of goals, which we refer to as context-monitoring. Using behavioral, neuroimaging, electrophysiological and computational approaches, we tested whether motoric stopping per se is the cognitively-controlled process supporting response inhibition, or whether context-monitoring may fill this role. Our results demonstrate that inhibition does not require control mechanisms beyond those involved in context-monitoring, and that such control mechanisms are the same regardless of stopping demands. These results challenge dominant accounts of inhibitory control, which posit that motoric stopping is the cognitively-controlled process of response inhibition, and clarify emerging debates on the frontal substrates of response inhibition by replacing the centrality of controlled mechanisms for motoric stopping with context-monitoring. PMID:22384038

  8. Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

    PubMed Central

    Walter, Nadine; Holweg, Carola L

    2008-01-01

    Background The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. Results We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar) F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. Conclusion The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of organelles such as peroxisomes

  9. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

    DeGiorgis, Joseph A.; Reese, Thomas S.; Bearer, Elaine L.

    2002-01-01

    Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor. PMID:11907281

  10. Functions of plant-specific myosin XI: from intracellular motility to plant postures.

    PubMed

    Ueda, Haruko; Tamura, Kentaro; Hara-Nishimura, Ikuko

    2015-12-01

    The plant-specific protein motor class myosin XI is known to function in rapid bulk flow of the cytoplasm (cytoplasmic streaming) and in organellar movements. Recent studies unveiled a wide range of physiological functions of myosin XI motors, from intracellular motility to organ movements. Arabidopsis thaliana has 13 members of myosin XI class. In vegetative organs, myosins XIk, XI1, and XI2 primarily contribute to dynamics and spatial configurations of endoplasmic reticulum that develops a tubular network in the cell periphery and thick strand-like structures in the inner cell regions. Myosin XI-i forms a nucleocytoplasmic linker and is responsible for nuclear movement and shape. In addition to these intracellular functions, myosin XIf together with myosin XIk is involved in the fundamental nature of plants; the actin-myosin XI cytoskeleton regulates organ straightening to adjust plant posture. PMID:26432645

  11. CORRELATIONS OF PESTICIDE-INDUCED CHOLINESTERASE INHIBITION AND MOTOR ACTIVITY CHANGES IN ADULT RATS.

    EPA Science Inventory

    The acute neurobehavioral effects of acetylcholinesterase-inhibiting pesticides are primarily due to overstimulation of the cholinergic system. Lowered motor activity levels represent a sensitive endpoint with which to monitor functional changes in laboratory animals exposed to ...

  12. Lipid Emulsion Inhibits Vasodilation Induced by a Toxic Dose of Bupivacaine via Attenuated Dephosphorylation of Myosin Phosphatase Target Subunit 1 in Isolated Rat Aorta

    PubMed Central

    Ok, Seong-Ho; Byon, Hyo-Jin; Kwon, Seong-Chun; Park, Jungchul; Lee, Youngju; Hwang, Yeran; Baik, Jiseok; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2015-01-01

    Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca2+]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca2+]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF. PMID:26664257

  13. Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking.

    PubMed

    Li, Jianchao; Lu, Qing; Zhang, Mingjie

    2016-08-01

    Unconventional myosins are a superfamily of actin-based molecular motors playing diverse roles including cellular trafficking, mechanical supports, force sensing and transmission, etc. The variable neck and tail domains of unconventional myosins function to bind to specific cargoes including proteins and lipid vesicles and thus are largely responsible for the diverse cellular functions of myosins in vivo. In addition, the tail regions, together with their cognate cargoes, can regulate activities of the motor heads. This review outlines the advances made in recent years on cargo recognition and cargo binding-induced regulation of the activity of several unconventional myosins including myosin-I, V, VI and X in cellular trafficking. We approach this topic by describing a series of high-resolution structures of the neck and tail domains of these unconventional myosins either alone or in complex with their specific cargoes, and by discussing potential implications of these structural studies on cellular trafficking of these myosin motors. PMID:26842936

  14. Myosins and cell dynamics in cellular slime molds.

    PubMed

    Yumura, Shigehiko; Uyeda, Taro Q P

    2003-01-01

    Myosin is a mechanochemical transducer and serves as a motor for various motile activities such as cell migration, cytokinesis, maintenance of cell shape, phagocytosis, and morphogenesis. Nonmuscle myosin in vivo does not either stay static at specific subcellular regions or construct highly organized structures, such as sarcomere in skeletal muscle cells. The cellular slime mold Dictyostelium discoideum is an ideal "model organism" for the investigation of cell movement and cytokinesis. The advantages of this organism prompted researchers to carry out pioneering cell biological, biochemical, and molecular genetic studies on myosin II, which resulted in elucidation of many fundamental features of function and regulation of this most abundant molecular motor. Furthermore, recent molecular biological research has revealed that many unconventional myosins play various functions in vivo. In this article, how myosins are organized and regulated in a dynamic manner in Dictyostelium cells is reviewed and discussed. PMID:12722951

  15. The role of myosin 1c and myosin 1b in surfactant exocytosis.

    PubMed

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-04-15

    Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  16. The role of myosin 1c and myosin 1b in surfactant exocytosis

    PubMed Central

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-01-01

    ABSTRACT Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  17. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  18. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

    PubMed Central

    Reisen, Daniel; Hanson, Maureen R

    2007-01-01

    Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1), myosin XI-6 (At MYA2), myosin XI-8 (At XI-B), myosin XI-15 (At XI-I), myosin XI-16 (At XI-J) and myosin XI-17 (At XI-K) were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2), previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and/or organelles when

  19. Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

    PubMed Central

    Kaiser, Christian M.; Bujalowski, Paul J.; Ma, Liang; Anderson, John; Epstein, Henry F.; Oberhauser, Andres F.

    2012-01-01

    Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail. PMID:22824286

  20. Demand on skillfulness modulates interhemispheric inhibition of motor cortices.

    PubMed

    Wischnewski, Miles; Kowalski, Greg M; Rink, Farrah; Belagaje, Samir R; Haut, Marc W; Hobbs, Gerald; Buetefisch, Cathrin M

    2016-06-01

    The role of primary motor cortex (M1) in the control of hand movements is still unclear. Functional magnetic resonance imaging (fMRI) studies of unimanual performance reported a relationship between level of precision of a motor task and additional ipsilateral M1 (iM1) activation. In the present study, we determined whether the demand on accuracy of a movement influences the magnitude of the inhibitory effect between primary motor cortices (IHI). We used transcranial magnetic stimulation (TMS) to measure active IHI (aIHI) of the iM1 on the contralateral M1 (cM1) in the premovement period of a left-hand motor task. Ten healthy participants manipulated a joystick to point to targets of two different sizes. For aIHI, the conditioning stimulus (CS) was applied to iM1, and the test stimulus (TS) to cM1, with an interstimulus interval of 10 ms. The amount of the inhibitory effect of the CS on the motor-evoked potential (MEP) of the subsequent TS was expressed as percentage of the mean MEP amplitude evoked by the single TS. Across different time points of aIHI measurements in the premovement period, there was a significant effect for target size on aIHI. Preparing to point to small targets was associated with weaker aIHI compared with pointing to large targets. The present findings suggest that, during the premovement period, aIHI from iM1 on cM1 is modulated by the demand on accuracy of the motor task. This is consistent with task fMRI findings showing bilateral M1 activation during high-precision movements but only unilateral M1 activity during low-precision movements. PMID:26961108

  1. 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.

  2. Motor Inhibition during Overt and Covert Actions: An Electrical Neuroimaging Study

    PubMed Central

    Angelini, Monica; Calbi, Marta; Ferrari, Annachiara; Sbriscia-Fioretti, Beatrice; Franca, Michele; Gallese, Vittorio; Umiltà, Maria Alessandra

    2015-01-01

    Given ample evidence for shared cortical structures involved in encoding actions, whether or not subsequently executed, a still unsolved problem is the identification of neural mechanisms of motor inhibition, preventing “covert actions” as motor imagery from being performed, in spite of the activation of the motor system. The principal aims of the present study were the evaluation of: 1) the presence in covert actions as motor imagery of putative motor inhibitory mechanisms; 2) their underlying cerebral sources; 3) their differences or similarities with respect to cerebral networks underpinning the inhibition of overt actions during a Go/NoGo task. For these purposes, we performed a high density EEG study evaluating the cerebral microstates and their related sources elicited during two types of Go/NoGo tasks, requiring the execution or withholding of an overt or a covert imagined action, respectively. Our results show for the first time the engagement during motor imagery of key nodes of a putative inhibitory network (including pre-supplementary motor area and right inferior frontal gyrus) partially overlapping with those activated for the inhibition of an overt action during the overt NoGo condition. At the same time, different patterns of temporal recruitment in these shared neural inhibitory substrates are shown, in accord with the intended overt or covert modality of action performance. The evidence that apparently divergent mechanisms such as controlled inhibition of overt actions and contingent automatic inhibition of covert actions do indeed share partially overlapping neural substrates, further challenges the rigid dichotomy between conscious, explicit, flexible and unconscious, implicit, inflexible forms of motor behavioral control. PMID:26000451

  3. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    ERIC Educational Resources Information Center

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  4. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  5. Modulation of myosin filament activation by telokin in smooth muscle liberation of myosin kinase and phosphatase from supramolecular complexes.

    PubMed

    Sobieszek, Apolinary; Andruchov, Oleg Y; Grabarek, Zenon; Kulikova, Natalia; Liebetrau, Claudia; Matusovsky, Oleg S

    2005-01-01

    The mechanism of telokin action on reversible phosphorylation of turkey gizzard myosin was investigated using a native-like filamentous myosin. This myosin contained endogenous calmodulin (CaM) and myosin light chain kinase (MLCK) at a molar ratio to myosin of about 1 to 40 or less depending on the initial extractions conditions. These levels were sufficient to fully phosphorylate myosin within 20-40 s or less after addition of [gamma-32P]ATP, but when the ATP was depleted, they became dephosphorylated indicating the presence of myosin light chain phosphatase (MLCP). Addition of telokin at the 1 to 1 or higher molar ratio to myosin caused a three- to five-fold inhibition of the initial phosphorylation rates (without reduction of the overall extent of phosphorylation) and produced a similar increase in the rate of dephosphorylation. The inhibition was also observed for myosin filaments free of MLCK and CaM together with constitutively active MLCKs produced by digestion, or by expression of a truncated mammalian kinase as well as for the wild-type enzyme. Thus, neither N- nor C-terminal of MLCK was necessary for interaction of myosin with telokin and the inhibition resulted from telokin-induced change of myosin head configuration within the filament that prevented their ordered, paracrystaline-like, aggregation. Sedimentation of the filamentous myosin in glycerol gradients showed that this change made the filaments less compact and facilitated release of the endogenous MLCK/CaM complex. For a mixture of the filaments with or without the complex, the configuration change resulted in an increase of the phosphorylation rate but not in its inhibition. The increase of the rate resulting from the liberation of the complex was also observed in mixtures of the filamentous myosin with added isolated regulatory light chain (ReLC) or soluble myosin head subfragment. This observation reinforces the above conclusions. The acceleration of the MLCP activity by telokin was shown to

  6. Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

    PubMed Central

    Stauffer, Eric A.; Scarborough, John D.; Hirono, Moritoshi; Miller, Emilie D.; Shah, Kavita; Mercer, John A.; Holt, Jeffrey R.; Gillespie, Peter G.

    2009-01-01

    Summary In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells. PMID:16102537

  7. A model of motor inhibition for a complex skill: baseball batting.

    PubMed

    Gray, Rob

    2009-06-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different inhibition signals (out of range launch angle, early expected-actual trajectory discrepancy, and late expected-actual trajectory discrepancy) resulting in four possible response outcomes for the batter's swing (full swing, inhibited swing, partial response, or interrupted swing). The predictions of the model were compared with the actual batting performance of 20 baseball players using a high-fidelity batting simulator. The proportions of the different response outcomes could be explained by the inhibition model for 17/20 of the batters in the study. These findings suggest that models of motor inhibition developed for simple, discrete tasks can be applied to complex, multistage behaviors. This batting inhibition model could be used to provide a quantitative measure of a player's bat control for training and player-screening purposes. (PsycINFO Database Record (c) 2009 APA, all rights reserved). PMID:19586249

  8. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  9. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments. PMID:25712372

  10. Renshaw cells are inactive during motor inhibition elicited by the pontine microinjection of carbachol.

    PubMed

    Morales, F R; Engelhardt, J K; Pereda, A E; Yamuy, J; Chase, M H

    1988-04-12

    The present study was undertaken to determine whether the postsynaptic inhibition of motoneurons that occurs following the pontine microinjection of carbachol in the decerebrate cat is due to the activity of Renshaw cells. Thirty-two out of 37 Renshaw cells (86%) were spontaneously active prior to the administration of carbachol, whereas only 2 out of 13 Renshaw cells (15%) discharged during carbachol-induced motor inhibition. In addition, discrete inhibitory synaptic potentials were observed in 33% of the Renshaw cells from which intracellular recordings were obtained after carbachol administration, indicating that these cells were actively inhibited. The finding that a population of Renshaw cells, which inhibit motoneurons, were themselves inhibited during a period of profound motoneuron inhibition was quite unexpected. These results support the conclusion that Renshaw cells are not the inhibitory interneurons that are responsible for the powerful inhibition of motoneurons that occurs following the pontine microinjection of carbachol. PMID:3380320

  11. Impaired inhibition of prepotent motor actions in patients with Tourette syndrome

    PubMed Central

    Wylie, Scott A.; Claassen, Daniel O.; Kanoff, Kristen E.; Ridderinkhof, K. Richard; van den Wildenberg, Wery P.M.

    2013-01-01

    Background Evidence that tic behaviour in individuals with Tourette syndrome reflects difficulties inhibiting prepotent motor actions is mixed. Response conflict tasks produce sensitive measures of response interference from prepotent motor impulses and the proficiency of inhibiting these impulses as an act of cognitive control. We tested the hypothesis that individuals with Tourette syndrome show a deficit in inhibiting prepotent motor actions. Methods Healthy controls and older adolescents/adults with persistent Tourette syndrome without a history of obsessive–compulsive disorder or attention-deficit/hyperactivity disorder and presenting with stable mood functioning (i.e., no history of well-treated anxiety or depression) participated in this study. They performed a Simon task that induced conflict between prepotent actions and goal-directed actions. A novel theoretical framework distinguished group differences in acting impulsively (i.e., fast motor errors) from the proficiency of inhibiting interference by prepotent actions (i.e., slope of interference reduction). Results We included 27 controls and 28 individuals with Tourette syndrome in our study. Both groups showed similar susceptibility to making fast, impulsive motor errors (Tourette syndrome 26% v. control 23%; p = 0.10). The slope (m) reduction of the interference effect was significantly less pronounced among participants with Tourette syndrome than controls (Tourette syndrome: m = −0.07 v. control: m = −0.23; p = 0.022), consistent with deficient inhibitory control over prepotent actions in Tourette syndrome. Limitations This study does not address directly the role of psychiatric comorbidities and medication effects on inhibitory control over impulsive actions in individuals with Tourette syndrome. Conclusion The results offer empirical evidence for deficient inhibitory control over prepotent motor actions in individuals with persistent Tourette syndrome with minimal to absent psychiatric

  12. Intracortical inhibition and facilitation in different representations of the human motor cortex.

    PubMed

    Chen, R; Tam, A; Bütefisch, C; Corwell, B; Ziemann, U; Rothwell, J C; Cohen, L G

    1998-12-01

    Intracortical inhibition and facilitation in different representations of the human motor cortex. J. Neurophysiol. 80: 2870-2881, 1998. Intracortical inhibition (ICI) and intracortical facilitation (ICF) of the human motor cortex can be studied with paired transcranial magnetic stimulation (TMS). Plastic changes and some neurological disorders in humans are associated with changes in ICI and ICF. Although well characterized in the hand representation, it is not known if ICI and ICF vary across different body part representations. Therefore we studied ICI and ICF in different motor representations of the human motor cortex. The target muscles were rectus abdominus (RA), biceps brachii (BB), abductor pollicis brevis (APB), quadriceps femoris (QF), and abductor hallucis (AH). For each muscle, we measured the rest and active motor thresholds (MTs), the motor-evoked potential (MEP) stimulus-response curve (MEP recruitment), ICI, and ICF. The effects of different interstimulus intervals (ISIs) were studied with a conditioning stimulus (CS) intensity of 80% active MT. The effects of different CS intensities were studied at ISI of 2 ms for ICI and ISI of 15 ms for ICF. MT was lowest for APB, followed by BB, AH, and QF, and was highest for RA. Except for BB, MEP recruitment was generally steeper for muscles with lower MT. ICI and ICF were present in all the motor representations tested. The stimulus intensity necessary to elicit ICI was consistently lower than that required to elicit ICF, suggesting that they are mediated by separate mechanisms. Despite wide differences in MT and MEP recruitment, the absolute CS intensities (expressed as percentage of the stimulator's output) required to elicit ICI and ICF appear unrelated to MT and MEP recruitment in the different muscles tested. These findings suggest that the intracortical mechanisms for inhibition and facilitation in different motor representations are not related to the strength of corticospinal projections. PMID

  13. On the kinetics that moves Myosin V

    NASA Astrophysics Data System (ADS)

    Maes, Christian; O'Kelly de Galway, Winny

    2015-10-01

    Molecular motor proteins such as Myosin V, Dynein or Kinesin are no ratchets, at least not with a flashing asymmetric potential; the crucial asymmetry is in the dynamical activity. We make that explicit in terms of a simple Markov model, emphasizing the kinetic (and non-thermodynamic) aspects of stochastic transport. The analysis shows the presence of a fluctuation symmetry in that part of the dynamical activity which is antisymmetric under reversal of trailing and leading head of the motor. The direction of the motor motion is determined by it.

  14. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

    PubMed Central

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2016-01-01

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin’s ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31–41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  15. Negative and positive masked-priming – implications for motor inhibition

    PubMed Central

    Sumner, Petroc

    2008-01-01

    Masked stimuli can prime responses to subsequent target stimuli, causing response benefits when the prime is similar to the target. However, one masked-prime paradigm has produced counter-intuitive negative compatibility effects (NCE), such that performance costs occur when prime and target are similar. This NCE has been interpreted as an index of an automatic self-inhibition mechanism that suppresses the partial motor activation caused by the prime. However, several alternative explanations for the NCE have been proposed and supported by new evidence. As a framework for discussion, I divide the original theory into five potentially separable issues and briefly examine each with regard to alternative theories and current evidence. These issues are: 1) whether the NCE is caused by motor inhibition or perceptual interactions; 2) whether inhibition is self-triggered or stimulus-triggered; 3) whether prime visibility plays a causal role; 4) whether there is a threshold for triggering inhibition; 5) whether inhibition is automatic. Lastly, I briefly consider why NCEs have not been reported in other priming paradigms, and what the neural substrate for any automatic motor inhibition might be. PMID:20517517

  16. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats

    PubMed Central

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session. PMID:27314672

  17. Effects of Aerobic Fitness on Aging-Related Changes of Interhemispheric Inhibition and Motor Performance

    PubMed Central

    McGregor, Keith M.; Nocera, Joe R.; Sudhyadhom, Atchar; Patten, Carolynn; Manini, Todd M.; Kleim, Jeffrey A.; Crosson, Bruce; Butler, Andrew J.

    2013-01-01

    Physical fitness has been long associated with maintenance and improvement of motor performance as we age. In particular, measures of psychomotor speed and motor dexterity tend to be higher in physically fit aging adults as compared to their sedentary counterparts. Using functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation (TMS), we explored the patterns of neural activity that may, in part, account for differences between individuals of varying physical fitness levels. In this study, we enrolled both sedentary and physically fit middle age (40–60) and younger (18–30) adults and measured upper extremity motor performance during behavioral testing. In a follow-up session, we employed TMS and fMRI to assess levels of interhemispheric communication during unimanual tasks. Results show that increased physical fitness is associated with better upper extremity motor performance on distal dexterity assessments and increased levels of interhemispheric inhibition in middle age adults. Further, the functional correlates of changes of ipsilateral activity appears to be restricted to the aging process as younger adults of varying fitness levels do not differ in hemispheric patterns of activity or motor performance. We conclude that sedentary aging confers a loss of interhemispheric inhibition that is deleterious to some aspects of motor function, as early as midlife, but these changes can be mediated by chronic engagement in aerobic exercise. PMID:24198784

  18. Model of Rho-Mediated Myosin Recruitment to the Cleavage Furrow during Cytokinesis

    NASA Astrophysics Data System (ADS)

    Veksler, Alexander; Vavylonis, Dimitrios

    2010-03-01

    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 cytokinesis, the myosin attached to the cell's cortex progressively disassembles at the flanking regions and concentrates in the equator [1]. This recruitment depends on myosin motor activity and activation by Rho proteins. Central spindle and astral microtubules establish a spatial pattern of differential Rho activity [2]. We propose a reaction-diffusion model for the dynamics of myosin and Rho proteins during cytokinesis. In the model, the mitotic spindle activates Rho at the equator. Active Rho promotes, in a switch-like manner, myosin assembly into cortical minifilaments. Mechanical stress by cortical myosin causes disassembly of myosin minifilaments and deactivates Rho. Our results explain both the recruitment of myosin to the cleavage furrow and the observed damped myosin oscillations in the cell's flanking regions [1]. Spatial extent, period and decay rate of myosin oscillations are calculated. Various regimes of myosin recruitment are predicted. [1] Zhou & Wang, Mol. Biol. Cell 19:318 (2008) [2] Murthy & Wadsworth, J. Cell Sci. 121:2350 (2008)

  19. Inhibition of a Mitotic Motor Protein: Where, How, and Conformational Consequences

    SciTech Connect

    Yan, Youwei; Sardana, Vinod; Xu, Bei; Homnick, Carl; Halczenko, Wasyl; Buser, Carolyn A.; Schaber, Michael; Hartman, George D.; Huber, Hans E.; Kuo, Lawrence C.

    2010-11-16

    We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 {angstrom} resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg{sup 2+} {center_dot} ADP, reveals that monastrol confers inhibition by 'induced-fitting' onto the protein some 12 {angstrom} away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.

  20. 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.

  1. Force-producing ADP state of myosin bound to actin

    PubMed Central

    Wulf, Sarah F.; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G.; Pylypenko, Olena; Sweeney, H. Lee; Houdusse, Anne M.; Schröder, Rasmus R.

    2016-01-01

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  2. Force-producing ADP state of myosin bound to actin.

    PubMed

    Wulf, Sarah F; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G; Pylypenko, Olena; Sweeney, H Lee; Houdusse, Anne M; Schröder, Rasmus R

    2016-03-29

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  3. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  4. Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases.

    PubMed

    Joo, Emily; Surka, Mark C; Trimble, William S

    2007-11-01

    Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis. PMID:17981136

  5. Expression, Splicing, and Evolution of the Myosin Gene Family in Plants1[W][OA

    PubMed Central

    Peremyslov, Valera V.; Mockler, Todd C.; Filichkin, Sergei A.; Fox, Samuel E.; Jaiswal, Pankaj; Makarova, Kira S.; Koonin, Eugene V.; Dolja, Valerian V.

    2011-01-01

    Plants possess two myosin classes, VIII and XI. The myosins XI are implicated in organelle transport, filamentous actin organization, and cell and plant growth. Due to the large size of myosin gene families, knowledge of these molecular motors remains patchy. Using deep transcriptome sequencing and bioinformatics, we systematically investigated myosin genes in two model plants, Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon). We improved myosin gene models and found that myosin genes undergo alternative splicing. We experimentally validated the gene models for Arabidopsis myosin XI-K, which plays the principal role in cell interior dynamics, as well as for its Brachypodium ortholog. We showed that the Arabidopsis gene dubbed HDK (for headless derivative of myosin XI-K), which emerged through a partial duplication of the XI-K gene, is developmentally regulated. A gene with similar architecture was also found in Brachypodium. Our analyses revealed two predominant patterns of myosin gene expression, namely pollen/stamen-specific and ubiquitous expression throughout the plant. We also found that several myosins XI can be rhythmically expressed. Phylogenetic reconstructions indicate that the last common ancestor of the angiosperms possessed two myosins VIII and five myosins XI, many of which underwent additional lineage-specific duplications. PMID:21233331

  6. Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation

    PubMed Central

    Ren, Yixin; West-Foyle, Hoku; Surcel, Alexandra; Miller, Christopher; Robinson, Douglas N.

    2014-01-01

    How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in wild-type cells. 3xAsp myosin II's localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting in parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system. PMID:25318674

  7. The negative compatibility effect with relevant masks: a case for automatic motor inhibition

    PubMed Central

    Ocampo, Brenda; Finkbeiner, Matthew

    2013-01-01

    For many years controversy has surrounded the so-called “negative compatibility effect” (NCE), a surprising phenomenon whereby responses to a target stimulus are delayed when the target is preceded by an unconscious, response-compatible prime. According to proponents of the “self-inhibition” hypothesis, the NCE occurs when a low-level self-inhibitory mechanism supresses early motor activations that are no longer supported by perceptual evidence. This account has been debated, however, by those who regard the NCE to be a stimulus-specific phenomenon that can be explained without recourse to a self-inhibitory mechanism. The present study used a novel reach-to-touch paradigm to test whether unconscious response priming would manifest as motor activation of the opposite-to-prime response (supporting mask-induced priming accounts), or motor inhibition of the primed response (supporting the notion of low-level self-inhibition). This paper presents new findings that show the emergence of positive and negative compatibility effects as they occur in stimulus processing time. In addition, evidence is provided suggesting that the NCE is not driven by the activation of the incorrect, “opposite-to-prime” response, but rather might reflect automatic motor inhibition. PMID:24265623

  8. Frontal and motor cortex contributions to response inhibition: evidence from electrocorticography.

    PubMed

    Fonken, Yvonne M; Rieger, Jochem W; Tzvi, Elinor; Crone, Nathan E; Chang, Edward; Parvizi, Josef; Knight, Robert T; Krämer, Ulrike M

    2016-04-01

    Changes in the environment require rapid modification or inhibition of ongoing behavior. We used the stop-signal paradigm and intracranial recordings to investigate response preparation, inhibition, and monitoring of task-relevant information. Electrocorticographic data were recorded in eight patients with electrodes covering frontal, temporal, and parietal cortex, and time-frequency analysis was used to examine power differences in the beta (13-30 Hz) and high-gamma bands (60-180 Hz). Over motor cortex, beta power decreased, and high-gamma power increased during motor preparation for both go trials (Go) and unsuccessful stops (US). For successful stops (SS), beta increased, and high-gamma was reduced, indexing the cancellation of the prepared response. In the middle frontal gyrus (MFG), stop signals elicited a transient high-gamma increase. The MFG response occurred before the estimated stop-signal reaction time but did not distinguish between SS and US trials, likely signaling attention to the salient stop stimulus. A postresponse high-gamma increase in MFG was stronger for US compared with SS and absent in Go, supporting a role in behavior monitoring. These results provide evidence for differential contributions of frontal subregions to response inhibition, including motor preparation and inhibitory control in motor cortex and cognitive control and action evaluation in lateral prefrontal cortex. PMID:26864760

  9. Association of kinesin and myosin with pigment granules in crustacean chromatophores.

    PubMed

    Boyle, Robert Tew; McNamara, John Campbell

    2006-02-01

    Chromatic adaptation in crustaceans results from the differential distribution of colored pigment granules within their chromatophores consequent to cell signaling by neurosecretory peptides. However, the force transducing, mechanochemical protein motors responsible for granule translocation, and their molecular mechanisms of action, are not well understood. The present study uses immunocytochemical techniques and a motility assay in vitro to demonstrate that protein motors from the kinesin and myosin superfamilies are stably associated with membrane-bounded pigment granules in the red, ovarian chromatophores of the freshwater, palaemonid shrimp, Macrobrachium olfersii. Monoclonal antibodies against conventional kinesin heavy chain, and an anti-myosin whole serum, labeled pigment-containing fragments prepared from homogenates of chromatophores with fully dispersed or aggregated pigments: this finding infers a permanent association between the protein motors and the pigment granules, and suggests that such motors may be regulated while bound to their cargos. The pigment aggregator appears to be a myosin since the anti-myosin whole serum attenuated hormonally triggered pigment aggregation in the motility assay in vitro, and induced pigment hyper-dispersion in some chromatophores. Western blots of the chromatophore-containing, ovarian tissue homogenate demonstrated protein bands consistent with myosin II and myosin XII, either of which may be the pigment aggregator. This study provides the first direct evidence for myosin and kinesin protein motors directly and stably associated with pigment granules in crustacean chromatophores, and may represent the first successful isolation of myosin class XII. PMID:16420248

  10. Motor Inhibition Affects the Speed But Not Accuracy of Aimed Limb Movements in an Insect

    PubMed Central

    Calas-List, Delphine; Clare, Anthony J.; Komissarova, Alexandra; Nielsen, Thomas A.

    2014-01-01

    When reaching toward a target, human subjects use slower movements to achieve higher accuracy, and this can be accompanied by increased limb impedance (stiffness, viscosity) that stabilizes movements against motor noise and external perturbation. In arthropods, the activity of common inhibitory motor neurons influences limb impedance, so we hypothesized that this might provide a mechanism for speed and accuracy control of aimed movements in insects. We recorded simultaneously from excitatory leg motor neurons and from an identified common inhibitory motor neuron (CI1) in locusts that performed natural aimed scratching movements. We related limb movement kinematics to recorded motor activity and demonstrate that imposed alterations in the activity of CI1 influenced these kinematics. We manipulated the activity of CI1 by injecting depolarizing or hyperpolarizing current or killing the cell using laser photoablation. Naturally higher levels of inhibitory activity accompanied faster movements. Experimentally biasing the firing rate downward, or stopping firing completely, led to slower movements mediated by changes at several joints of the limb. Despite this, we found no effect on overall movement accuracy. We conclude that inhibitory modulation of joint stiffness has effects across most of the working range of the insect limb, with a pronounced effect on the overall velocity of natural movements independent of their accuracy. Passive joint forces that are greatest at extreme joint angles may enhance accuracy and are not affected by motor inhibition. PMID:24872556

  11. Time-course of motor inhibition during hypnotic paralysis: EEG topographical and source analysis.

    PubMed

    Cojan, Yann; Archimi, Aurélie; Cheseaux, Nicole; Waber, Lakshmi; Vuilleumier, Patrik

    2013-02-01

    Cognitive hypotheses of hypnotic phenomena have proposed that executive attentional systems may be either inhibited or overactivated to produce a selective alteration or disconnection of some mental operations. Recent brain imaging studies have reported changes in activity in both medial (anterior cingulate) and lateral (inferior) prefrontal areas during hypnotically induced paralysis, overlapping with areas associated with attentional control as well as inhibitory processes. To compare motor inhibition mechanisms responsible for paralysis during hypnosis and those recruited by voluntary inhibition, we used electroencephalography (EEG) to record brain activity during a modified bimanual Go-Nogo task, which was performed either in a normal baseline condition or during unilateral paralysis caused by hypnotic suggestion or by simulation (in two groups of participants, each tested once with both hands valid and once with unilateral paralysis). This paradigm allowed us to identify patterns of neural activity specifically associated with hypnotically induced paralysis, relative to voluntary inhibition during simulation or Nogo trials. We used a topographical EEG analysis technique to investigate both the spatial organization and the temporal sequence of neural processes activated in these different conditions, and to localize the underlying anatomical generators through minimum-norm methods. We found that preparatory activations were similar in all conditions, despite left hypnotic paralysis, indicating preserved motor intentions. A large P3-like activity was generated by voluntary inhibition during voluntary inhibition (Nogo), with neural sources in medial prefrontal areas, while hypnotic paralysis was associated with a distinctive topography activity during the same time-range and specific sources in right inferior frontal cortex. These results add support to the view that hypnosis might act by enhancing executive control systems mediated by right prefrontal areas, but

  12. Review: The ATPase mechanism of myosin and actomyosin.

    PubMed

    Geeves, Michael A

    2016-08-01

    Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016. PMID:27061920

  13. Myosin assembly critical for the enzyme activity of smooth muscle myosin phosphatase: effects of MgATP, ionic strength, and Mg(2+).

    PubMed

    Sato, O; Ogawa, Y

    2001-06-01

    We suggested that an assembled form of phosphorylated myosin (P-myosin) might exhibit higher affinity for smooth muscle myosin phosphatase (SMMP) than dissociated P-myosin on the basis of the effect of MgATP [Sato and Ogawa (1999) J. Biochem. 126, 787-797]. To further deepen our understanding, we examined the SMMP activity and P-myosin assembly with various ionic strengths and Mg(2+) concentrations, with and without MgATP, all of which are well known to be critical for myosin assembly. The structure of myosin molecules was directly observed by electron microscopy using a rotary shadowing procedure, which was found to be consistent with the sedimentation assay. We found that the SMMP activity was always high when P-myosin was assembled. MgATP, which disassembled P-myosin mostly into a folded conformation, in contrast, decreased the enzyme activity. We also found that glycerol had a dissociating action on P-myosin, primarily dissociating it into an extended conformation, resulting in reduced SMMP activity, and that increases in the ionic strength and Mg(2+) (>5 mM) inhibited SMMP. These results indicate that myosin assembly is essential for SMMP activity. PMID:11388902

  14. The effect of motor load on planning and inhibition in developmental coordination disorder.

    PubMed

    Pratt, Michelle L; Leonard, Hayley C; Adeyinka, Hanna; Hill, Elisabeth L

    2014-07-01

    Previous research has reported mixed findings regarding executive function (EF) abilities in developmental coordination disorder (DCD), which is diagnosed on the basis of significant impairments in motor skills. The current study aimed to assess whether these differences in study outcomes could result from the relative motor loads of the tasks used to assess EF in DCD. Children with DCD had significant difficulties on measures of inhibition and planning compared to a control group, although there were no significant correlations between motor skills and EF task performance in either group. The complexity of the response, as well as the component skills required in EF tasks, should be considered in future research to ensure easier comparison across studies and a better understanding of EF in DCD over development. PMID:24770468

  15. Analogous corticocortical inhibition and facilitation in ipsilateral and contralateral human motor cortex representations of the tongue.

    PubMed

    Muellbacher, W; Boroojerdi, B; Ziemann, U; Hallett, M

    2001-11-01

    How the human brain controls activation of the ipsilateral part of midline muscles is unknown. We studied corticospinal and corticocortical network excitability of both ipsilateral and contralateral motor representations of the tongue to determine whether they are under analogous or disparate inhibitory and facilitatory corticocortical control. Motor evoked potentials (MEPs) to unilateral focal transcranial magnetic stimulation (TMS) of the tongue primary motor cortex were recorded simultaneously from the ipsilateral and contralateral lingual muscles. Single-pulse TMS was used to assess motor threshold (MT) and MEP recruitment. Paired-pulse TMS was used to study intracortical inhibition (ICI) and intracortical facilitation (ICF) at various interstimulus intervals (ISIs) between the conditioning stimulus (CS) and the test stimulus (TS), and at different CS and TS intensities, respectively. Focal TMS invariably produced MEPs in both ipsilateral and contralateral lingual muscles. MT was lower and MEP recruitment was steeper when recorded from the contralateral muscle group. ICI and ICF were identical in the ipsilateral and contralateral representations, with inhibition occurring at short ISIs (2 and 3 ms) and facilitation occurring at longer ISIs (10 and 15 ms). Moreover, changing one stimulus parameter regularly produced analogous changes in MEP size bilaterally, revealing strong linear correlations between ipsilateral and contralateral ICI and ICF (P < 0.0001). These findings indicate that the ipsilateral and contralateral representations of the tongue are under analogous inhibitory and facilitatory control, possibly by a common intracortical network. PMID:11779968

  16. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    PubMed

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc. PMID:27220038

  17. Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

    PubMed

    Sari-Hassoun, Meryem; Clement, Marie-Jeanne; Hamdi, Imane; Bollot, Guillaume; Bauvais, Cyril; Joshi, Vandana; Toma, Flavio; Burgo, Andrea; Cailleret, Michel; Rosales-Hernández, Martha Cecilia; Macias Pérez, Martha Edith; Chabane-Sari, Daoudi; Curmi, Patrick A

    2016-02-15

    Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research. PMID:26707799

  18. Contribution of myosin II activity to cell spreading dynamics.

    PubMed

    Nisenholz, Noam; Paknikar, Aishwarya; Köster, Sarah; Zemel, Assaf

    2016-01-14

    Myosin II activity and actin polymerization at the leading edge of the cell are known to be essential sources of cellular stress. However, a quantitative account of their separate contributions is still lacking; so is the influence of the coupling between the two phenomena on cell spreading dynamics. We present a simple analytic elastic theory of cell spreading dynamics that quantitatively demonstrates how actin polymerization and myosin activity cooperate in the generation of cellular stress during spreading. Consistent with experiments, myosin activity is assumed to polarize in response to the stresses generated during spreading. The characteristic response time and the overall spreading time are predicted to determine different evolution profiles of cell spreading dynamics. These include, a (regular) monotonic increase of cell projected area with time, a non-monotonic (overshooting) profile with a maximum, and damped oscillatory modes. In addition, two populations of myosin II motors are distinguished based on their location in the lamella; those located above the major adhesion zone at the cell periphery are shown to facilitate spreading whereas those in deeper regions of the lamella are shown to oppose spreading. We demonstrate that the attenuation of myosin activity in the two regions may result in reciprocal effects on spreading. These findings provide important new insight into the function of myosin II motors in the course of spreading. PMID:26481613

  19. Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures

    PubMed Central

    Jaenisch, Nadine; Liebmann, Lutz; Guenther, Madlen; Hübner, Christian A.; Frahm, Christiane; Witte, Otto W.

    2016-01-01

    Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors. PMID:27188341

  20. Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures.

    PubMed

    Jaenisch, Nadine; Liebmann, Lutz; Guenther, Madlen; Hübner, Christian A; Frahm, Christiane; Witte, Otto W

    2016-01-01

    Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors. PMID:27188341

  1. Dual role of myosin II during Drosophila imaginal disc metamorphosis.

    PubMed

    Aldaz, Silvia; Escudero, Luis M; Freeman, Matthew

    2013-01-01

    The motor protein non-muscle myosin II is a major driver of the movements that sculpt three-dimensional organs from two-dimensional epithelia. The machinery of morphogenesis is well established but the logic of its control remains unclear in complex organs. Here we use live imaging and ex vivo culture to report a dual role of myosin II in regulating the development of the Drosophila wing. First, myosin II drives the contraction of a ring of cells that surround the squamous peripodial epithelium, providing the force to fold the whole disc through about 90°. Second, myosin II is needed to allow the squamous cells to expand and then retract at the end of eversion. The combination of genetics and live imaging allows us to describe and understand the tissue dynamics, and the logic of force generation needed to transform a relatively simple imaginal disc into a more complex and three-dimensional adult wing. PMID:23612302

  2. Still and rotating myosin clusters determine cytokinetic ring constriction

    PubMed Central

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  3. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  4. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  5. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated. PMID:23791790

  6. Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells

    PubMed Central

    Han, Young-Eun; Lim, Ajin; Park, Sun-Hyun; Chang, Sunghoe; Lee, Suk-Ho; Ho, Won-Kyung

    2015-01-01

    AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic β-cells. PMID:26471000

  7. Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

    In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles. PMID:18799629

  8. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    SciTech Connect

    Vu, N.D.; Wagner, P.D.

    1987-07-28

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of /sup 32/P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca/sup 2 +/- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation.

  9. Comparison of Motor Inhibition in Variants of the Instructed-Delay Choice Reaction Time Task.

    PubMed

    Quoilin, Caroline; Lambert, Julien; Jacob, Benvenuto; Klein, Pierre-Alexandre; Duque, Julie

    2016-01-01

    Using instructed-delay choice reaction time (RT) paradigms, many previous studies have shown that the motor system is transiently inhibited during response preparation: motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex are typically suppressed during the delay period. This effect has been observed in both selected and non-selected effectors, although MEP changes in selected effectors have been more inconsistent across task versions. Here, we compared changes in MEP amplitudes in three different variants of an instructed-delay choice RT task. All variants required participants to choose between left and right index finger movements but the responses were either provided "in the air" (Variant 1), on a regular keyboard (Variant 2), or on a response device designed to control from premature responses (Variant 3). The task variants also differed according to the visual layout (more concrete in Variant 3) and depending on whether participants received a feedback of their performance (absent in Variant 1). Behavior was globally comparable between the three variants of the task although the propensity to respond prematurely was highest in Variant 2 and lowest in Variant 3. MEPs elicited in a non-selected hand were similarly suppressed in the three variants of the task. However, significant differences emerged when considering MEPs elicited in the selected hand: these MEPs were suppressed in Variants 1 and 3 whereas they were often facilitated in Variant 2, especially in the right dominant hand. In conclusion, MEPs elicited in selected muscles seem to be more sensitive to small variations to the task design than those recorded in non-selected effectors, probably because they reflect a complex combination of inhibitory and facilitatory influences on the motor output system. Finally, the use of a standard keyboard seems to be particularly inappropriate because it encourages participants to respond promptly with no

  10. Different subcellular localizations and functions of Arabidopsis myosin VIII

    PubMed Central

    Golomb, Lior; Abu-Abied, Mohamad; Belausov, Eduard; Sadot, Einat

    2008-01-01

    Background Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. Results In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding. Conclusion Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity. PMID:18179725

  11. Impairment of Procedural Learning and Motor Intracortical Inhibition in Neurofibromatosis Type 1 Patients

    PubMed Central

    Zimerman, Máximo; Wessel, Maximilian J.; Timmermann, Jan E.; Granström, Sofia; Gerloff, Christian; Mautner, Victor F.; Hummel, Friedhelm C.

    2015-01-01

    Background Cognitive difficulties are the most common neurological complications in neurofibromatosis type 1 (NF1) patients. Recent animal models proposed increased GABA-mediated inhibition as one underlying mechanism directly affecting the induction of long-term potentiation (LTP) and learning. In most adult NF1 patients, apparent cognitive and attentional deficits, tumors affecting the nervous system and other confounding factors for neuroscientific studies are difficult to control for. Here we used a highly specific group of adult NF1 patients without cognitive or nervous system impairments. Such selected NF1 patients allowed us to address the following open questions: Is the learning process of acquiring a challenging motor skill impaired in NF1 patients? And is such an impairment in relation to differences in intracortical inhibition? Methods We used an established non-invasive, double-pulse transcranial magnetic stimulation (dp-TMS) paradigm to assess practice-related modulation of intracortical inhibition, possibly mediated by gamma-minobutyric acid (GABA)ergic-neurotransmission. This was done during an extended learning paradigm in a group of NF1 patients without any neuropsychological deficits, functioning normally in daily life and compared them to healthy age-matched controls. Findings NF1 patients experienced substantial decline in motor skill acquisition (F = 9.2, p = 0.008) over five-consecutives training days mediated through a selective reduction in the early acquisition (online) and the consolidation (offline) phase. Furthermore, there was a consistent decrease in task-related intracortical inhibition as a function of the magnitude of learning (T = 2.8, p = 0.014), especially evident after the early acquisition phase. Interpretations Collectively, the present results provide evidence that learning of a motor skill is impaired even in clinically intact NF1 patients based, at least partially, on a GABAergic-cortical dysfunctioning as

  12. Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

    PubMed Central

    Watanabe, Toshiyuki; Hosoya, Hiroshi

    2007-01-01

    Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase. PMID:17151359

  13. Hypoactivation in right inferior frontal cortex is specifically associated with motor response inhibition in adult ADHD

    PubMed Central

    Morein-Zamir, Sharon; Dodds, Chris; van Hartevelt, Tim J; Schwarzkopf, Wolfgang; Sahakian, Barbara; Müller, Ulrich; Robbins, Trevor

    2014-01-01

    Adult ADHD has been linked to impaired motor response inhibition and reduced associated activation in the right inferior frontal cortex (IFC). However, it is unclear whether abnormal inferior frontal activation in adult ADHD is specifically related to a response inhibition deficit or reflects a more general deficit in attentional processing. Using functional magnetic resonance imaging, we tested a group of 19 ADHD patients with no comorbidities and a group of 19 healthy control volunteers on a modified go/no-go task that has been shown previously to distinguish between cortical responses related to response inhibition and attentional shifting. Relative to the healthy controls, ADHD patients showed increased commission errors and reduced activation in inferior frontal cortex during response inhibition. Crucially, this reduced activation was observed when controlling for attentional processing, suggesting that hypoactivation in right IFC in ADHD is specifically related to impaired response inhibition. The results are consistent with the notion of a selective neurocognitive deficit in response inhibition in adult ADHD associated with abnormal functional activation in the prefrontal cortex, whilst ruling out likely group differences in attentional orienting, arousal and motivation. Hum Brain Mapp 35:5141–5152, 2014. PMID:24819224

  14. Automatic ultrarapid activation and inhibition of cortical motor systems in spoken word comprehension.

    PubMed

    Shtyrov, Yury; Butorina, Anna; Nikolaeva, Anastasia; Stroganova, Tatiana

    2014-05-01

    To address the hotly debated question of motor system involvement in language comprehension, we recorded neuromagnetic responses elicited in the human brain by unattended action-related spoken verbs and nouns and scrutinized their timecourse and neuroanatomical substrates. We found that already very early on, from ∼80 ms after disambiguation point when the words could be identified from the available acoustic information, both verbs and nouns produced characteristic somatotopic activations in the motor strip, with words related to different body parts activating the corresponding body representations. Strikingly, along with this category-specific activation, we observed suppression of motor-cortex activation by competitor words with incompatible semantics, documenting operation of the neurophysiological principles of lateral/surround inhibition in neural word processing. The extremely early onset of these activations and deactivations, their emergence in the absence of attention, and their similar presence for words of different lexical classes strongly suggest automatic involvement of motor-specific circuits in the perception of action-related language. PMID:24753617

  15. Acute response of airway muscle to extreme temperature includes disruption of actin-myosin interaction.

    PubMed

    Dyrda, Peter; Tazzeo, Tracy; DoHarris, Lindsay; Nilius, Berndt; Roman, Horia Nicolae; Lauzon, Anne-Marie; Aziz, Tariq; Lukic, Dusan; Janssen, Luke J

    2011-02-01

    Despite the emerging use of bronchial thermoplasty in asthma therapy, the response of airway smooth muscle (ASM) to extreme temperatures is unknown. We investigated the immediate effects of exposing ASM to supraphysiologic temperatures. Isometric contractions were studied in bovine ASM before and after exposure to various thermal loads and/or pharmacologic interventions. Actin-myosin interactions were investigated using a standard in vitro motility assay. We found steep thermal sensitivity for isometric contractions evoked by acetylcholine, with threshold and complete inhibition at less than 50°C and greater than 55°C, respectively. Contractile responses to serotonin or KCl were similarly affected, whereas isometric relaxations evoked by the nitric oxide donor S-nitrosyl-N-acetylpenicillamine or the β-agonist isoproterenol were unaffected. This thermal sensitivity developed within 15 minutes, but did not evolve further over the course of several days (such a rapid time-course rules out heat shock proteins, apoptosis, autophagy, and necrosis). Although heat-sensitive transient receptor potential (TRPV2) channels and the calmodulin-dependent (Cam) kinase-II-induced inactivation of myosin light chain kinase are both acutely thermally sensitive, with a temperature producing half-maximal effect (T(1/2)) of 52.5°C, the phenomenon we describe was not prevented by blockers of TRPV2 channels (e.g., ruthenium red, gadolinium, zero-Ca(2+) or zero-Na(+)/zero-Ca(2+) media, and cromakalim) or of Cam kinase-II (e.g., W7, trifluoperazine, and KN-93). However, direct measurements of actin-myosin interactions showed the same steep thermal profile. The functional changes preceded any histologic evidence of necrosis or apoptosis. We conclude that extreme temperatures (such as those used in bronchial thermoplasty) directly disrupt actin-myosin interactions, likely through a denaturation of the motor protein, leading to an immediate loss of ASM cell function. PMID:20395634

  16. Hold on tightly, let go lightly: myosin functions at adherens junctions

    PubMed Central

    Sandquist, Joshua C.; Bement, William M.

    2016-01-01

    Adherens junctions, the sites of cadherin-dependent cell–cell adhesion, are also important for dynamic tension sensing, force transduction and signalling. Different myosin motors contribute to adherens junction assembly and versatility in distinct ways. PMID:20596044

  17. Effects of pathogenic proline mutations on myosin assembly.

    PubMed

    Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie A

    2012-02-01

    Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the β-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease. PMID:22155079

  18. 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

  19. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions.

    PubMed

    Zult, Tjerk; Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-04-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp (2) = 0.005; ECR: P = 0.712, ηp (2) = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp (2) = 0.049; ECR: P = 0.343, ηp (2) = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions. PMID:25632077

  20. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions

    PubMed Central

    Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-01-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp2 = 0.005; ECR: P = 0.712, ηp2 = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp2 = 0.049; ECR: P = 0.343, ηp2 = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions. PMID:25632077

  1. Modeling Hand-Over-Hand and Inchworm Steps in Myosin VI

    NASA Astrophysics Data System (ADS)

    Jack, Amanda; Lowe, Ian; Tehver, Riina

    Myosin VI is a molecular motor protein that moves along actin filaments to transport cargo within a cell. There is much experimental evidence that the myosin VI dimer moves ``hand-over-hand'' along actin; however, recent experiments suggest that the protein can also move via an ``inchworm'' mechanism. We created a mechanochemical kinetic model to predict myosin VI's behavior under different ATP, ADP, and force conditions, taking these alternative mechanisms into account. Our model's calculations agree well with experimental results and can also be used to predict myosin VI's behavior outside experimentally tested regimes, such as under forward force. We also predict an optimized motor function for the protein around physiological (-2 pN) load and anchoring under -3 pN load. By using our model to predict myosin VI's response to environmental change, we can gain insight into the behavior of a protein that can be difficult to observe experimentally.

  2. Abnormal Dorsal Premotor-Motor Inhibition in Writer’s Cramp

    PubMed Central

    Richardson, Sarah Pirio; Beck, Sandra; Bliem, Barbara; Hallett, Mark

    2014-01-01

    Background We hypothesize that a deficient premotor-motor inhibitory network contributes to the unwanted involuntary movements in dystonia. Methods We studied nine controls and nine patients with writer’s cramp (WC). Dorsal premotor-motor cortical inhibition (dPMI) was tested by applying conditioning transcranial magnetic stimulation (TMS) to the dorsal premotor cortex and then a test pulse to the ipsilateral motor cortex at an interval of 6ms. We used an H-reflex in flexor carpi radialis paired with TMS over the premotor cortex to assess for spinal cord excitability change. Finally, we interrupted a choice reaction time task with TMS over dorsal premotor cortex to assess performance in a non-dystonic task. Results Our results showed that WC patients exhibited dPMI at rest (88.5%, the ratio of conditioned to unconditioned test pulse) in contrast to our controls who did not show dPMI (109.6%) (p=0.0198). This difference between patients and controls persisted during contraction (100% vs. 112%) and pen-holding (95.6% vs. 111%). The H-reflex in the arm was not modulated by the premotor cortex stimulation. WC patients made more errors and the error rate improved with TMS over the premotor cortex. Conclusions These results suggest that abnormal premotor-motor interactions may play a role in the pathophysiology of focal dystonia. dPMI was not modulated by task in either group, but was constantly greater in the patients. The significance of the increased inhibition is likely to be compensatory. It appears to be a robust finding and, in combination with other features, could be further explored as a biomarker. PMID:24710852

  3. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  4. In vitro and in vivo single myosin step-sizes in striated muscle.

    PubMed

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

    2015-12-01

    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

  5. Functional networks of motor inhibition in conversion disorder patients and feigning subjects.

    PubMed

    Hassa, Thomas; de Jel, Esther; Tuescher, Oliver; Schmidt, Roger; Schoenfeld, Mircea Ariel

    2016-01-01

    The neural correlates of motor inhibition leading to paresis in conversion disorder are not well known. The key question is whether they are different of those of normal subjects feigning the symptoms. Thirteen conversion disorder patients with hemiparesis and twelve healthy controls were investigated using functional magnetic resonance tomography under conditions of passive motor stimulation of the paretic/feigned paretic and the non-paretic hand. Healthy controls were also investigated in a non-feigning condition. During passive movement of the affected right hand conversion disorder patients exhibited activations in the bilateral triangular part of the inferior frontal gyri (IFG), with a left side dominance compared to controls in non-feigning condition. Feigning controls revealed for the same condition a weak unilateral activation in the right triangular part of IFG and an activity decrease in frontal midline areas, which couldn't be observed in patients. The results suggest that motor inhibition in conversion disorder patients is mediated by the IFG that was also involved in inhibition processes in normal subjects. The activity pattern in feigning controls resembled that of conversion disorder patients but with a clear difference in the medial prefrontal cortex. Healthy controls showed decreased activity in this region during feigning compared to non-feigning conditions suggesting a reduced sense of self-agency during feigning. Remarkably, no activity differences could be observed in medial prefrontal cortex for patients vs healthy controls in feigning or non-feigning conditions suggesting self-agency related activity in patients to be in between those of non-feigning and feigning healthy subjects. PMID:27330971

  6. Loss of Striatonigral GABAergic Presynaptic Inhibition Enables Motor Sensitization in Parkinsonian Mice

    PubMed Central

    Borgkvist, Anders; Avegno, Elizabeth M.; Wong, Minerva Y.; Kheirbek, Mazen A.; Sonders, Mark S.; Hen, Rene; Sulzer, David

    2015-01-01

    SUMMARY Degeneration of dopamine (DA) neurons in Parkinson’s disease (PD) causes hypokinesia, but DA replacement therapy can elicit exaggerated voluntary and involuntary behaviors that have been attributed to enhanced DA receptor sensitivity in striatal projection neurons. Here we reveal that in hemiparkinsonian mice, striatal D1 receptor-expressing medium spiny neurons (MSNs) directly projecting to the substantia nigra reticulata (SNr) lose tonic presynaptic inhibition by GABAB receptors. The absence of presynaptic GABAB response potentiates evoked GABA release from MSN efferents to the SNr and drives motor sensitization. This alternative mechanism of sensitization suggests a synaptic target for PD pharmacotherapy. PMID:26335644

  7. A Perspective on the Role of Myosins as Mechanosensors.

    PubMed

    Greenberg, Michael J; Arpağ, Göker; Tüzel, Erkan; Ostap, E Michael

    2016-06-21

    Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms. PMID:27332116

  8. Engineering myosins for long-range transport on actin filaments.

    PubMed

    Schindler, Tony D; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1). PMID:24240432

  9. Engineering myosins for long-range transport on actin filaments

    PubMed Central

    Schindler, Tony D.; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2013-01-01

    Cytoskeletal motors act as cargo transporters in cells1 and may be harnessed for directed transport applications in molecular detection and diagnostic devices2. High processivity — the ability to take many steps along a track before dissociating3 — is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances of microns in vivo and in vitro. Natural processive myosins4,5 are dimeric and use internal tension to coordinate the detachment cycles of the two heads6–8. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (i) formation of three-headed and four-headed myosins; and (ii) introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both the forward and backward direction, and also produce the fastest processive cytoskeletal motor measured to date, reaching a speed of 10 μm/s. PMID:24240432

  10. Engineering myosins for long-range transport on actin filaments

    NASA Astrophysics Data System (ADS)

    Schindler, Tony D.; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s-1.

  11. Molecular Modulation of Actomyosin Function by Cardiac Myosin-Binding Protein C

    PubMed Central

    Previs, Michael J.; Michalek, Arthur J.; Warshaw, David M.

    2014-01-01

    Cardiac myosin-binding protein C is a key regulator of cardiac contractility and is capable of both activating the thin filament to initiate actomyosin motion generation and governing maximal sliding velocities. While MyBP-C’s C-terminus localizes the molecule within the sarcomere the N-terminus appears to confer regulatory function by binding to the myosin motor domain and/or actin. Literature pertaining to how MyBP-C binding to the myosin motor domain and or actin leads to MyBP-C’s dual modulatory roles that can impact actomyosin interactions are discussed. PMID:24407948

  12. The modulatory effect of MgATP on heterotrimeric smooth muscle myosin phosphatase activity.

    PubMed

    Sato, O; Ogawa, Y

    1999-10-01

    Regulation of the enzymatic activity of heterotrimeric smooth muscle myosin phosphatase (SMMP) by MgATP was examined using phosphorylated myosin (P-myosin), heavy meromyosin (P-HMM), subfragment-1 (P-S1), and 20 kDa myosin light chain (P-MLC(20)) as substrates. The activity toward P-myosin and P-HMM was dose-dependently reduced by MgATP, whereas that toward P-S1 or P-MLC(20) was unchanged. The reduction was mainly due to a decrease in the affinity of SMMP for the substrate with the unchanged maximum activity. This regulation is entirely new in the respect that the responsible molecule is the substrate, not SMMP. Because P-myosin derived from myosin stored in 50% glycerol at -20 degrees C was insensitive to MgATP, the proper integrity of P-myosin is required. Coexisting myosin did not affect this regulation, but it inhibited the SMMP activity in the absence of MgATP. With P-myosin, the enzyme activity was biphasically steeply dependent on the ionic strength. This requires that determinations are conducted with a fixed ionic strength. The Q(10) value was about 2, which was quite similar to that for myosin light chain kinase. These results suggest that the rate of dephosphorylation of P-myosin is lowered at rest, but that it may reach a value comparable to the rate of phosphorylation of myosin in the sarcoplasm with the increased level of P-myosin during muscle activation. This regulation by MgATP may underlie the "latch mechanism" in some respects. PMID:10502690

  13. The effect of dual-task difficulty on the inhibition of the motor cortex.

    PubMed

    Corp, Daniel T; Rogers, Mark A; Youssef, George J; Pearce, Alan J

    2016-02-01

    Dual-tasking is intrinsic to many daily activities, including walking and driving. However, the activity of the primary motor cortex (M1) in response to dual-tasks (DT) is still not well characterised. A recent meta-analysis (Corp in Neurosci Biobehav Rev 43:74-87, 2014) demonstrated a reduction in M1 inhibition during dual-tasking, yet responses were not consistent between studies. It was suggested that DT difficulty might account for some of this between-study variability. The aim of this study was to investigate whether corticospinal excitability and M1 inhibition differed between an easier and more difficult dual-task. Transcranial magnetic stimulation (TMS) was applied to participants' abductor pollicis brevis muscle representation during a concurrent pincer grip task and stationary bike-riding. The margin of error in which to maintain pincer grip force was reduced to increase task difficulty. Compared to ST conditions, significantly increased M1 inhibition was demonstrated for the easier, but not more difficult, DT. However, there was no significant difference in M1 inhibition between easy and difficult DTs. The difference in difficulty between the two tasks may not have been wide enough to result in significant differences in M1 inhibition. Increased M1 inhibition for the easy DT condition was in opposition to the reduction in M1 inhibition found in our meta-analysis (Corp in Neurosci Biobehav Rev 43:74-87, 2014). We propose that this may be partially explained by differences in the timing of the TMS pulse between DT studies. PMID:26514811

  14. The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation

    PubMed Central

    Fusi, Luca; Huang, Zhe; Irving, Malcolm

    2015-01-01

    In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads. PMID:26287630

  15. The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.

    PubMed

    Fusi, Luca; Huang, Zhe; Irving, Malcolm

    2015-08-18

    In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads. PMID:26287630

  16. Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein

    PubMed Central

    Bentea, Eduard; Van der Perren, Anke; Van Liefferinge, Joeri; El Arfani, Anissa; Albertini, Giulia; Demuyser, Thomas; Merckx, Ellen; Michotte, Yvette; Smolders, Ilse; Baekelandt, Veerle; Massie, Ann

    2015-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 μg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets. PMID:25873870

  17. 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

  18. Peripheral antagonistic action of trimebutine and kappa opioid substances on acoustic stress-induced gastric motor inhibition in dogs.

    PubMed

    Gué, M; Pascaud, X; Hondé, C; Junien, J L; Buéno, L

    1988-01-27

    The effects of intracerebroventricular (i.c.v.), intravenous (i.v.) and oral (p.o.) administration of trimebutine on the gastric motor inhibition induced by acoustic stress were investigated in fasted dogs fitted with strain-gauge transducers on the antrum and proximal jejunum. Started 40-50 min after the last migrating motor complex, a 1 h acoustic stress delayed by 111% the occurrence of the next gastric migrating motor complex without affecting the jejunal motor pattern. This inhibition of gastric migrating motor complex induced by acoustic stress was abolished by previous p.o. administration of trimebutine (1 mg/kg) but not by its i.v. (0.1 mg/kg) or i.c.v. (0.01 mg/kg) injection. The trimebutine blockade of gastric motor alterations induced by acoustic stress was suppressed after previous i.v. treatment with MR 2266 (0.3 mg/kg) but was unaffected by naloxone (0.3 mg/kg). Furthermore oral administration of U-50488H (10 micrograms/kg) and ethylketocyclazocine (10 micrograms/kg) respectively abolished and reduced the acoustic stress-induced delay of the occurrence of the gastric migrating motor complex. We concluded that trimebutine is able to antagonize the gastric motor disturbances induced in dogs by acoustic stress, probably by acting selectively on peripheral kappa receptors located in the wall of the proximal gut and directly stimulated from a mucosal site. PMID:2895010

  19. Late Recovery from Stuttering: The Role of Hand Dominancy, Fine Motor and ‎Inhibition Control

    PubMed Central

    Mohammadi, Hiwa; Khazaie, Habibolah; Rezaei, Mansour; Joghataei, Mohammad Taghi

    2016-01-01

    Objective: There are controversial reports about factors that affect recovery from stuttering. In the ‎present study, the effect of hand dominancy, fine motor and inhibition control on late ‎recovery from stuttering was investigated among a group of Kurdish-Persian children who ‎stuttered in Iran.‎ Method: Twenty-two Kurdish-Persian children aged 7-14 years who stuttered were followed for 6 ‎years. Based on the evaluation of three experienced speech therapists and parental judgments, ‎these children were classified into recovered or persistent groups. Data about fine motor ‎control of hand and inhibition control were obtained, using Purdue Pegboard and Victoria ‎Strop Color Word Tests, respectively. Risk factors including sex, age, and family history of ‎stuttering, handedness, inhibitory control and fine motor control of hand were compared ‎between the groups and modeled to predict recovery from stuttering using logistic regression.‎ Results: From the 22 participants, 5 (22.7%) recovered from stuttering. The recovered and persistent ‎groups did not show significant differences in the interference effect. By dividing the scores ‎of the Purdue Pegboard tests to the right and left hand, we created a new Handedness Index ‎‎(HI). HI was significantly higher in the recovered group. The score of right hand was higher ‎than the left in the recovered group, but no difference was found between the two hands in ‎the persistent group. Among the investigated risk factors, only HI could predict the recovery ‎from or persistency of stuttering with 94% sensitivity and 84% specificity.‎ Conclusion: Handedness Index can predict the recovery from stuttering significantly among children who ‎stutter.‎ PMID:27252769

  20. Analysis of the interactions between Rab GTPases and class V myosins.

    PubMed

    Lindsay, Andrew J; Miserey-Lenkei, Stéphanie; Goud, Bruno

    2015-01-01

    Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli's Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid "living chip" assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation. PMID:25800833

  1. A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

    PubMed Central

    Dror, Amiel A.; Song, Lin; Ron, Uri; Tan, Joshua T.; Shitrit, Alina Starovolsky; Fuchs, Helmut; Hasson, Tama; Ben-Tal, Nir; Sweeney, H. Lee; de Angelis, Martin Hrabe; Steel, Karen P.; Avraham, Karen B.

    2008-01-01

    Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness. PMID:18833301

  2. Isoforms Confer Characteristic Force Generation and Mechanosensation by Myosin II Filaments

    PubMed Central

    Stam, Samantha; Alberts, Jon; Gardel, Margaret L.; Munro, Edwin

    2015-01-01

    Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) arrays to generate contractile forces in muscle and nonmuscle cells. How myosin II force production is shaped by isoform-specific motor properties and environmental stiffness remains poorly understood. Here, we used computer simulations to analyze force production by an ensemble of myosin motors against an elastically tethered actin filament. We found that force output depends on two timescales: the duration of F-actin attachment, which varies sharply with the ensemble size, motor duty ratio, and external load; and the time to build force, which scales with the ensemble stall force, gliding speed, and environmental stiffness. Although force-dependent kinetics were not required to sense changes in stiffness, the myosin catch bond produced positive feedback between the attachment time and force to trigger switch-like transitions from transient attachments, generating small forces, to high-force-generating runs. Using parameters representative of skeletal muscle myosin, nonmuscle myosin IIB, and nonmuscle myosin IIA revealed three distinct regimes of behavior, respectively: 1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness; 2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force buildup times that exceed physiological timescales; and 3) small assemblies of low-duty ratio motors operating at intermediate speeds are poised to respond sharply to changes in mechanical context—at low force or stiffness, they serve as low-affinity cross-links, but they can transition to force production via the positive-feedback mechanism described above. Together, these results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment. PMID:25902439

  3. Analysis of persistence during intracellular actin-based transport mediated by molecular motors

    NASA Astrophysics Data System (ADS)

    Pallavicini, C.; Despósito, M. A.; Levi, V.; Bruno, L.

    2010-09-01

    The displacement of particles or probes in the cell cytoplasm as a function of time is characterized by different anomalous diffusion regimes. The transport of large cargoes, such as organelles, vesicles or large proteins, involves the action of ATP-consuming molecular motors. We investigate the motion of pigment organelles driven by myosin-V motors in Xenopus laevis melanocytes using a high spatio-temporal resolution tracking technique. By analyzing the turning angles (phi) of the obtained 2D trajectories as a function of the time lag, we determine the critical time of the transition between anticorrelated and directed motion as the time when the turning angles begin to concentrate around phi = 0. We relate this transition with the crossover from subdiffusive to superdiffusive behavior observed in a previous work [5]. We also assayed the properties of the trajectories in cells with inhibited myosin activity, and we can compare the results in the presence and absence of active motors.

  4. Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

    PubMed

    Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

    2014-12-01

    The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean

  5. Effects of volitional contraction on intracortical inhibition and facilitation in the human motor cortex.

    PubMed

    Ortu, Enzo; Deriu, Franca; Suppa, Antonio; Tolu, Eusebio; Rothwell, John C

    2008-11-01

    Short-interval intracortical inhibition (SICI), intracortical facilitation (ICF) and short-interval intracortical facilitation (SICF) were assessed in the cortical motor area of the first dorsal interosseous muscle (FDI) of 16 healthy subjects. Paired-pulse TMS was delivered to the left hemisphere at the following interstimulus intervals (ISIs): 2 and 3 ms for SICI, 10 and 15 ms for ICF and 1-5 ms for SICF. Motor-evoked potentials were recorded from the resting and active right FDI. The effects exerted on SICI and ICF by four intensities (60-90% of active motor threshold, AMT) of the conditioning stimulus (S1) and by three levels of muscle contraction (10%, 25%, 50% of maximal voluntary contraction, MVC) were evaluated. The effects exerted on SICF were evaluated with two intensities (90% and 70% of AMT) of the test stimulus (S2) and with the same levels of muscle contraction. Results showed that: (i) during 10% MVC, maximum SICI was observed with S1 = 70% AMT; (ii) the amount of SICI obtained with S1 = 70% AMT was the same at rest as during 10% MVC, but decreased at higher contraction levels; (iii) ICF was observed only at rest with S1 = 90% AMT; (iv) SICF was facilitated at 10% and 25% MVC, but not at 50% MVC. We conclude that during muscle activation, intracortical excitability reflects a balance between activation of SICI and SICF systems. Part of the reduction in SICI during contraction is due to superimposed recruitment of SICF. Low intensity (70% AMT) conditioning stimuli can test SICI independently of effects on SICF at low contraction levels. PMID:18787036

  6. Axonal isoforms of myosin-I.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2005-05-13

    We have examined spinal motor neurons in Sprague-Dawley rats to further characterize a mechanoenzyme, myosin-Igamma (myr4), which is found in high concentration during axon tract formation in neonates. We raised an antibody to myr4 and made riboprobes for in situ hybridization. Myr4 mRNA was abundant in spinal cord motor neurons (particularly during axon regrowth). Nerves undergoing Wallerian degeneration (from a crush 7 days earlier) showed anti-myr4 labeling of the axolemma and SER--after microtubules, neurofilaments, and F-actin had already been degraded--which is consistent with a described lipid-binding domain in the tail region of myosin-Is. Newly synthesized myr4 was carried in axons by the slow component (SC) of axonal transport at 1-8 mm/day, whereas, none was carried by the fast component (FC). We conclude that SC delivers myr4 to the cytoplasmic surfaces of stationary axonal membranes (SER and axolemma). This positioning would anchor the tail domain of myr4 and leave the catalytic head domain free to interact with F-actin. PMID:15809075

  7. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  8. Structural Basis for Myosin V Discrimination Between Distinct Cargoes

    SciTech Connect

    Pashkova,N.; Jin, Y.; Ramaswamy, S.; Weisman, L.

    2006-01-01

    Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 {angstrom} of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180{sup o}. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.

  9. 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

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

    PubMed Central

    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. PMID:19039101

  11. Fewer fluctuations, higher maximum concentration and better motor response of levodopa with catechol-O-methyltransferase inhibition.

    PubMed

    Muhlack, Siegfried; Herrmann, Lennard; Salmen, Stephan; Müller, Thomas

    2014-11-01

    Catechol-O-methyltransferase inhibitor addition to levodopa/carbidopa formulations improves motor symptoms and reduces levodopa fluctuations in patients with Parkinson's disease. Objectives were to investigate the effects of entacapone and tolcapone on plasma behaviour of levodopa, its metabolite 3-O-methyldopa and on motor impairment. 22 patients orally received levodopa/carbidopa first, then levodopa/carbidopa/entacapone and finally levodopa/carbidopa plus tolcapone within a 4.5 h interval twice. Maximum concentration, time to maximum level and bioavailability of levodopa did not differ between all conditions each with 200 mg levodopa application as a whole. Catechol-O-methyltransferase inhibition caused less fluctuations and higher baseline levels of levodopa after the first intake and less 3-O-methyldopa appearance. The maximum levodopa concentrations were higher after the second levodopa intake, particularly with catechol-O-methyltransferase inhibition. The motor response to levodopa was better with catechol-O-methyltransferase inhibition than without, tolcapone was superior to entacapone. More continuous levodopa brain delivery and lower 3-O-methyldopa bioavailability caused a better motor response during catechol-O-methyltransferase inhibition. PMID:24770794

  12. TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule*

    PubMed Central

    Balchand, Sai K.; Mann, Barbara J.; Titus, Janel; Ross, Jennifer L.; Wadsworth, Patricia

    2015-01-01

    The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays. PMID:26018074

  13. Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease.

    PubMed

    Zhai, Jinbin; Zhang, Lei; Mojsilovic-Petrovic, Jelena; Jian, Xiaoying; Thomas, Jeffrey; Homma, Kengo; Schmitz, Anton; Famulok, Michael; Ichijo, Hidenori; Argon, Yair; Randazzo, Paul A; Kalb, Robert G

    2015-06-17

    Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS. PMID:26085633

  14. TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule.

    PubMed

    Balchand, Sai K; Mann, Barbara J; Titus, Janel; Ross, Jennifer L; Wadsworth, Patricia

    2015-07-10

    The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays. PMID:26018074

  15. Mechanochemical tuning of myosin-I by the N-terminal region

    PubMed Central

    Greenberg, Michael J.; Lin, Tianming; Shuman, Henry; Ostap, E. Michael

    2015-01-01

    Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post–power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms. PMID:26056287

  16. Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin.

    PubMed

    Capitanio, M; Canepari, M; Cacciafesta, P; Lombardi, V; Cicchi, R; Maffei, M; Pavone, F S; Bottinelli, R

    2006-01-01

    During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the cross-bridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ( approximately 300 mus) and precisely ( approximately 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ( approximately 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ( approximately 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family. PMID:16371472

  17. The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V-Actin Bond

    PubMed Central

    Gebhardt, J. Christof M.; Ökten, Zeynep; Rief, Matthias

    2010-01-01

    Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense. PMID:20338849

  18. Myosin Vs organize actin cables in fission yeast

    PubMed Central

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G.

    2012-01-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  19. Myosin Vs organize actin cables in fission yeast.

    PubMed

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G

    2012-12-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  20. The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

    PubMed Central

    Hung, Pei-Fang; Hong, Tse-Ming; Hsu, Yi-Chiung; Chen, Hsuan-Yu; Chang, Yih-Leong; Wu, Chen-Tu; Chang, Gee-Chen; Jou, Yuh-Shan

    2013-01-01

    The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas. PMID:23626713

  1. The motor protein KIF14 inhibits tumor growth and cancer metastasis in lung adenocarcinoma.

    PubMed

    Hung, Pei-Fang; Hong, Tse-Ming; Hsu, Yi-Chiung; Chen, Hsuan-Yu; Chang, Yih-Leong; Wu, Chen-Tu; Chang, Gee-Chen; Jou, Yuh-Shan; Pan, Szu-Hua; Yang, Pan-Chyr

    2013-01-01

    The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas. PMID:23626713

  2. Identification and characterization of multiple novel Rab–myosin Va interactions

    PubMed Central

    Lindsay, Andrew J.; Jollivet, Florence; Horgan, Conor P.; Khan, Amir R.; Raposo, Graça; McCaffrey, Mary W.; Goud, Bruno

    2013-01-01

    Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes. PMID:24006491

  3. Cloning and characterization of a novel RING finger protein that interacts with class V myosins.

    PubMed

    El-Husseini, A E; Vincent, S R

    1999-07-01

    We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport. PMID:10391919

  4. Evidence that myosin does not contribute to force production in chromosome movement

    PubMed Central

    1982-01-01

    Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement. PMID:6181080

  5. A Role for Myosin 1e in Cortical Granule Exocytosis in Xenopus Oocytes*s

    PubMed Central

    Schietroma, Cataldo; Yu, Hoi-Ying; Wagner, Mark C.; Umbach, Joy A.; Bement, William M.; Gundersen, Cameron B.

    2010-01-01

    Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin1e in cortical granule exocytosis. Myosin1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin1e relocates to the surface of cortical granules. Overexpression of myosin1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis. PMID:17702742

  6. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  7. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  8. High Resolution Characterization of Myosin IIC Protein Tailpiece and Its Effect on Filament Assembly

    PubMed Central

    Rosenberg, Masha M.; Ronen, Daniel; Lahav, Noa; Nazirov, Elvira; Ravid, Shoshana; Friedler, Assaf

    2013-01-01

    The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr1957 or Thr1960 inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology. PMID:23426373

  9. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

    PubMed

    Chang, Ching-Wei; Kumar, Sanjay

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces. PMID:26336830

  10. Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes.

    PubMed

    Dhekne, Herschel S; Hsiao, Nai-Hua; Roelofs, Pieter; Kumari, Meena; Slim, Christiaan L; Rings, Edmond H H M; van Ijzendoorn, Sven C D

    2014-03-01

    Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function. PMID:24413175

  11. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  12. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics

    PubMed Central

    Chang, Ching-Wei; Kumar, Sanjay

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces. PMID:26336830

  13. Cell-sized liposome doublets reveal active tension build-up driven by acto-myosin dynamics.

    PubMed

    Caorsi, V; Lemière, J; Campillo, C; Bussonnier, M; Manzi, J; Betz, T; Plastino, J; Carvalho, K; Sykes, C

    2016-07-20

    Cells modulate their shape to fulfill specific functions, mediated by the cell cortex, a thin actin shell bound to the plasma membrane. Myosin motor activity, together with actin dynamics, contributes to cortical tension. Here, we examine the individual contributions of actin polymerization and myosin activity to tension increase with a non-invasive method. Cell-sized liposome doublets are covered with either a stabilized actin cortex of preformed actin filaments, or a dynamic branched actin network polymerizing at the membrane. The addition of myosin II minifilaments in both cases triggers a change in doublet shape that is unambiguously related to a tension increase. Preformed actin filaments allow us to evaluate the effect of myosin alone while, with dynamic actin cortices, we examine the synergy of actin polymerization and myosin motors in driving shape changes. Our assay paves the way for a quantification of tension changes triggered by various actin-associated proteins in a cell-sized system. PMID:27378156

  14. Use of Fluorescent Techniques to Study the In Vitro Movement of Myosins

    PubMed Central

    Toepfer, Christopher

    2014-01-01

    Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories. There are two basic versions of the assay which involve different geometries. In the sliding actin in vitro motility assay, myosin molecules are bound to a coverslip surface in a simply constructed microscopic flow chamber. Fluorescently labeled actin filaments are added to the flow chamber in the presence of ATP, and the movement of these actin filaments powered by the surface-bound myosins is observed. This assay has been used widely for a variety of myosins including both processive and nonprocessive ones. From this assay, one can easily measure the rate at which myosin is translocating actin. The single-molecule motility assay uses an inverted geometry compared to the sliding actin in vitro motility assay. It is most useful for processive myosins. Here, actin filaments are affixed to the coverslip surface. Fluorescently labeled single molecules of myosins (usually ones with processive kinetics) are introduced, and the movement of single molecules along the actin filaments is observed. This assay typically uses total internal reflection fluorescent (TIRF) microscopy to reduce the background signal arising from myosins in solution. From this assay, one can measure the velocity of movement, the frequency of movement, and the run length. If sufficient photons can be collected, one can use Gaussian fitting of the point spread function to determine the position of the labeled

  15. Head-to-tail regulation is critical for the in vivo function of myosin V

    PubMed Central

    Donovan, Kirk W.

    2015-01-01

    Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor. PMID:25940346

  16. Inhibition of the pontine Kölliker-Fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat.

    PubMed

    Bautista, T G; Dutschmann, M

    2014-05-16

    The pontine Kölliker-Fuse nucleus (KF) has established functions in the regulation of inspiratory-expiratory phase transition and the regulation of upper airway patency via laryngeal valving mechanisms. Here we studied the role of the KF in the gating and modulation of eupneic hypoglossal motor activity (HNA) using the in situ perfused brainstem preparation, which displays robust inspiratory HNA. Microinjection of glutamate into the KF area triggered complex and often biphasic modulation (excitation/inhibition or inhibition/excitation) of HNA. Subsequent transient pharmacological inhibition of KF by unilateral microinjection of GABA-A receptor agonist isoguvacine reduced HNA and while bilateral microinjections completely abolished HNA. Our results indicate that mixed and overlapping KF pre-motor neurons provide eupneic drive for inspiratory HNA and postinspiratory vagal nerve activity. Both motor activities have important functions in the regulation of upper airway patency during eupnea but also during various oro-pharyngeal behaviors. These results have potential implications in the contribution of state-dependent modulation of KF hypoglossal pre-motor neurons during sleep-wake cycle to obstructive sleep apnea. PMID:24603053

  17. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-01

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning. PMID:25065759

  18. Better Working Memory and Motor Inhibition in Children Who Delayed Gratification

    PubMed Central

    Yu, Junhong; Kam, Chi-Ming; Lee, Tatia M. C.

    2016-01-01

    Background: Despite the extensive research on delayed gratification over the past few decades, the neurocognitive processes that subserve delayed gratification remains unclear. As an exploratory step in studying these processes, the present study aims to describe the executive function profiles of children who were successful at delaying gratification and those who were not. Methods: A total of 138 kindergarten students (65 males, 73 females; Mage = 44 months, SD = 3.5; age range = 37–53 months) were administered a delayed gratification task, a 1-back test, a Day/night Stroop test and a Go/no-go test. The outcome measures of these tests were then analyzed between groups using a Multivariate Analysis of Variance, and subsequently a Multivariate Analysis of Covariance incorporating age as a covariate. Results: Children who were successful in delaying gratification were significantly older and had significantly better outcomes in the 1-back test and go/no-go test. With the exception of the number of hits in the go/no-go test, all other group differences remained significant after controlling for age. Conclusion: Children who were successful in delaying gratification showed better working memory and motor inhibition relative to those who failed the delayed gratification task. The implications of these findings are discussed. PMID:27493638

  19. Calcium can mobilize and activate myosin-VI

    PubMed Central

    Batters, Christopher; Brack, Dario; Ellrich, Heike; Averbeck, Beate; Veigel, Claudia

    2016-01-01

    The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux. PMID:26811464

  20. Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

    PubMed

    Yao, Lin-Lin; Shen, Mei; Lu, Zekuan; Ikebe, Mitsuo; Li, Xiang-dong

    2016-04-01

    Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5. PMID:26912658

  1. Temperature dependent measurements reveal similarities between muscle and non-muscle myosin motility

    PubMed Central

    Yengo, Christopher M.; Takagi, Yasuharu; Sellers, James R.

    2013-01-01

    We examined the temperature dependence of muscle and non-muscle myosin (heavy meromyosin, HMM) with in vitro motility and actin-activated ATPase assays. Our results indicate that myosin V (MV) has a temperature dependence that is similar in both ATPase and motility assays. We demonstrate that skeletal muscle myosin (SK), smooth muscle myosin (SM), and non-muscle myosin IIA (NM) have a different temperature dependence in ATPase compared to in vitro motility assays. In the class II myosins we examined (SK, SM, and NM) the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while for in vitro motility assays it is controversial. In myosin V the rate-limiting step for both in vitro motility and ATPase assays is known to be ADP release. Consequently, in MV the temperature dependence of the ADP release rate constant is similar to the temperature dependence of in vitro motility. Interestingly, the temperature dependence of the ADP release rate constant of SM and NM was shifted toward the in vitro motility temperature dependence. Our results suggest that the rate-limiting step in SK, SM, and NM may shift from attachment-limited in solution to detachment-limited in the in vitro motility assay. Internal strain within the myosin molecule or by neighboring myosin motors may slow ADP release which becomes rate-limiting in the in vitro motility assay. Within this small subset of myosins examined, the in vitro sliding velocity correlates reasonably well with actin-activated ATPase activity, which was suggested by the original study by Barany et al. (Barany 1967). PMID:22930330

  2. Hybrid and non-hybrid actomyosins reconstituted with actin, myosin and tropomyosin from skeletal and catch muscles.

    PubMed

    Shelud'ko, Nikolay S; Vyatchin, Ilya G; Lazarev, Stanislav S; Shevchenko, Ulyana V

    2015-08-21

    In this study, we investigated hybrid and non-hybrid actomyosin models including key contractile proteins: actin, myosin, and tropomyosin. These proteins were isolated from the rabbit skeletal muscle and the catch muscle of the mussel Crenomytilus grayanus. Our results confirmed literature data on an unusual ability of bivalve's tropomyosin to inhibit Mg-ATPase activity of skeletal muscle actomyosin. We have shown that the degree of inhibition depends on the environmental conditions and may vary within a wide range. The inhibitory effect of mussel tropomyosin was not detected in non-hybrid model (mussel myosin + mussel actin + mussel tropomyosin). This effect was revealed only in hybrid models containing mussel tropomyosin + rabbit (or mussel) actin + rabbit myosin. We assume that mussel and rabbit myosins have mismatched binding sites for actin. In addition, mussel tropomyosin interacting with actin is able to close the binding sites of rabbit myosin with actin, which leads to inhibition of Mg-ATPase activity. PMID:26166820

  3. Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

    PubMed Central

    Yu, Yan; Fay, Nicole C.; Smoligovets, Alexander A.; Wu, Hung-Jen; Groves, Jay T.

    2012-01-01

    Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL. PMID:22347397

  4. Intra-axonal myosin and actin in nerve regeneration.

    PubMed

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  5. 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

  6. Naloxone reduces the amplitude of IPSPs evoked in lumbar motoneurons by reticular stimulation during carbachol-induced motor inhibition.

    PubMed

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1999-02-20

    During active sleep or carbachol-induced motor inhibition, electrical stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) evoked large amplitude, glycinergic inhibitory postsynaptic potentials (IPSPs) in cat motoneurons. The present study was directed to determine whether these IPSPs, that are specific to the state of active sleep, are modulated by opioid peptides. Accordingly, intracellular recordings were obtained from lumbar motoneurons of acute decerebrate cats during carbachol-induced motor inhibition while an opiate receptor antagonist, naloxone, was microiontophoretically released next to the recorded cells. Naloxone reversibly reduced by 26% the mean amplitude of NRGc-evoked IPSPs (1.9+/-0.2 mV (S.E.M.) vs. 1.4+/-0.2 mV; n=11, control and naloxone, respectively, p<0.05), but had no effect on the other waveform parameters of these IPSPs (e.g., latency-to-onset, latency-to-peak, duration, etc.). The mean resting membrane potential, input resistance and membrane time constant of motoneurons following naloxone ejection were not statistically different from those of the control. These data indicate that opioid peptides have a modulatory effect on NRGc-evoked IPSPs during carbachol-induced motor inhibition. We therefore suggest that endogenous opioid peptides may act as neuromodulators to regulate inhibitory glycinergic synaptic transmission at motoneurons during active sleep. PMID:10082872

  7. Specific Inhibition of Cyclin-dependent Kinase 5 Activity Induces Motor Neuron Development in vivo

    PubMed Central

    Kanungo, Jyotshnabala; Zheng, Ya-Li; Amin, Niranjana D.; Kaur, Sukhbir; Ramchandran, Ramani; Pant, Harish C.

    2009-01-01

    Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in motor neuron development remains unexplored. Here, using gain and loss-of-function analyses in developing zebrafish embryos, we report that cdk5 plays a critical role in spinal and cranial motor neuron development. Cdk5 knockdown results in supernumerary spinal and cranial motor neurons. While a dominant negative, kinase-dead cdk5 promotes the generation of supernumerary motor neurons; over-expression of cdk5 suppresses motor neuron development. Thus, modulating cdk5 activity seems promising in inducing motor neuron development in vivo. PMID:19523926

  8. 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.

  9. 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.

  10. Approaches to myosin modelling in a two-phase flow model for cell motility

    NASA Astrophysics Data System (ADS)

    Kimpton, L. S.; Whiteley, J. P.; Waters, S. L.; Oliver, J. M.

    2016-04-01

    A wide range of biological processes rely on the ability of cells to move through their environment. Mathematical models have been developed to improve our understanding of how cells achieve motion. Here we develop models that explicitly track the cell's distribution of myosin within a two-phase flow framework. Myosin is a small motor protein which is important for contracting the cell's actin cytoskeleton and enabling cell motion. The two phases represent the actin network and the cytosol in the cell. We start from a fairly general description of myosin kinetics, advection and diffusion in the two-phase flow framework, then identify a number of sub-limits of the model that may be relevant in practice, two of which we investigate further via linear stability analyses and numerical simulations. We demonstrate that myosin-driven contraction of the actin network destabilizes a stationary steady state leading to cell motion, but that rapid diffusion of myosin and rapid unbinding of myosin from the actin network are stabilizing. We use numerical simulation to investigate travelling-wave solutions relevant to a steadily gliding cell and we consider a reduction of the model in which the cell adheres strongly to the substrate on which it is crawling. This work demonstrates that a number of existing models for the effect of myosin on cell motility can be understood as different sub-limits of our two-phase flow model.

  11. Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium.

    PubMed

    Uyeda, T Q; Yumura, S

    2000-04-15

    The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. PMID:10816252

  12. Two-headed binding of a processive myosin to F-actin.

    PubMed

    Walker, M L; Burgess, S A; Sellers, J R; Wang, F; Hammer, J A; Trinick, J; Knight, P J

    2000-06-15

    Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation. PMID:10866203

  13. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI.

    PubMed

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-14

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  14. Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

    PubMed

    Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A

    2016-04-01

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly

  15. "How Did I Make It?": Uncertainty about Own Motor Performance after Inhibition of the Premotor Cortex.

    PubMed

    Bolognini, Nadia; Zigiotto, Luca; Carneiro, Maíra Izzadora Souza; Vallar, Giuseppe

    2016-07-01

    Optimal motor performance requires the monitoring of sensorimotor input to ensure that the motor output matches current intentions. The brain is thought to be equipped with a "comparator" system, which monitors and detects the congruence between intended and actual movement; results of such a comparison can reach awareness. This study explored in healthy participants whether the cathodal transcranial direct current stimulation (tDCS) of the right premotor cortex (PM) and right posterior parietal cortex (PPC) can disrupt performance monitoring in a skilled motor task. Before and after tDCS, participants underwent a two-digit sequence motor task; in post-tDCS session, single-pulse TMS (sTMS) was applied to the right motor cortex, contralateral to the performing hand, with the aim of interfering with motor execution. Then, participants rated on a five-item questionnaire their performance at the motor task. Cathodal tDCS of PM (but not sham or PPC tDCS) impaired the participants' ability to evaluate their motor performance reliably, making them unconfident about their judgments. Congruently with the worsened motor performance induced by sTMS, participants reported to have committed more errors after sham and PPC tDCS; such a correlation was not significant after PM tDCS. In line with current computational and neuropsychological models of motor control and awareness, the present results show that a mechanism in the PM monitors and compares intended versus actual movements, evaluating their congruence. Cathodal tDCS of the PM impairs the activity of such a "comparator," disrupting self-confidence about own motor performance. PMID:26967945

  16. Lamellipodial actin mechanically links myosin activity with adhesion-site formation.

    PubMed

    Giannone, Grégory; Dubin-Thaler, Benjamin J; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P

    2007-02-01

    Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  17. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    SciTech Connect

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  18. Development and initial assessment of a new paradigm for assessing cognitive and motor inhibition: the bimodal virtual-reality Stroop.

    PubMed

    Henry, Mylène; Joyal, Christian C; Nolin, Pierre

    2012-09-30

    Assessing and predicting inhibition in adults is a common assignment for clinicians. However, there is no single measure of inhibition that is complete, sensitive and enjoyable. The main goal of this study was to develop a virtual reality neuropsychological task (the bimodal VR-Stroop) capable of measuring both cognitive (control of internal and external interference) and motor inhibition (a go no-go paradigm with reaction time variation, commission errors and omissions). Preliminary data obtained with 71 healthy adult participants confirmed that the VR-Stroop is capable of eliciting the Stroop effect with bimodal stimuli. Initial validation data also suggested that measures of the VR-Stroop significantly correlate with measures of the Elevator counting with distracters, the Continuous Performance Task (CPT-II), and the Stop-it task. Finally, regression analyses indicated that commission errors and variability of reaction times at the VR-Stroop were significantly predicted by scores of the Elevator task and the CPT-II. These preliminary results suggest that the VR-Stroop is an interesting measure of cognitive and motor inhibition for adults, although confirmatory investigations are warranted. PMID:22897988

  19. Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype.

    PubMed

    Schubert, Timm; Gleiser, Corinna; Heiduschka, Peter; Franz, Christoph; Nagel-Wolfrum, Kerstin; Sahaboglu, Ayse; Weisschuh, Nicole; Eske, Gordon; Rohbock, Karin; Rieger, Norman; Paquet-Durand, François; Wissinger, Bernd; Wolfrum, Uwe; Hirt, Bernhard; Singer, Wibke; Rüttiger, Lukas; Zimmermann, Ulrike; Knipper, Marlies

    2015-10-01

    The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms. PMID:25939269

  20. The effect of age on in vitro motility speed of slow myosin extracted from single rat soleus fibres.

    PubMed

    Höök, P; Li, X; Sleep, J; Hughes, S; Larsson, L

    1999-12-01

    The effect of age on the motor protein myosin was examined in a novel in vitro motility assay. Myosin was extracted from soleus fibres of young (3-6 month) and old (20-24 month) rats. All fibres expressed the type I myosin heavy chain (MyHC) and the slow isoforms of the myosin light chains (MyLCs). In vitro motility speed was significantly (P < 0.001) faster in the young adult (1.43 +/- 0.23 microm s-1) than in the aged group (1.27 +/- 0.23 microm s-1). The result indicates that the age-related decrease in contractile speed observed in slow fibres may be the effect of a change in the properties of myosin with age. PMID:10632634

  1. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity. PMID:25733667

  2. Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

    PubMed Central

    Smith, Cody J.; Morris, Angela D.; Welsh, Taylor G.; Kucenas, Sarah

    2014-01-01

    Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition. PMID:25268888

  3. Premotor spinal network with balanced excitation and inhibition during motor patterns has high resilience to structural division.

    PubMed

    Petersen, Peter C; Vestergaard, Mikkel; Jensen, Kristian H R; Berg, Rune W

    2014-02-19

    Direct measurements of synaptic inhibition (I) and excitation (E) to spinal motoneurons can provide an important insight into the organization of premotor networks. Such measurements of flexor motoneurons participating in motor patterns in turtles have recently demonstrated strong concurrent E and I as well as stochastic membrane potentials and irregular spiking in the adult turtle spinal cord. These findings represent a departure from the widespread acceptance of feedforward reciprocal rate models for spinal motor function. The apparent discrepancy has been reviewed as an experimental artifact caused by the distortion of local networks in the transected turtle spinal cord. We tested this assumption in the current study by performing experiments to assess the integrity of motor functions in the intact spinal cord and the cord transected at segments D9/D10. Excitatory and inhibitory synaptic inputs to motoneurons were estimated during rhythmic motor activity and demonstrated primarily intense inputs that consisted of qualitatively similar mixed E/I before and after the transection. To understand this high functional resilience, we used mathematical modeling of networks with recurrent connectivity that could potentially explain the balanced E/I. Both experimental and modeling data support the concept of a locally balanced premotor network consisting of recurrent E/I connectivity, in addition to the well known reciprocal network activity. The multifaceted synaptic connections provide spinal networks with a remarkable ability to remain functional after structural divisions. PMID:24553920

  4. A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton.

    PubMed

    Yuan, Yanggang; Zhao, Chuanyan; An, Xiaofei; Wu, Lin; Wang, Hui; Zhao, Min; Bai, Mi; Duan, Suyan; Zhang, Bo; Zhang, Aihua; Xing, Changying

    2016-06-01

    Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. PMID:26902808

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

    SciTech Connect

    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.

  6. Molecular mechanism regulating myosin and cardiac functions by ELC.

    PubMed

    Lossie, Janine; Köhncke, Clemens; Mahmoodzadeh, Shokoufeh; Steffen, Walter; Canepari, Monica; Maffei, Manuela; Taube, Martin; Larchevêque, Oriane; Baumert, Philipp; Haase, Hannelore; Bottinelli, Roberto; Regitz-Zagrosek, Vera; Morano, Ingo

    2014-07-18

    The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 μm/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3±3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1(E56G)≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations. PMID:24911555

  7. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division

    PubMed Central

    Uhart, Perrine; Tassan, Jean-Pierre; Michaux, Grégoire

    2015-01-01

    During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis. PMID:26416962

  8. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.

    PubMed

    Pacquelet, Anne; Uhart, Perrine; Tassan, Jean-Pierre; Michaux, Grégoire

    2015-09-28

    During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis. PMID:26416962

  9. Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

    PubMed Central

    Noguchi, Tatsuhiko; Frank, Deborah J.; Isaji, Mamiko

    2009-01-01

    Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. PMID:19005209

  10. Evolution of Premotor Cortical Excitability after Cathodal Inhibition of the Primary Motor Cortex: A Sham-Controlled Serial Navigated TMS Study

    PubMed Central

    Bathe-Peters, Rouven; Irlbacher, Kerstin; Brandt, Stephan A.

    2013-01-01

    Background Premotor cortical regions (PMC) play an important role in the orchestration of motor function, yet their role in compensatory mechanisms in a disturbed motor system is largely unclear. Previous studies are consistent in describing pronounced anatomical and functional connectivity between the PMC and the primary motor cortex (M1). Lesion studies consistently show compensatory adaptive changes in PMC neural activity following an M1 lesion. Non-invasive brain modification of PMC neural activity has shown compensatory neurophysiological aftereffects in M1. These studies have contributed to our understanding of how M1 responds to changes in PMC neural activity. Yet, the way in which the PMC responds to artificial inhibition of M1 neural activity is unclear. Here we investigate the neurophysiological consequences in the PMC and the behavioral consequences for motor performance of stimulation mediated M1 inhibition by cathodal transcranial direct current stimulation (tDCS). Purpose The primary goal was to determine how electrophysiological measures of PMC excitability change in order to compensate for inhibited M1 neural excitability and attenuated motor performance. Hypothesis Cathodal inhibition of M1 excitability leads to a compensatory increase of ipsilateral PMC excitability. Methods We enrolled 16 healthy participants in this randomized, double-blind, sham-controlled, crossover design study. All participants underwent navigated transcranial magnetic stimulation (nTMS) to identify PMC and M1 corticospinal projections as well as to evaluate electrophysiological measures of cortical, intracortical and interhemispheric excitability. Cortical M1 excitability was inhibited using cathodal tDCS. Finger-tapping speeds were used to examine motor function. Results Cathodal tDCS successfully reduced M1 excitability and motor performance speed. PMC excitability was increased for longer and was the only significant predictor of motor performance. Conclusion The PMC

  11. Glycinergic and GABA(A)-mediated inhibition of somatic motoneurons does not mediate rapid eye movement sleep motor atonia.

    PubMed

    Brooks, Patricia L; Peever, John H

    2008-04-01

    A hallmark of rapid eye movement (REM) sleep is a potent suppression of postural muscle tone. Motor control in REM sleep is unique because it is characterized by flurries of intermittent muscle twitches that punctuate muscle atonia. Because somatic motoneurons are bombarded by strychnine-sensitive IPSPs during REM sleep, it is assumed that glycinergic inhibition underlies REM atonia. However, it has never been determined whether glycinergic inhibition of motoneurons is indeed responsible for triggering the loss of postural muscle tone during REM sleep. Therefore, we used reverse microdialysis, electrophysiology, and pharmacological and histological methods to determine whether glycinergic and/or GABA(A)-mediated neurotransmission at the trigeminal motor pool mediates masseter muscle atonia during REM sleep in rats. By antagonizing glycine and GABA(A) receptors on trigeminal motoneurons, we unmasked a tonic glycinergic/GABAergic drive at the trigeminal motor pool during waking and non-rapid eye movement (NREM) sleep. Blockade of this drive potently increased masseter muscle tone during both waking and NREM sleep. This glycinergic/GABAergic drive was immediately switched-off and converted into a phasic glycinergic drive during REM sleep. Blockade of this phasic drive potently provoked muscle twitch activity in REM sleep; however, it did not prevent or reverse REM atonia. Muscle atonia in REM even persisted when glycine and GABA(A) receptors were simultaneously antagonized and trigeminal motoneurons were directly activated by glutamatergic excitation, indicating that a powerful, yet unidentified, inhibitory mechanism overrides motoneuron excitation during REM sleep. Our data refute the prevailing hypothesis that REM atonia is caused by glycinergic inhibition. The inhibitory mechanism mediating REM atonia therefore requires reevaluation. PMID:18385312

  12. Evidence That the Cortical Motor Command for the Initiation of Dynamic Plantarflexion Consists of Excitation followed by Inhibition

    PubMed Central

    Taube, Wolfgang; Lundbye-Jensen, Jesper; Schubert, Martin; Gollhofer, Albert; Leukel, Christian

    2011-01-01

    At the onset of dynamic movements excitation of the motor cortex (M1) is spatially restricted to areas representing the involved muscles whereas adjacent areas are inhibited. The current study elucidates whether the cortical motor command for dynamic contractions is also restricted to a certain population of cortical neurons responsible for the fast corticospinal projections. Therefore, corticospinal transmission was assessed with high temporal resolution during dynamic contractions after both, magnetic stimulation over M1 and the brainstem. The high temporal resolution could be obtained by conditioning the soleus H-reflex with different interstimulus intervals by cervicomedullary stimulation (CMS-conditioning) and transcranial magnetic stimulation (TMS) of M1 (M1-conditioning). This technique provides a precise time course of facilitation and inhibition. CMS- and M1-conditioning produced an ‘early facilitation’ of the H-reflex, which occurred around 3 ms earlier with CMS-conditioning. The early facilitation is believed to be caused by activation of direct monosynaptic projections to the spinal motoneurons. CMS-conditioning resulted in a subsequent ‘late facilitation’, which is considered to reflect activity of slow-conducting and/or indirect corticospinal pathways. In contrast, M1-conditioning produced a ‘late dis-facilitation’ or even ‘late inhibition’. As the late dis-facilitation was only seen following M1- but not CMS-conditioning, it is argued that cortical activation during dynamic tasks is restricted to fast, direct corticospinal projections whereas corticomotoneurons responsible for slow and/or indirectly projecting corticospinal pathways are inhibited. The functional significance of restricting the descending cortical drive to fast corticospinal pathways may be to ensure a temporally focused motor command during the execution of dynamic movements. PMID:22003402

  13. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    PubMed Central

    Wu, Shenping; Liu, Jun; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2010-01-01

    Background 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. Methodology 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 filament 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°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° 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. Conclusion 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

  14. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    SciTech Connect

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    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 filament 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

  15. Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex

    PubMed Central

    Le, Kang; Li, Chun-Chun; Ye, Guan; Moss, Joel; Vaughan, Martha

    2013-01-01

    Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells. PMID:23918382

  16. 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

  17. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    PubMed Central

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-01

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor’s oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  18. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

    PubMed Central

    Yang, Long; Qin, Li; Liu, Guosheng; Peremyslov, Valera V.; Dolja, Valerian V.; Wei, Yangdou

    2014-01-01

    The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity. PMID:25201952

  19. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

  20. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  1. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  2. Electrophysiological manifestations of stimulus evaluation, response inhibition and motor processing in Tourette syndrome patients

    PubMed Central

    Thibault, Genevieve; O’Connor, Kieron P.; Stip, Emmanuel; Lavoie, Marc E.

    2013-01-01

    Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder with childhood onset presenting with multiple fluctuating motor tics and one or more phonic tics. A significant proportion of people suffering from GTS are still symptomatic in adulthood and present other emotional and cognitive difficulties, along with motor problems that often accompany these comorbid conditions. The nature of these difficulties is still poorly understood and multiple comorbidities are often inadequately controlled. The current study investigates both stimulus evaluation and motor processing in GTS while controlling for comorbidity. Fifteen adults with GTS and 20 control participants were matched on gender, laterality and intelligence. The P300 component, the no-go anteriorization (NGA) as well as the stimulus and response-locked lateralized-readiness potentials (S-LRP, R-LRP) were elicited during a stimulus–response compatibility (SRC) paradigm. The standard version of the Stroop Color–Word Test (SCWT) was also administered. Reaction times showed that participants with GTS processed both the SRC and the SCWT more rapidly than the control group, while producing a delayed P300 peak latency. The GTS group also showed faster S-LRP onset in response to the incompatible and faster processing of interference in the SCWT. There was also a tendency toward a greater frontal shift of the NGA in the GTS group. The P300 latency showed that with GTS patients, stimulus evaluation occurs later whereas the overlapping pre-motor response selection processes occur faster. Our findings are congruent with a probable cortical motor over-activation hypothesis of GTS involving faster motor program selection in processing conflicting SR configuration. PMID:19395047

  3. Electron microscopic recording of myosin head power stroke in hydrated myosin filaments

    PubMed Central

    Sugi, Haruo; Chaen, Shigeru; Akimoto, Tsuyoshi; Minoda, Hiroki; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Sugiura, Seiryo

    2015-01-01

    Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed. PMID:26498981

  4. Diversity and Similarity of Motor Function and Cross-Bridge Kinetics in Papillary Muscles of Transgenic Mice Carrying Myosin Regulatory Light Chain Mutations D166V and R58Q

    PubMed Central

    Wang, Li; Muthu, Priya; Szczesna-Cordary, Danuta; Kawai, Masataka

    2013-01-01

    Mechanical properties of skinned papillary muscle fibers from transgenic mice expressing familial hypertrophic cardiomyopathy associated mutations D166V and R58Q in myosin regulatory light chain were investigated. Elementary steps and the apparent rate constants of the cross-bridge cycle were characterized from the tension transients induced by sinusoidal length changes during maximal Ca2+ activation, together with ATP, ADP, and Pi studies. The tension-pCa relation was also tested in two sets of solutions with differing Pi and ionic strength. Our results showed that in both mutants, the fast apparent rate constant 2πc and the rate constants of the cross-bridge detachment step (k2) were smaller than those of wild type (WT), demonstrating the slower cross-bridge kinetics. D166V showed significantly smaller ATP (K1) and ADP (K0) association constants than WT, displaying weaker ATP binding and easier ADP release, whereas those of R58Q were not significantly different from WT. In tension-pCa study, both D166V and R58Q mutations exhibited increased Ca2+ sensitivity and less cooperativity. We conclude that, while the two FHC mutations have similar clinical manifestations and prognosis, some of the mechanical parameters of cross-bridges (K0, K1) are differently modified, whereas some others (Ca2+-sensitivity, cooperativity, k2) are similarly modified by these two FHC associated mutations. PMID:23727233

  5. Mapping interactions between myosin relay and converter domains that power muscle function.

    PubMed

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

    2014-05-01

    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 (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). 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

  6. Myosin VI and its cargo adaptors – linking endocytosis and autophagy

    PubMed Central

    Tumbarello, David A.; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Summary The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway – both of which are involved in cargo trafficking and sorting – through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein–protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy. PMID:23781020

  7. Delineating cooperative responses of processive motors in living cells

    PubMed Central

    Efremov, Artem K.; Radhakrishnan, Anand; Tsao, David S.; Bookwalter, Carol S.; Trybus, Kathleen M.; Diehl, Michael R.

    2014-01-01

    Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances, and position noise on these parameters suggests that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared with those of other cytoskeletal motors. PMID:24402168

  8. INVESTIGATIONS OF AMITRAZ NEUROTOXICITY IN RATS. III. EFFECTS ON MOTOR ACTIVITY AND INHIBITION OF MONOAMINE OXIDASE

    EPA Science Inventory

    The formamidine pesticide amitraz (AMZ) produces many behavioral and physiological changes in rats. o explore possible neurochemical mechanisms for the behavioral effects of AMZ, we examined the dose effect and time course of AMZ on motor activity, monoamine oxidase (MAO) and ace...

  9. Impaired Inhibition of Prepotent Motor Tendencies in Friedreich Ataxia Demonstrated by the Simon Interference Task

    ERIC Educational Resources Information Center

    Corben, L. A.; Akhlaghi, H.; Georgiou-Karistianis, N.; Bradshaw, J. L.; Egan, G. F.; Storey, E.; Churchyard, A. J.; Delatycki, M. B.

    2011-01-01

    Friedreich ataxia (FRDA) is the most common of the genetically inherited ataxias. We recently demonstrated that people with FRDA have impairment in motor planning--most likely because of pathology affecting the cerebral cortex and/or cerebello-cortical projections. We used the Simon interference task to examine how effective 13 individuals with…

  10. Involvement of unconventional myosin VI in myoblast function and myotube formation.

    PubMed

    Karolczak, Justyna; Pavlyk, Iuliia; Majewski, Łukasz; Sobczak, Magdalena; Niewiadomski, Paweł; Rzhepetskyy, Yuriy; Sikorska, Agata; Nowak, Natalia; Pomorski, Paweł; Prószyński, Tomasz; Ehler, Elisabeth; Rędowicz, Maria Jolanta

    2015-07-01

    The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation. PMID:25896210

  11. 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

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

    PubMed

    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

    2016-06-14

    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

  13. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator. PMID:27273580

  14. Reconciling the influence of task-set switching and motor inhibition processes on stop signal after-effects

    PubMed Central

    Anguera, Joaquin A.; Lyman, Kyle; Zanto, Theodore P.; Bollinger, Jacob; Gazzaley, Adam

    2013-01-01

    Executive response functions can be affected by preceding events, even if they are no longer associated with the current task at hand. For example, studies utilizing the stop signal task have reported slower response times to “GO” stimuli when the preceding trial involved the presentation of a “STOP” signal. However, the neural mechanisms that underlie this behavioral after-effect are unclear. To address this, behavioral and electroencephalography (EEG) measures were examined in 18 young adults (18–30 years) on “GO” trials following a previously “Successful Inhibition” trial (pSI), a previously “Failed Inhibition” trial (pFI), and a previous “GO” trial (pGO). Like previous research, slower response times were observed during both pSI and pFI trials (i.e., “GO” trials that were preceded by a successful and unsuccessful inhibition trial, respectively) compared to pGO trials (i.e., “GO” trials that were preceded by another “GO” trial). Interestingly, response time slowing was greater during pSI trials compared to pFI trials, suggesting executive control is influenced by both task set switching and persisting motor inhibition processes. Follow-up behavioral analyses indicated that these effects resulted from between-trial control adjustments rather than repetition priming effects. Analyses of inter-electrode coherence (IEC) and inter-trial coherence (ITC) indicated that both pSI and pFI trials showed greater phase synchrony during the inter-trial interval compared to pGO trials. Unlike the IEC findings, differential ITC was present within the beta and alpha frequency bands in line with the observed behavior (pSI > pFI > pGO), suggestive of more consistent phase synchrony involving motor inhibition processes during the ITI at a regional level. These findings suggest that between-trial control adjustments involved with task-set switching and motor inhibition processes influence subsequent performance, providing new insights into the

  15. Isolation of myosin from starfish sperm heads.

    PubMed

    Mabuchi, I

    1976-08-01

    Myosin was extracted and partially purified from the head portion of spermatozoa of the starfish, Asterias amurensis. The sperm myosin showed a specific Ca2+-activated ATPase [EC 3.6.1.3] activity of 0.2 mumoles Pi/min/mg at high ionic strength and pH 6.5. It resembled egg myosin in forming thick filaments, becoming attached to actin filaments. subunit composition, and serological properties. PMID:12147

  16. 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

  17. Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.

    PubMed

    Katta, Santharam S; Sahasrabuddhe, Amogh A; Gupta, Chhitar M

    2009-04-01

    Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids. PMID:19121339

  18. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  19. Identification of T. gondii Myosin Light Chain-1 as a Direct Target of TachypleginA-2, a Small-Molecule Inhibitor of Parasite Motility and Invasion

    PubMed Central

    Leung, Jacqueline M.; Tran, Fanny; Pathak, Ravindra B.; Poupart, Séverine; Heaslip, Aoife T.; Ballif, Bryan A.; Westwood, Nicholas J.; Ward, Gary E.

    2014-01-01

    Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite’s life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by “click” chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite’s myosin motor. PMID:24892871

  20. Insights from the supplementary motor area syndrome in balancing movement initiation and inhibition

    PubMed Central

    Potgieser, A. R. E.; de Jong, B. M.; Wagemakers, M.; Hoving, E. W.; Groen, R. J. M.

    2014-01-01

    The supplementary motor area (SMA) syndrome is a characteristic neurosurgical syndrome that can occur after unilateral resection of the SMA. Clinical symptoms may vary from none to a global akinesia, predominantly on the contralateral side, with preserved muscle strength and mutism. A remarkable feature is that these symptoms completely resolve within weeks to months, leaving only a disturbance in alternating bimanual movements. In this review we give an overview of the old and new insights from the SMA syndrome and extrapolate these findings to seemingly unrelated diseases and symptoms such as Parkinson’s disease (PD) and tics. Furthermore, we integrate findings from lesion, stimulation and functional imaging studies to provide insight in the motor function of the SMA. PMID:25506324

  1. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

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

    PubMed

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, H Daniel; 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

  3. Electrophysiological properties of lumbar motoneurons in the alpha-chloralose-anesthetized cat during carbachol-induced motor inhibition.

    PubMed

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1997-07-01

    The present study was undertaken 1) to examine the neuronal mechanisms responsible for the inhibition of spinal cord motoneurons that occurs in alpha-chloralose-anesthetized cats following the microinjection of carbachol into the nucleus pontis oralis (NPO), and 2) to determine whether the inhibitory mechanisms are the same as those that are responsible for the postsynaptic inhibition of motoneurons that is present during naturally occurring active sleep. Accordingly, the basic electrophysiological properties of lumbar motoneurons were examined, with the use of intracellular recording techniques, in cats anesthetized with alpha-chloralose and compared with those present during naturally occurring active sleep. The intrapontine administration of carbachol resulted in a sustained reduction in the amplitude of the spinal cord Ia monosynaptic reflex. Discrete large-amplitude inhibitory postsynaptic potentials (IPSPs), which are only present during the state of active sleep in the chronic cat, were also observed in high-gain recordings from lumbar motoneurons after the injection of carbachol. During carbachol-induced motor inhibition, lumbar motoneurons exhibited a statistically significant decrease in input resistance, membrane time constant and a reduction in the amplitude of the action potential's afterhyperpolarization. In addition, there was a statistically significant increase in rheobase and in the delay between the initial-segment (IS) and somadendritic (SD) portions of the action potential (IS-SD delay). There was a significant increase in the mean motoneuron resting membrane potential (i.e., hyperpolarization). The preceding changes in the electrophysiological properties of motoneurons, as well as the development of discrete IPSPs, indicate that lumbar motoneurons are postsynaptically inhibited after the intrapontine administration of carbachol in cats that are anesthetized with alpha-chloralose. These changes in the electrophysiological properties of lumbar

  4. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

    The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures. PMID:27060364

  5. Intraduodenal capsaicin inhibits gastric migrating motor complex via an extrinsic neural reflex in conscious dogs.

    PubMed

    Shibata, C; Naito, H; Ueno, T; Jin, X-L; Funayama, Y; Fukushima, K; Matsuno, S; Sasaki, I

    2002-10-01

    The aim was to study the effect of intraduodenal capsaicin on interdigestive gastric contractions. Mongrel dogs were equipped with strain-gauge force transducers to measure gastroduodenal motility. The effects of intraduodenal capsaicin with or without pharmacological antagonists on spontaneous and motilin-induced interdigestive gastric contractions and on plasma motilin were studied in dogs with intact stomachs. The effect of intraduodenal capsaicin on gastric contractions was also studied in vagally denervated gastric (Heidenhain) pouch and vagally innervated antral pouch. Intraduodenal capsaicin inhibited spontaneous and motilin-induced gastric contractions. The spontaneous peak in plasma motilin was inhibited by intraduodenal capsaicin. The effect of intraduodenal capsaicin on motilin-induced gastric contractions was not affected by blockade of nitric oxide synthase, or by beta-adrenoceptor antagonist. Administration of alpha-adrenergic blocker inhibited basal interdigestive gastric motility. Intraduodenal capsaicin had no effect on contractions in the Heidenhain pouch but inhibited those in vagally innervated antral pouch. Duodenal afferent fibres stimulated by capsaicin inhibit gastric contractions via a nitric oxide-independent extrinsic neural reflex. PMID:12358683

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

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    2015-02-15

    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 aggregated 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.

  7. 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. PMID:26586917

  8. 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

  9. Suppression of motor protein KIF3C expression inhibits tumor growth and metastasis in breast cancer by inhibiting TGF-β signaling.

    PubMed

    Wang, Chengqin; Wang, Chenggang; Wei, Zhimin; Li, Yujun; Wang, Wenhong; Li, Xia; Zhao, Jing; Zhou, Xuan; Qu, Xun; Xiang, Fenggang

    2015-11-01

    Breast cancer is the most common cause of death among women. KIF3C, a member of kinesin superfamily, functions as a motor protein involved in axonal transport in neuronal cells. To explore the expression, regulation and mechanism of KIF3C in breast cancer, 4 breast cancer cell lines and 93 cases of primary breast cancer and paired adjacent tissues were examined. Immunohistochemistry, Real Time Polymerase Chain Reaction (RT-PCR), Western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation techniques and xenograft mice model were used. We found that KIF3C was over-expressed in breast cancer tissues and such high KIF3C expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3C by shRNA inhibited epithelial-mesenchymal transition and metastasis by inhibiting TGF-β signaling and suppressed breast cancer cell proliferation through inducing G2/M phase arrest. The tumor size was smaller and the number of lung metastatic nodules was less in KIF3C depletion MDA-MB-231 cell xenograft mice than in negative control group. These results suggested that high expression of KIF3C in breast cancer may be associated with the tumor progression and metastasis. PMID:26272184

  10. Cytoskeletal coherence requires myosin-IIA contractility

    PubMed Central

    Cai, Yunfei; Rossier, Olivier; Gauthier, Nils C.; Biais, Nicolas; Fardin, Marc-Antoine; Zhang, Xian; Miller, Lawrence W.; Ladoux, Benoit; Cornish, Virginia W.; Sheetz, Michael P.

    2010-01-01

    Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in ‘C’ or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading. PMID:20067993

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

    PubMed

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

    2016-03-29

    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 [Ca(2+)]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 Ca(2+)-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

  12. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

    PubMed

    Linari, Marco; Brunello, Elisabetta; Reconditi, Massimo; Fusi, Luca; Caremani, Marco; Narayanan, Theyencheri; Piazzesi, Gabriella; Lombardi, Vincenzo; Irving, Malcolm

    2015-12-10

    Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart. PMID:26560032

  13. Radial glia inhibit peripheral glial infiltration into the spinal cord at motor exit point transition zones.

    PubMed

    Smith, Cody J; Johnson, Kimberly; Welsh, Taylor G; Barresi, Michael J F; Kucenas, Sarah

    2016-07-01

    In the mature vertebrate nervous system, central and peripheral nervous system (CNS and PNS, respectively) GLIA myelinate distinct motor axon domains at the motor exit point transition zone (MEP TZ). How these cells preferentially associate with and myelinate discrete, non-overlapping CNS versus PNS axonal segments, is unknown. Using in vivo imaging and genetic cell ablation in zebrafish, we demonstrate that radial glia restrict migration of PNS glia into the spinal cord during development. Prior to development of radial glial endfeet, peripheral cells freely migrate back and forth across the MEP TZ. However, upon maturation, peripherally located cells never enter the CNS. When we ablate radial glia, peripheral glia ectopically migrate into the spinal cord during developmental stages when they would normally be restricted. These findings demonstrate that radial glia contribute to both CNS and PNS development and control the unidirectional movement of glial cell types across the MEP TZ early in development. GLIA 2016. GLIA 2016;64:1138-1153. PMID:27029762

  14. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  15. Response Inhibition in Motor and Oculomotor Conflict Tasks: Different Mechanisms, Different Dynamics?

    ERIC Educational Resources Information Center

    Wijnen, Jasper G.; Ridderinkhof, K. Richard

    2007-01-01

    Previous research has shown that the appearance of task-irrelevant abrupt onsets influences saccadic eye movements during visual search and may slow down manual reactions to target stimuli. Analysis of reaction time distributions in the present study offers evidence suggesting that top-down inhibition processes actively suppress oculomotor or…

  16. Motoneuron properties during motor inhibition produced by microinjection of carbachol into the pontine reticular formation of the decerebrate cat.

    PubMed

    Morales, F R; Engelhardt, J K; Soja, P J; Pereda, A E; Chase, M H

    1987-04-01

    It is well established that cholinergic agonists, when injected into the pontine reticular formation in cats, produce a generalized suppression of motor activity (1, 3, 6, 14, 18, 27, 33, 50). The responsible neuronal mechanisms were explored by measuring ventral root activity, the amplitude of the Ia-monosynaptic reflex, and the basic electrophysiological properties of hindlimb motoneurons before and after carbachol was microinjected into the pontine reticular formation of decerebrate cats. Intrapontine microinjections of carbachol (0.25-1.0 microliter, 16 mg/ml) resulted in the tonic suppression of ventral root activity and a decrease in the amplitude of the Ia-monosynaptic reflex. An analysis of intracellular records from lumbar motoneurons during the suppression of motor activity induced by carbachol revealed a considerable decrease in input resistance and membrane time constant as well as a reduction in motoneuron excitability, as evidenced by a nearly twofold increase in rheobase. Discrete inhibitory postsynaptic potentials were also observed following carbachol administration. The changes in motoneuron properties (rheobase, input resistance, and membrane time constant), as well as the development of discrete inhibitory postsynaptic potentials, indicate that spinal cord motoneurons were postsynaptically inhibited following the pontine administration of carbachol. In addition, the inhibitory processes that arose after carbachol administration in the decerebrate cat were remarkably similar to those that are present during active sleep in the chronic cat. These findings suggest that the microinjection of carbachol into the pontine reticular formation activates the same brain stem-spinal cord system that is responsible for the postsynaptic inhibition of alpha-motoneurons that occurs during active sleep. PMID:3585456

  17. Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum.

    PubMed

    Katta, Santharam S; Tammana, Trinadh V Satish; Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2010-06-15

    Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum. PMID:20501700

  18. 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. PMID:27166421

  19. Molecular dynamics simulation for the reversed power stroke motion of a myosin subfragment-1.

    PubMed

    Masuda, Tadashi

    2015-06-01

    Myosins are typical molecular motor proteins that convert the chemical energy from the ATP hydrolysis into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results already obtained, Masuda has proposed a hypothesis called the "Driven by Detachment" theory for the working principle of the myosins. This theory insists that the energy used during the power stroke of the myosins does not directly originate from the chemical energy of ATP, but is converted from the elastic energy within the molecule at the joint between the head and neck domains. One method for demonstrating the validity of this theory is a computational simulation using the molecular dynamics (MD) method. The MD software used was GROMACS. The target of the MD simulations was myosin subfragment-1 (S1), for which the initial structure was obtained from the Protein Data Bank entry 1M8Q. The AFM pull code of GROMACS was used to apply an external force of 17 pN at the end of the neck domain in the direction opposite to the power stroke to observe whether the myosin S1 takes the pre-power stroke conformation. The residues assumed to be engaged in the docking with an actin filament were fixed to the space. Starting from exactly the same initial position, 10 simulations were repeated by varying the random seeds for generating the initial velocities of the atoms. After 64ns of calculations, the myosin S1 took the conformation of the pre-power stroke state in which the neck domain was bent around the joint between the head and the neck domains. This result agrees with the prediction expected by the DbD theory, the validity of which may be established by conducting similar simulations for the other steps of the myosin working processes. PMID:25864376

  20. The kinetics underlying the velocity of smooth muscle myosin filament sliding on actin filaments in vitro.

    PubMed

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

    2014-07-25

    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

  1. 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

  2. Resolution of three structural states of spin-labeled myosin in contracting muscle.

    PubMed Central

    Ostap, E M; Barnett, V A; Thomas, D D

    1995-01-01

    We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle. Images FIGURE 7 PMID:7669895

  3. Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron

    PubMed Central

    Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

    2016-01-01

    Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons. PMID:27570482

  4. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    SciTech Connect

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K.

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  5. Reverse actin sliding triggers strong myosin binding that moves tropomyosin.

    PubMed

    Bekyarova, T I; Reedy, M C; Baumann, B A J; Tregear, R T; Ward, A; Krzic, U; Prince, K M; Perz-Edwards, R J; Reconditi, M; Gore, D; Irving, T C; Reedy, M K

    2008-07-29

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions. PMID:18658238

  6. The role of myosin-II in force generation of DRG filopodia and lamellipodia

    PubMed Central

    Sayyad, Wasim A.; Amin, Ladan; Fabris, Paolo; Ercolini, Erika; Torre, Vincent

    2015-01-01

    Differentiating neurons process the mechanical stimulus by exerting the protrusive forces through lamellipodia and filopodia. We used optical tweezers, video imaging and immunocytochemistry to analyze the role of non-muscle myosin-II on the protrusive force exerted by lamellipodia and filopodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. When the activity of myosin-II was inhibited by 30 μM Blebbistatin protrusion/retraction cycles of lamellipodia slowed down and during retraction lamellipodia could not lift up axially as in control condition. Inhibition of actin polymerization with 25 nM Cytochalasin-D and of microtubule polymerization with 500 nM Nocodazole slowed down the protrusion/retraction cycles, but only Cytochalasin-D decreased lamellipodia axial motion. The force exerted by lamellipodia treated with Blebbistatin decreased by 50%, but, surprisingly, the force exerted by filopodia increased by 20-50%. The concomitant disruption of microtubules caused by Nocodazole abolished the increase of the force exerted by filopodia treated with Blebbistatin. These results suggest that; i- Myosin-II controls the force exerted by lamellipodia and filopodia; ii- contractions of the actomyosin complex formed by filaments of actin and myosin have an active role in ruffle formation; iii- myosin-II is an essential component of the structural stability of GCs architecture. PMID:25598228

  7. Transcranial direct current stimulation reverses neurophysiological and behavioural effects of focal inhibition of human pharyngeal motor cortex on swallowing

    PubMed Central

    Vasant, Dipesh H; Mistry, Satish; Michou, Emilia; Jefferson, Samantha; Rothwell, John C; Hamdy, Shaheen

    2014-01-01

    The human cortical swallowing system exhibits bilateral but functionally asymmetric representation in health and disease as evidenced by both focal cortical inhibition (pre-conditioning with 1 Hz repetitive transcranial magnetic stimulation; rTMS) and unilateral stroke, where disruption of the stronger (dominant) pharyngeal projection alters swallowing neurophysiology and behaviour. Moreover, excitatory neurostimulation protocols capable of reversing the disruptive effects of focal cortical inhibition have demonstrated therapeutic promise in post-stroke dysphagia when applied contralaterally. In healthy participants (n = 15, 8 males, mean age (±SEM) 35 ± 9 years), optimal parameters of transcranial direct current stimulation (tDCS) (anodal, 1.5 mA, 10 min) were applied contralaterally after 1 Hz rTMS pre-conditioning to the strongest pharyngeal projection. Swallowing neurophysiology was assessed in both hemispheres by intraluminal recordings of pharyngeal motor-evoked responses (PMEPs) to single-pulse TMS as a measure of cortical excitability. Swallowing behaviour was examined using a pressure-based reaction time protocol. Measurements were made before and for up to 60 min post intervention. Subjects were randomised to active or sham tDCS after 1 Hz rTMS on separate days and data were compared using repeated measures ANOVA. Active tDCS increased PMEPs bilaterally (F1,14 = 7.4, P = 0.017) reversing the inhibitory effects of 1 Hz rTMS in the pre-conditioned hemisphere (F1,14 = 10.1, P = 0.007). Active tDCS also enhanced swallowing behaviour, increasing the number of correctly timed challenge swallows compared to sham (F1,14 = 6.3, P = 0.025). Thus, tDCS to the contralateral pharyngeal motor cortex reverses the neurophysiological and behavioural effects of focal cortical inhibition on swallowing in healthy individuals and has therapeutic potential for dysphagia rehabilitation. PMID:24247983

  8. Myosin-II repression favors pre/proplatelets but shear activation generates platelets and fails in macrothrombocytopenia

    PubMed Central

    Spinler, Kyle R.; Shin, Jae-Won; Lambert, Michele P.

    2015-01-01

    Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation and after treatment with a myosin-II inhibitor. MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent with MIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41+ fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of a MYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets in MYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number. PMID:25395423

  9. Cross-reactivity of termite myosin; a potential allergen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Opioid Peptides Inhibit Excitatory But Not Inhibitory Synaptic Transmission in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Browning, Kirsteen N.; Kalyuzhny, Alexander E.; Travagli, R. Alberto

    2011-01-01

    Opioid peptides produce gastrointestinal inhibition and increase feeding when applied to the brainstem. The present studies were designed to determine the actions of opioid peptides on synaptic transmission within the dorsal motor nucleus of the vagus (DMV) and the localization of μ-opioid receptors. Whole-cell recordings were made from identified gastrointestinal-projecting DMV neurons in thin brainstem slices of the rat. Electrical stimulation of the nucleus of the tractus solitarius evoked EPSCs and IPSCs. In all neurons tested, methionine (Met)-enkephalin (0.003–30 μm) inhibited the peak amplitude of the EPSCs. The effect was prevented by naloxone (1 μm) as well as by naloxonazine (0.2 μm). An increase in the ratio of the evoked paired pulses indicated that the inhibition was attributable to actions at presynaptic receptors. This presynaptic inhibitory action was mimicked by [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (0.1 μm) and the analgesic dipeptide kyotorphin (10 μm) but not by cyclic[d-Pen2, d-Pen5]-enkephalin (1 μm) and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (1 μm). In contrast, the amplitude of evoked IPSCs was not altered either by Met-enkephalin or by any of the opioid receptor-selective agonists. Immunohistochemical studies revealed that nerve terminals apposing DMV neurons showed immunoreactivity to μ-opioid receptors colocalized with glutamate immunoreactivity but not glutamic acid decarboxylase immunoreactivity. These results suggest that within the DMV, μ-opioid receptors are present on the nerve terminals of excitatory but not inhibitory inputs to GI motoneurons. Such specificity may imply that the central inhibitory action of opioid peptides on gastrointestinal function targets selected pathways. PMID:11943802

  11. Preventive effect of rikkunshito on gastric motor function inhibited by L-dopa in rats.

    PubMed

    Wang, Lixin; Mogami, Sachiko; Karasawa, Hiroshi; Yamada, Chihiro; Yakabi, Seiichi; Yakabi, Koji; Hattori, Tomohisa; Taché, Yvette

    2014-05-01

    We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinson's disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mgkg(-1)) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg(-1)) reduced the LD/CD (20/2 mg kg(-1)) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys(3)]-GHRP-6 (1 mg kg(-1)) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg(-1)) blocked LD/CD (20/2 mg kg(-1))-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinson's disease rat model, rikkunshito (1.0 g kg(-1), og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms. PMID:24631952

  12. Inhibition of the colonic motor response to eating by pinaverium bromide in irritable bowel syndrome patients.

    PubMed

    Fioramonti, J; Frexinos, J; Staumont, G; Bueno, L

    1988-01-01

    The effect of pinaverium bromide on the colonic motor response to eating was investigated in 10 irritable bowel syndrome patients, by means of an intraluminal probe supporting 8 groups of electrodes. At each site examined from transverse to sigmoid colon, the electromyograms exhibited 2 kinds of spike bursts: short spike bursts (SSB) localized at one electrode, and long spike bursts (LSB), isolated, propagated orally or aborally over a few centimeters, or aborally propagated over the whole length of the colon investigated (migrating long spike bursts, MLSB). Recordings were continuously performed over 24 hr. Each patient received at 7.00 p.m. on day 1 and at noon on day 2 an 800-1000 Kcal meal preceded by IV administration of pinaverium bromide (4 mg) or placebo. After placebo administration, the duration of LSB activity and the number of MLSB were significantly increased over 3 postprandial hr by comparison with the 2 hr preceding the meal. After pinaverium injection no significant postprandial change in LSB and MLSB activity was noted. The SSB activity was not modified after the meals preceded by placebo or pinaverium injection. These results suggest that the inhibitory action of pinaverium bromide on postprandial colonic motility may support the clinical efficacy of this agent in the treatment of the irritable bowel syndrome. PMID:3371838

  13. Arginine Vasopressin Injected into the Dorsal Motor Nucleus of the Vagus Inhibits Gastric Motility in Rats

    PubMed Central

    Zhu, Jianping; Chang, Lanlan; Xie, Jinlu; Ai, Hongbin

    2016-01-01

    Background. Until now, the effect of arginine vasopressin (AVP) in the DMV on gastric motility and the possible modulating pathway between the DMV and the gastrointestinal system remain poorly understood. Objectives. We aimed to explore the role of AVP in the DMV in regulating gastric motility and the possible central and peripheral pathways. Material and Methods. Firstly, we microinjected different doses of AVP into the DMV and investigated its effects on gastric motility in rats. Then, the possible central and peripheral pathways that regulate gastric motility were also discussed by microinjecting SR49059 (a specific AVP receptor antagonist) into the DMV and intravenous injection of hexamethonium (a specific neuronal nicotinic cholinergic receptor antagonist) before AVP microinjection. Results. Following microinjection of AVP (180 pmol and 18 pmol) into the DMV, the gastric motility (including total amplitude, total duration, and motility index of gastric contraction) was significantly inhibited (P < 0.05). Moreover, the inhibitory effect of AVP (180 pmol) on gastric motility could be blocked completely by both SR49059 (320 pmol) and hexamethonium (8 μmol). Conclusions. It is concluded that AVP inhibits the gastric motility by acting on the specific AVP receptor in the DMV, with the potential involvement of the parasympathetic preganglionic cholinergic fibers. PMID:26843857

  14. 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.

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

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    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 the 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.

  16. Study on the tripolyphosphatase (TPPase) property of bighead carp ( Aristichthys nobilis) myosin subfragment-1

    NASA Astrophysics Data System (ADS)

    Gao, Ruichang; Xue, Changhu; Yuan, Li; Zhang, Yongqin; Xue, Yong; Sun, Yan; Feng, Hui

    2007-10-01

    Myosin subfragment-1 was prepared from the myofibrils of bighead carp ( Aristichthys nobilis). The myosin subfragment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30°C and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2+ was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2+ under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL-1 under the condition of 17 mmolL-1 Mg2+. The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a K M of 3.2mmolL-1.

  17. Arp2/3 complex-dependent actin networks constrain myosin II function in driving retrograde actin flow.

    PubMed

    Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D; Forscher, Paul

    2012-06-25

    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

  18. Non-muscle myosin-II-B filament regulation of paracellular resistance in cervical epithelial cells is associated with modulation of the cortical acto-myosin

    PubMed Central

    Li, Xin; Gorodeski, George

    2007-01-01

    Objective To understand myosin regulation of epithelial permeability. Methods Experimental study, using human cervical epithelial cells CaSki. Endpoints were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B – actin interaction (determined in-vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in-vitro using purified NMM-II-B filaments in terms of filaments disassembly / assembly ratios. Results Treatment of cells with the ROCK inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the Resistance of the Lateral Intercellular Space (RLIS), and increased phosphorylation of non-muscle myosin-II-B (NMM-II-B) on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, CK2 inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with DRB did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the RLIS was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in-vivo with Y-27632, okadaic acid, or DRB. Conclusions The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the RLIS and increase the

  19. Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment

    PubMed Central

    Chibalina, Margarita V.; Seaman, Matthew N.J.; Miller, Christopher C.; Kendrick-Jones, John; Buss, Folma

    2009-01-01

    Summary Myosin VI is an actin-based retrograde motor protein, which plays a crucial role in both endocytic and secretory membrane trafficking pathways. Myosin VI’s targeting to and function in these intracellular pathways is mediated by a number of specific binding partners. In this paper we have identified a new myosin VI binding partner, Lemur tyrosine kinase 2 (LMTK2), which is the first transmembrane protein and kinase that directly binds to myosin VI. LMTK2 binds to the WWY site in the C-terminal myosin VI tail, the same site as the endocytic adaptor protein Dab2. When either myosin VI or LMTK2 is depleted by siRNA, the transferrin receptor (TfR) is trapped in swollen endosomes and tubule formation in the endocytic recycling pathway is dramatically reduced, showing that both proteins are required for the transport of cargo such as the TfR from early endosomes to the endocytic recycling compartment. PMID:18029400

  20. Ketogenic diet restores aberrant cortical motor maps and excitation-to-inhibition imbalance in the BTBR mouse model of autism spectrum disorder.

    PubMed

    Smith, Jacklyn; Rho, Jong M; Teskey, G Campbell

    2016-05-01

    Autism spectrum disorder (ASD) is an increasingly prevalent neurodevelopmental disorder characterized by deficits in sociability and communication, and restricted and/or repetitive motor behaviors. Amongst the diverse hypotheses regarding the pathophysiology of ASD, one possibility is that there is increased neuronal excitation, leading to alterations in sensory processing, functional integration and behavior. Meanwhile, the high-fat, low-carbohydrate ketogenic diet (KD), traditionally used in the treatment of medically intractable epilepsy, has already been shown to reduce autistic behaviors in both humans and in rodent models of ASD. While the mechanisms underlying these effects remain unclear, we hypothesized that this dietary approach might shift the balance of excitation and inhibition towards more normal levels of inhibition. Using high-resolution intracortical microstimulation, we investigated basal sensorimotor excitation/inhibition in the BTBR T+Itpr(tf)/J (BTBR) mouse model of ASD and tested whether the KD restores the balance of excitation/inhibition. We found that BTBR mice had lower movement thresholds and larger motor maps indicative of higher excitation/inhibition compared to C57BL/6J (B6) controls, and that the KD reversed both these abnormalities. Collectively, our results afford a greater understanding of cortical excitation/inhibition balance in ASD and may help expedite the development of therapeutic approaches aimed at improving functional outcomes in this disorder. PMID:26876011

  1. Changes in electrophysiological properties of cat hypoglossal motoneurons during carbachol-induced motor inhibition.

    PubMed

    Fung, S J; Yamuy, J; Xi, M C; Engelhardt, J K; Morales, F R; Chase, M H

    2000-12-01

    The control of hypoglossal motoneurons during sleep is important from a basic science perspective as well as to understand the bases for pharyngeal occlusion which results in the obstructive sleep apnea syndrome. In the present work, we used intracellular recording techniques to determine changes in membrane properties in adult cats in which atonia was produced by the injection of carbachol into the pontine tegmentum (AS-carbachol). During AS-carbachol, 86% of the recorded hypoglossal motoneurons were found to be postsynaptically inhibited on the basis of analyses of their electrical properties; the electrical properties of the remaining 14% were similar to motoneurons recorded during control conditions. Those cells that exhibited changes in their electrical properties during AS-carbachol also displayed large-amplitude inhibitory synaptic potentials. Following sciatic nerve stimulation, hypoglossal motoneurons which responded with a depolarizing potential during control conditions exhibited a hyperpolarizing potential during AS-carbachol. Both spontaneous and evoked inhibitory potentials recorded during AS-carbachol were comparable to those that have been previously observed in trigeminal and spinal cord motoneurons under similar experimental conditions as well as during naturally occurring active sleep. Calculations based on modeling the changes that we found in input resistance and membrane time constant with a three-compartment neuron model suggest that shunts are present in all three compartments of the hypoglossal motoneuron model. Taken together, these data indicate that postsynaptic inhibitory drives are widely distributed on the soma-dendritic tree of hypoglossal motoneurons during AS-carbachol. These postsynaptic inhibitory actions are likely to be involved in the pathophysiology of obstructive sleep apnea. PMID:11102580

  2. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  3. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD. PMID:27440420

  4. Single Myosin Cross-Bridge Orientation in Cardiac Papillary Muscle Detects Lever-Arm Shear Strain in Transduction

    PubMed Central

    Burghardt, Thomas P.; Josephson, Matthew P.; Ajtai, Katalin

    2011-01-01

    Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley–Simmons model for myosin based contraction [Huxley and Simmons (1971) Nature 233, 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution. PMID:21819137

  5. Distinct Roles of Myosins in Aspergillus fumigatus Hyphal Growth and Pathogenesis.

    PubMed

    Renshaw, Hilary; Vargas-Muñiz, José M; Richards, Amber D; Asfaw, Yohannes G; Juvvadi, Praveen R; Steinbach, William J

    2016-05-01

    Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the ΔmyoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoE strain showed fungal growth in the murine lung, while the ΔmyoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies. PMID:26953327

  6. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    PubMed

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro

    2016-06-01

    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  7. Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking.

    PubMed

    Carvalho, Kevin; Lemière, Joël; Faqir, Fahima; Manzi, John; Blanchoin, Laurent; Plastino, Julie; Betz, Timo; Sykes, Cécile

    2013-01-01

    Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an 'outside geometry'. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin-streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications. PMID:24062578

  8. 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

  9. Unregulated smooth-muscle myosin in human intestinal neoplasia.

    PubMed

    Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Järvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S; Lucassen, Anneke; Tomlinson, Ian P M; Launonen, Virpi; Ristimäki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H Lee; Aaltonen, Lauri A

    2008-04-01

    A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia. PMID:18391202

  10. Par-4: A New Activator of Myosin Phosphatase

    PubMed Central

    Vetterkind, Susanne; Lee, Eunhee; Sundberg, Eric; Poythress, Ransom H.; Tao, Terence C.; Preuss, Ute

    2010-01-01

    Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a “padlock” model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires “unlocking” of Par-4 by phosphorylation and displacement of Par-4 from the MP complex. PMID:20130087

  11. Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1*

    PubMed Central

    Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2014-01-01

    Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (Vmax = 4 s−1), although their affinities for actin were high (Kactin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s−1, respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

  12. Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge and Specific Domains of the Myosin Rod

    PubMed Central

    2004-01-01

    Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) “head” is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines. PMID:15492777

  13. Dictyostelium myosin bipolar thick filament formation: importance of charge and specific domains of the myosin rod.

    PubMed

    Hostetter, Daniel; Rice, Sarah; Dean, Sara; Altman, David; McMahon, Peggy M; Sutton, Shirley; Tripathy, Ashutosh; Spudich, James A

    2004-11-01

    Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) "head" is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines. PMID:15492777

  14. X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

    PubMed

    Yamaguchi, Maki; Kimura, Masako; Li, Zhao-Bo; Ohno, Tetsuo; Takemori, Shigeru; Hoh, Joseph F Y; Yagi, Naoto

    2016-04-15

    The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments. PMID:26911280

  15. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-01-01

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV. PMID:24517254

  16. Sensory Motor Inhibition as a Prerequisite for Theory-of-Mind: A Comparison of Clinical and Normal Preschoolers Differing in Sensory Motor Abilities

    ERIC Educational Resources Information Center

    Chasiotis, Athanasios; Kiessling, Florian; Winter, Vera; Hofer, Jan

    2006-01-01

    After distinguishing between neocortical abilities for executive control and subcortical sensory motor skills for proprioceptive and vestibular integration, we compare a sample of 116 normal preschoolers with a sample of 31 preschoolers receiving occupational therapeutical treatment. This is done in an experimental design controlled for age (mean:…

  17. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    PubMed

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion. PMID:27498562

  18. 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

  19. Myosin-II sets the optimal response time scale of chemotactic amoeba

    NASA Astrophysics Data System (ADS)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Bodenschatz, Eberhard; Beta, Carsten

    2014-03-01

    The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba. Institute of Physics und Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany.

  20. Specific Myosins Control Actin Organization, Cell Morphology, and Migration in Prostate Cancer Cells.

    PubMed

    Makowska, Katarzyna A; Hughes, Ruth E; White, Kathryn J; Wells, Claire M; Peckham, Michelle

    2015-12-15

    We investigated the myosin expression profile in prostate cancer cell lines and found that Myo1b, Myo9b, Myo10, and Myo18a were expressed at higher levels in cells with high metastatic potential. Moreover, Myo1b and Myo10 were expressed at higher levels in metastatic tumors. Using an siRNA-based approach, we found that knockdown of each myosin resulted in distinct phenotypes. Myo10 knockdown ablated filopodia and decreased 2D migration speed. Myo18a knockdown increased circumferential non-muscle myosin 2A-associated actin filament arrays in the lamella and reduced directional persistence of 2D migration. Myo9b knockdown increased stress fiber formation, decreased 2D migration speed, and increased directional persistence. Conversely, Myo1b knockdown increased numbers of stress fibers but did not affect 2D migration. In all cases, the cell spread area was increased and 3D migration potential was decreased. Therefore, myosins not only act as molecular motors but also directly influence actin organization and cell morphology, which can contribute to the metastatic phenotype. PMID:26670045

  1. Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

    PubMed Central

    Mecklenburg, Kirk L.; Freed, Stephanie A.; Raval, Manmeet; Quintero, Omar A.; Yengo, Christopher M.; O'Tousa, Joseph. E.

    2015-01-01

    In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior. PMID:25822849

  2. Myosin head orientation and mobility during isometric contraction: effects of osmotic compression.

    PubMed Central

    Adhikari, B B; Fajer, P G

    1996-01-01

    We have correlated the mobility and the generation of force of myosin heads by applying radial compression to isometrically contracting muscle fibers. Osmotic pressure was produced by dextran T-500, and its effect on the orientation and mobility of myosin heads labeled with N-(1-oxy-2,2,5,5-tetramethyl-4-pyperidinyl)maleimide was observed by conventional and saturation-transfer electron paramagnetic resonance methods. A biphasic behavior is spectral changes coinciding with the tension dependence was observed as the fibers were compressed. At diameters above the equilibrium spacing, the large myosin head disorder characteristic during contraction in the absence of compression was largely maintained, whereas the mobility decreased threefold, from tauR approximately 25 microseconds to approximately 80-90 microseconds. The inhibition of fast microsecond motions was not accompanied by tension loss, implying that these motions are not necessary for force generation. At diameters below the equilibrium spacing, both the disorder and the mobility decreased dramatically in parallel with the tension inhibition, suggesting that slower microsecond motions and the disorder of the myosin head are necessary for muscle function. PMID:8785347

  3. Correlation of dysfunction of nonmuscle myosin IIA with increased induction of Cyp1a1 in Hepa-1 cells.

    PubMed

    Ebina, Masayuki; Shibazaki, Masahiko; Kudo, Kyoko; Kasai, Shuya; Kikuchi, Hideaki

    2011-03-01

    The aryl hydrocarbon receptor (AhR) is one of the best known ligand-activated transcription factors and it induces Cyp1a1 transcription by binding with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent focus has been on the relationship of AhR with signaling pathways that modulate cell shape and migration. In nonmuscle cells, nonmuscle myosin II is one of the key determinants of cell morphology, but it has not been investigated whether its function is related to Cyp1a1 induction. In this study, we observed that (-)-blebbistatin, which is a specific inhibitor of nonmuscle myosin II, increased the level of CYP1A1-mRNA in Hepa-1 cells. Comparison of (-)-blebbistatin with (+)-blebbistatin, which is an inactive enantiomer, indicated that the increase of CYP1A1-mRNA was due to nonmuscle myosin II inhibition. Subsequent knockdown experiments observed that reduction of nonmuscle myosin IIA, which is only an isoform of nonmuscle myosin II expressed in Hepa-1 cells, was related to the enhancement of TCDD-dependent Cyp1a1 induction. Moreover, chromatin immunoprecipitation assay indicated that the increase of Cyp1a1 induction was the result of transcriptional activation due to increased binding of AhR and RNA polymerase II to the enhancer and proximal promoter regions of Cyp1a1, respectively. These findings provide a new insight into the correlation between the function of nonmuscle myosin II and gene induction. PMID:21216307

  4. Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.

    PubMed

    Saber, Walid; Begin, Kelly J; Warshaw, David M; VanBuren, Peter

    2008-06-01

    The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head. PMID:18482734

  5. A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition.

    PubMed

    Philbrook, Alison; Barbagallo, Belinda; Francis, Michael M

    2013-07-01

    Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation. PMID:24778941

  6. Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.

    PubMed Central

    Jancso, A; Szent-Györgyi, A G

    1994-01-01

    Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin. Images PMID:8090720

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

    PubMed Central

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.

    2009-01-01

    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 modifying 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. PMID:19450484

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

    SciTech Connect

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.

    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 modifying 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.

  9. Optogenetic activation of nigral inhibitory inputs to motor thalamus in the mouse reveals classic inhibition with little potential for rebound activation.

    PubMed

    Edgerton, Jeremy R; Jaeger, Dieter

    2014-01-01

    The inhibitory output from the internal pallidum and substantia nigra to the thalamus forms an important link in the transmission of basal ganglia processing to cortex. Two hypotheses consider either inhibition of thalamic activity or thalamic excitation via post-inhibitory rebound burst firing as the functional mode of this link. We used optogenetics to characterize the synaptic properties of nigral input to motor thalamus in adult mouse brain slices, and to determine in what conditions the nigral inhibition of motor thalamus is transmitted via inhibition or rebound firing. Our results are more consistent with graded inhibition of spiking for conditions expected in normal awake animals, because inhibitory potentials from nigral input were generally not sufficient to elicit rebound spikes when the thalamic neurons were actively firing. However, with bursty or fast trains of nigral input low-threshold rebound spike bursts could be triggered for low levels of excitation. This may form the basis of pathological burst generation and transmission in parkinsonian conditions. PMID:24574972

  10. Inhibition of the Motor Protein Eg5/Kinesin-5 in Amyloid β-Mediated Impairment of Hippocampal Long-Term Potentiation and Dendritic Spine Loss.

    PubMed

    Freund, Ronald K; Gibson, Emily S; Potter, Huntington; Dell'Acqua, Mark L

    2016-05-01

    Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aβ) 1-42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, includingN-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aβ(1-42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aβ-induced synaptic dysfunction, but we recently found that Aβ(1-42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aβ(1-42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aβ, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aβ(1-42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aβ(1-42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5-1.0μM Aβ(1-42) or 1-5μM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aβon LTP and synapse loss, suggesting that Aβinduces acute and chronic synaptic dysfunction in part through inhibiting Eg5. PMID:26957206

  11. Unconventional Myosins in Inner-Ear Sensory Epithelia

    PubMed Central

    Hasson, Tama; Gillespie, Peter G.; Garcia, Jesus A.; MacDonald, Richard B.; Zhao, Yi-dong; Yee, Ann G.; Mooseker, Mark S.; Corey, David P.

    1997-01-01

    To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Iβ is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Iβ is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Iβ, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Iβ probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles. PMID:9182663

  12. Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division

    PubMed Central

    Wu, Shu-Zon; Bezanilla, Magdalena

    2014-01-01

    Plant cells divide using the phragmoplast, a microtubule-based structure that directs vesicles secretion to the nascent cell plate. The phragmoplast forms at the cell center and expands to reach a specified site at the cell periphery, tens or hundreds of microns distant. The mechanism responsible for guiding the phragmoplast remains largely unknown. Here, using both moss and tobacco, we show that myosin VIII associates with the ends of phragmoplast microtubules and together with actin plays a role in guiding phragmoplast expansion to the cortical division site. Our data lead to a model whereby myosin VIII links phragmoplast microtubules to the cortical division site via actin filaments. Myosin VIII's motor activity along actin provides a molecular mechanism for steering phragmoplast expansion. DOI: http://dx.doi.org/10.7554/eLife.03498.001 PMID:25247701

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

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    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 chain 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

  14. Myosin motors fragment and compact membrane-bound actin filaments

    PubMed Central

    Vogel, Sven K; Petrasek, Zdenek; Heinemann, Fabian; Schwille, Petra

    2013-01-01

    Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis. DOI: http://dx.doi.org/10.7554/eLife.00116.001 PMID:23326639

  15. An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

    PubMed Central

    Elangovan, R; Capitanio, M; Melli, L; Pavone, F S; Lombardi, V; Piazzesi, G

    2012-01-01

    A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4–30°C) and substrate concentration. VF was 8.88 ± 0.26 μm s−1 at 30.6°C and decreased to 1.60 ± 0.09 μm s−1 at 4.5°C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40–50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5°C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads. PMID:22199170

  16. Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

    SciTech Connect

    Grishin, M.N.; Kodentsova, V.M.; Abdraimova, U.A.; Nikolaeva, O.P.; Petushkova, E.V.

    1986-03-20

    It was established that the dialdehyde derivative of ATP (oxo-ATP) is a good substrate of the Ca-ATPase of heavy meromyosin: (1.2-1.4) x 10/sup -4/ M; V = V/sub ATP/. At the same time, it is capable of inducing irreversible inhibition of the enzyme. Since oxo-ATP is rapidly digested by myosin, forming oxo-ADP, this inhibition is a consequence of the interaction of the enzyme with oxo-ADP. It was shown that the inhibition of heavy meromyosin (HMM), by oxo-ADP occurs according to the kinetics characteristic of affinity modification; moreover, ATP entirely protects HMM from the loss of activity. Similar data on the irreversible inhibition of ATPase activity under the action of oxo-ADP were obtained in the case of myosin, heavy meromyosin, subfragment-1, and natural actomyosin, as well as in the absence of divalent cations, which is evidence of modification of the active site of myosin ATPase.

  17. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species

    PubMed Central

    Odronitz, Florian; Kollmar, Martin

    2007-01-01

    Background The evolutionary history of organisms is expressed in phylogenetic trees. The most widely used phylogenetic trees describing the evolution of all organisms have been constructed based on single-gene phylogenies that, however, often produce conflicting results. Incongruence between phylogenetic trees can result from the violation of the orthology assumption and stochastic and systematic errors. Results Here, we have reconstructed the tree of eukaryotic life based on the analysis of 2,269 myosin motor domains from 328 organisms. All sequences were manually annotated and verified, and were grouped into 35 myosin classes, of which 16 have not been proposed previously. The resultant phylogenetic tree confirms some accepted relationships of major taxa and resolves disputed and preliminary classifications. We place the Viridiplantae after the separation of Euglenozoa, Alveolata, and Stramenopiles, we suggest a monophyletic origin of Entamoebidae, Acanthamoebidae, and Dictyosteliida, and provide evidence for the asynchronous evolution of the Mammalia and Fungi. Conclusion Our analysis of the myosins allowed combining phylogenetic information derived from class-specific trees with the information of myosin class evolution and distribution. This approach is expected to result in superior accuracy compared to single-gene or phylogenomic analyses because the orthology problem is resolved and a strong determinant not depending on any technical uncertainties is incorporated, the class distribution. Combining our analysis of the myosins with high quality analyses of other protein families, for example, that of the kinesins, could help in resolving still questionable dependencies at the origin of eukaryotic life. PMID:17877792

  18. 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

  19. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  20. Preparation and Characterization of Myosin Proteins.

    ERIC Educational Resources Information Center

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  1. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro. PMID:25403653

  2. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  3. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    PubMed

    Ueda, Shuji; Blee, Alexandra M; Macway, Katherine G; Renner, Derrick J; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  4. Myosin 1e is a component of the invadosome core that contributes to regulation of invadosome dynamics

    PubMed Central

    Ouderkirk, Jessica L.; Krendel, Mira

    2014-01-01

    Myosin 1e (myo1e) is an actin-based motor protein that has been implicated in cell adhesion and migration. We examined the role of myo1e in invadosomes, actin-rich adhesion structures that are important for degradation and invasion of the extracellular matrix. RSV-transformed BHK-21 cells, which readily form invadosomes and invadosome rosettes, were used as the experimental model. Myo1e localization to the actin-rich core of invadosomes required the proline-rich Tail Homology 2 (TH2) domain. During invadosome rosette expansion, we observed myo1e recruitment to newly forming invadosomes via Tail Homology 1 (TH1)-dependent interactions with the plasma membrane, where it preceded actin and paxillin. Dominant-negative inhibition of myo1e resulted in mislocalized invadosome formation, usually at the center of the rosette. We propose that TH2 domain of myo1e provides the key signal for localization to invadosomes, while TH1 domain interactions facilitate myo1e targeting to the plasma membrane-proximal locations within the rosettes. Myo1e may then act as a scaffold, linking the plasma membrane with the actin cytoskeleton and helping direct new invadosome formation to the periphery of the rosette. PMID:24462457

  5. Temperature effect on the chemomechanical regulation of substeps within the power stroke of a single Myosin II

    PubMed Central

    Dong, Chenling; Chen, Bin

    2016-01-01

    Myosin IIs in the skeletal muscle are highly efficient nanoscale machines evolved in nature. Understanding how they function can not only bring insights into various biological processes but also provide guidelines to engineer synthetic nanoscale motors working in the vicinity of thermal noise. Though it was clearly demonstrated that the behavior of a skeletal muscle fiber, or that of a single myosin was strongly affected by the temperature, how exactly the temperature affects the kinetics of a single myosin is not fully understood. By adapting the newly developed transitional state model, which successfully explained the intriguing motor force regulation during skeletal muscle contraction, here we systematically explain how exactly the power stroke of a single myosin proceeds, with the consideration of the chemomechanical regulation of sub-steps within the stroke. The adapted theory is then utilized to investigate the temperature effect on various aspects of the power stroke. Our analysis suggests that, though swing rates, the isometric force, and the maximal stroke size all strongly vary with the temperature, the temperature can have a very small effect on the releasable elastic energy within the power stroke. PMID:26786569

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

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    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 Glut4 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.

  7. Molecular motors and their functions in plants

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.

    2001-01-01

    Molecular motors that hydrolyze ATP and use the derived energy to generate force are involved in a variety of diverse cellular functions. Genetic, biochemical, and cellular localization data have implicated motors in a variety of functions such as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. In non-plant systems three families of molecular motors (kinesins, dyneins, and myosins) have been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials intracellularly. During the last decade tremendous progress has been made in understanding the structure and function of various motors in animals. These studies are yielding interesting insights into the functions of molecular motors and the origin of different families of motors. Furthermore, the paradigm that motors bind cargo and move along cytoskeletal tracks does not explain the functions of some of the motors. Relatively little is known about the molecular motors and their roles in plants. In recent years, by using biochemical, cell biological, molecular, and genetic approaches a few molecular motors have been isolated and characterized from plants. These studies indicate that some of the motors in plants have novel features and regulatory mechanisms. The role of molecular motors in plant cell division, cell expansion, cytoplasmic streaming, cell-to-cell communication, membrane trafficking, and morphogenesis is beginning to be understood. Analyses of the Arabidopsis genome sequence database (51% of genome) with conserved motor domains of kinesin and myosin families indicates the presence of a large number (about 40) of molecular motors and the functions of many of these motors remain to be discovered. It is likely that many more motors with novel regulatory

  8. 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

  9. 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. PMID:27446684

  10. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    PubMed Central

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

    2008-01-01

    Summary Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free-head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens reveals that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal here has been to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction reveals intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC, and fitted to the reconstruction an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 crystal structure. The fitting suggests an intramolecular interaction between the cardiomyopathy loop of the free-head and its own S2 and two intermolecular interactions—between the cardio-loop of the free head and the ELC of the blocked head, and between the Leu-305 - Gln-327 “interaction loop” (loop I) of the free-head and the N-terminal fragment of the RLC of the blocked-head. These interactions, added to those previously described, would help to switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament. PMID:18951904

  11. Long single [alpha]-helical tail domains bridge the gap between structure and function of myosin VI

    SciTech Connect

    Spink, Benjamin J.; Sivaramakrishnan, Sivaraj; Lipfert, Jan; Doniach, Sebastian; Spudich, James A.

    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 the 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.

  12. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  13. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  14. Clonidine, an alpha-2 adrenoceptor agonist relieves mechanical allodynia in oxaliplatin-induced neuropathic mice; potentiation by spinal p38 MAPK inhibition without motor dysfunction and hypotension.

    PubMed

    Yeo, Ji-Hee; Yoon, Seo-Yeon; Kim, Sol-Ji; Oh, Seog-Bae; Lee, Jang-Hern; Beitz, Alvin J; Roh, Dae-Hyun

    2016-05-15

    Cancer chemotherapy with platinum-based antineoplastic agents including oxaliplatin frequently results in a debilitating and painful peripheral neuropathy. We evaluated the antinociceptive effects of the alpha-2 adrenoceptor agonist, clonidine on oxaliplatin-induced neuropathic pain. Specifically, we determined if (i) the intraperitoneal (i.p.) injection of clonidine reduces mechanical allodynia in mice with an oxaliplatin-induced neuropathy and (ii) concurrent inhibition of p38 mitogen-activated protein kinase (MAPK) activity by the p38 MAPK inhibitor SB203580 enhances clonidine's antiallodynic effect. Clonidine (0.01-0.1 mg kg(-1), i.p.), with or without SB203580(1-10 nmol, intrathecal) was administered two weeks after oxaliplatin injection(10 mg kg(-1), i.p.) to mice. Mechanical withdrawal threshold, motor coordination and blood pressure were measured. Postmortem expression of p38 MAPK and ERK as well as their phosphorylated forms(p-p38 and p-ERK) were quantified 30 min or 4 hr after drug injection in the spinal cord dorsal horn of treated and control mice. Clonidine dose-dependently reduced oxaliplatin-induced mechanical allodynia and spinal p-p38 MAPK expression, but not p-ERK. At 0.1 mg kg(-1), clonidine also impaired motor coordination and decreased blood pressure. A 10 nmol dose of SB203580 alone significantly reduced mechanical allodynia and p-p38 MAPK expression, while a subeffective dose(3 nmol) potentiated the antiallodynic effect of 0.03 mg kg(-1) clonidine and reduced the increased p-p38 MAPK. Coadministration of SB203580 and 0.03 mg kg(-1) clonidine decreased allodynia similar to that of 0.10 mg kg(-1) clonidine, but without significant motor or vascular effects. These findings demonstrate that clonidine treatment reduces oxaliplatin-induced mechanical allodynia. The concurrent administration of SB203580 reduces the dosage requirements for clonidine, thereby alleviating allodynia without producing undesirable motor or cardiovascular effects. PMID

  15. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    PubMed

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  16. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments.

    PubMed

    Heaslip, Aoife T; Nelson, Shane R; Warshaw, David M

    2016-07-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  17. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

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

    PubMed

    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

  19. 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

  20. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  1. Nuclear myosin I regulates cell membrane tension

    PubMed Central

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  2. The On-off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

    SciTech Connect

    Himmel, D.; Mui, S; O' Neall-Hennessey, E; Szent-Györgyi, A; Cohen, C

    2009-01-01

    In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long {alpha}-helical 'heavy chain' stabilized by a 'regulatory' light chain (RLC) and an 'essential' light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca{sup 2+} to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca{sup 2+}. Our results indicate that loss of Ca{sup 2+} abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.

  3. Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Lin, Yifeng; Stone, Sarrabeth; Cvetanovic, Marija; Lin, Wensheng

    2016-01-01

    Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE. PMID:27466819

  4. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  5. Inhibition of Pathogenic Mutant SOD1 Aggregation in Cultured Motor Neuronal Cells by Prevention of Its SUMOylation on Lysine 75.

    PubMed

    Dangoumau, Audrey; Marouillat, Sylviane; Burlaud Gaillard, Julien; Uzbekov, Rustem; Veyrat-Durebex, Charlotte; Blasco, Hélène; Arnoult, Christophe; Corcia, Philippe; Andres, Christian R; Vourc'h, Patrick

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons. Mutations in the SOD1 gene encoding the superoxide dismutase 1 are present in 15% of familial ALS cases and in 2% of sporadic cases. These mutations are associated with the formation of SOD1-positive aggregates. The mechanisms of aggregation remain unknown, but posttranslational modifications of SOD1 may be involved. Here, we report that NSC-34 motor neuronal cells expressing mutant SOD1 contained aggregates positive for small ubiquitin modifier-1 (SUMO-1), and in parallel a reduced level of free SUMO-1. CLEM (correlative light and electron microscopy) analysis showed nonorganized cytosolic aggregates for all mutations tested (SOD1A4V, SOD1V31A, and SOD1G93C). We next show that preventing the SUMOylation of mutant SOD1 by the substitution of lysine 75, the SUMOylation site of SOD1, significantly reduces the number of motor neuronal cells with aggregates. These results support the need for further research on the SUMOylation pathways, which may be a potential therapeutic target in ALS. PMID:26605782

  6. 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

  7. Application of molecular modeling to analysis of inhibition of kinesin motor proteins of the BimC subfamily by monastrol and related compounds.

    PubMed

    Bevan, David R; Garst, James F; Osborne, Caroline K; Sims, Angela M

    2005-11-01

    Application of molecular modeling approaches has potential to contribute to rational drug design. These approaches may be especially useful when attempting to elucidate the structural features associated with novel drug targets. In this study, molecular docking and molecular dynamics were applied to studies of inhibition of the human motor protein denoted HsEg5 and other homologues in the BimC subfamily. These proteins are essential for mitosis, so compounds that inhibit their activity may have potential as anticancer therapeutics. The discovery of a small-molecule cell-permeable inhibitor, monastrol, has stimulated research in this area. Interestingly, monastrol is reported to inhibit the human and Xenopus forms of Eg5, but not those from Drosophila and Aspergillus. In this study, homology modeling was used to generate models of the Xenopus, Drosophila, and Aspergillus homologues, using the crystal structure of the human protein in complex with monastrol as a template. A series of known inhibitors was docked into each of the homologues, and the differences in binding energies were consistent with reported experimental data. Molecular dynamics revealed significant changes in the structure of the Aspergillus homologue that may contribute to its relative insensitivity to monastrol and related compounds. PMID:17191952

  8. Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.

    PubMed

    Spiró, Zoltán; Thyagarajan, Kalyani; De Simone, Alessandro; Träger, Sylvain; Afshar, Katayoun; Gönczy, Pierre

    2014-07-01

    Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension. PMID:24961801

  9. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style. PMID:19648394

  10. Myosin-V, Kinesin-1, and Kinesin-3 Cooperate in Hyphal Growth of the Fungus Ustilago maydisD⃞

    PubMed Central

    Schuchardt, Isabel; Aßmann, Daniela; Thines, Eckhard; Schuberth, Christian; Steinberg, Gero

    2005-01-01

    Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. Δkin1 and Δkin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in Δkin1rkin3 double mutants, but polarity was lost in Δmyo5rkin1 and Δmyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenkörper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth. PMID:16120650

  11. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    SciTech Connect

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony; Bernstein, Sanford I.

    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 the 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.

  12. An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle.

    PubMed

    Smith, D A; Stephenson, D G

    2011-06-01

    The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl(-) binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small. PMID:21641314

  13. Distinct inhibition of acute cocaine-stimulated motor activity following microinjection of a group III metabotropic glutamate receptor agonist into the dorsal striatum of rats.

    PubMed

    Mao, L; Wang, J Q

    2000-09-01

    Group III metabotropic glutamate receptors (mGluRs) are negatively coupled to adenylate cyclase through G-proteins. Activation of this group of mGluRs shows an inhibition of dopaminergic transmission in the forebrain. To define the role of striatal group III mGluRs in the regulation of basal and dopamine-stimulated motor behavior, the recently developed agonist and antagonist relatively selective for group III mGluRs were utilized to pharmacologically enhance and reduce group III mGluR glutamatergic tone in the dorsal striatum of chronically cannulated rats. Bilateral injections of a group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), did not alter basal levels of motor activity at three doses surveyed (1, 10, and 100 nmol). Neither did intracaudate injection of a group III antagonist, alpha-methyl-4-phosphonophenylglycine (MPPG), at 10, 30, and 100 nmol. However, pretreatment with L-AP4 (10 and 100 nmol) dose dependently blocked hyperlocomotion induced by acute injection of cocaine (20 mg/kg, i.p.), amphetamine (2.5 mg/kg, i.p.), or apomorphine (1 mg/kg, s.c.). The behavioral activity induced by cocaine was much more sensitive to L-AP4 than that induced by amphetamine and apomorphine. At 100 nmol, L-AP4 completely blocked cocaine effect whereas amphetamine- and apomorphine-stimulated behaviors were blocked only by 28% and 31%, respectively. The blocking effect of L-AP4 on cocaine action was reversed by pretreatment with MPPG. MPPG itself did not modify behavioral responses to cocaine, amphetamine, or apomorphine. These data indicate that the glutamatergic tone on the group III mGluRs is not active in the regulation of basal and acute dopamine-stimulated motor activity. However, enhanced group III mGluR glutamatergic transmission by an exogenous ligand is capable of suppressing behavioral responses to acute exposure of dopamine stimulants. PMID:11113488

  14. Influence of chronic dopamine transporter inhibition by RTI-336 on motor behavior, sleep, and hormone levels in rhesus monkeys.

    PubMed

    Andersen, Monica L; Sawyer, Eileen K; Carroll, F Ivy; Howell, Leonard L

    2012-04-01

    Dopamine transporter (DAT) inhibitors have been developed as a promising treatment approach for cocaine dependence. However, the stimulant effects of DAT inhibitors have the potential to disrupt sleep patterns, and the influence of long-term treatment on dopamine neurochemistry is still unknown. The objectives of this study were to (1) explore the stimulant-related effects of chronic DAT inhibitor (RTI-336) treatment on motor activity and sleep-like measures in male rhesus monkeys (Macaca mulatta; n = 4) and (2) to determine the effect of drug treatment on prolactin and cortisol levels. Subjects were fitted with a collar-mounted activity monitor to evaluate their motor activity, with 4 days of baseline recording preceding 21 days of daily saline or RTI-336 (1 mg/kg/day; intramuscular) injections. Blood samples were collected immediately prior to and following chronic treatment to assess hormone levels. RTI-336 produced a significant increase in locomotor activity at the end of the daytime period compared to saline administration. During the 3-week treatment period, sleep efficiency was decreased and the fragmentation index and latency to sleep onset were significantly increased. Hormone levels were not changed throughout the study. Chronic treatment with RTI-336 has a mild but significant stimulant effect, as evidenced by the significant increase in activity during the evening period which may cause minor disruptions in sleep measures. PMID:22023668

  15. Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii.

    PubMed

    Xu, Hong; Boychuk, Jeffery A; Boychuk, Carie R; Uteshev, Victor V; Smith, Bret N

    2015-02-15

    The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments. PMID:25429117

  16. The optical trapping dumbbell assay for nonprocessive motors or motors that turn around filaments.

    PubMed

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

    2011-11-01

    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 protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments. PMID:22046050

  17. The Optical Trapping Dumbbell Assay for Nonprocessive Motors or Motors That Turn around Filaments

    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 protocol describes the preparation of biotin–actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments. PMID:22046050

  18. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  19. 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

  20. Myosin filament 3D structure in mammalian cardiac muscle☆

    PubMed Central

    AL-Khayat, Hind A.; Morris, Edward P.; Kensler, Robert W.; Squire, John M.

    2008-01-01

    A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac). PMID:18472277

  1. 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. PMID:25770245

  2. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle.

    PubMed

    Kampourakis, Thomas; Irving, Malcolm

    2015-08-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  3. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

    PubMed Central

    Kampourakis, Thomas; Irving, Malcolm

    2015-01-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  4. Neuromuscular Development and Regulation of Myosin Expression

    NASA Technical Reports Server (NTRS)

    Bodine, Sue

    1997-01-01

    The proposed experiments were designed to determine whether the absence of gravity during embryogenesis influences the postnatal development of the neuromuscular system. Further, we examined the effects of reduced gravity on hindlimb muscles of the pregnant rats. Microgravity may have short and long-term effects on the development of muscle fiber type differentiation and force producing capabilities. Microgravity will reduce muscle fiber size and cause a shift in myosin heavy chain expression from slow to fast in hindlimb muscles of the adult pregnant rats.

  5. Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

    PubMed Central

    Andrecka, Joanna; Ortega Arroyo, Jaime; Takagi, Yasuharu; de Wit, Gabrielle; Fineberg, Adam; MacKinnon, Lachlan; Young, Gavin; Sellers, James R; Kukura, Philipp

    2015-01-01

    Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps. DOI: http://dx.doi.org/10.7554/eLife.05413.001 PMID:25748137

  6. Release from myosin V via regulated recruitment of an E3 Ub ligase controls organelle localization

    PubMed Central

    Yau, Richard G.; Peng, Yutian; Valiathan, Rajeshwari R.; Birkeland, Shanda R.; Wilson, Thomas E.; Weisman, Lois S.

    2014-01-01

    Summary Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin-V, Myo2, binds the vacuole specific adaptor, Vac17, to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 which recruits the E3 ligase, Dma1, to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism which detaches myosin-V from select cargoes. PMID:24636257

  7. Rab11a and myosin Vb regulate recycling of the M4 muscarinic acetylcholine receptor.

    PubMed

    Volpicelli, Laura A; Lah, James J; Fang, Guofu; Goldenring, James R; Levey, Allan I

    2002-11-15

    Agonist-induced internalization followed by subsequent return to the cell surface regulates G-protein-coupled receptor (GPCR) activity. Because the cellular responsiveness to ligand depends on the balance between receptor degradation and recycling, it is crucial to identify the molecules involved in GPCR recovery to the cell surface. In this study, we identify mechanisms involved in the recycling of the M4 subtype of muscarinic acetylcholine receptor. M4 is highly expressed in the CNS, plays a role in locomotor activity, and is a novel therapeutic target for neurologic and psychiatric disorders. Previous studies show that, after cholinergic stimulation, M4 internalizes from the cell surface to endosomes in cell culture and the rat brain. Here, we show that, after activation, M4 traffics to transferrin receptor- and Rab11a-positive perinuclear endosomes. Expression of the constitutively GDP-bound, inactive mutant Rab11aS25N inhibits M4 trafficking to recycling endosomes. Expression of the C-terminal tail of myosin Vb, a Rab11a effector, enhances M4 accumulation in perinuclear endosomes. Both Rab11aS25N and the myosin Vb tail impair M4 recycling. The results demonstrate that GPCR recycling is mediated through a discrete pathway using both Rab11a and myosin Vb. PMID:12427833

  8. Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

    NASA Astrophysics Data System (ADS)

    Sung, Jongmin; Nag, Suman; Mortensen, Kim I.; Vestergaard, Christian L.; Sutton, Shirley; Ruppel, Kathleen; Flyvbjerg, Henrik; Spudich, James A.

    2015-08-01

    Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using `harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's p