Journal articles: 'Myosine-X'

1

Berg, J. S., B. H. Derfler, C. M. Pennisi, D. P. Corey, and R. E. Cheney. "Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin." Journal of Cell Science 113, no. 19 (October 1, 2000): 3439–51. http://dx.doi.org/10.1242/jcs.113.19.3439.

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Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.

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Novakovic, Valerie A., and Gary E. Gilbert. "Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid." Blood 136, no. 21 (November 19, 2020): 2469–72. http://dx.doi.org/10.1182/blood.2020005930.

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Abstract Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine–binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

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Rogers, Michael S., and Emanuel E. Strehler. "The Tumor-sensitive Calmodulin-like Protein Is a Specific Light Chain of Human Unconventional Myosin X." Journal of Biological Chemistry 276, no. 15 (January 22, 2001): 12182–89. http://dx.doi.org/10.1074/jbc.m010056200.

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Human calmodulin-like protein (CLP) is an epithelial-specific Ca2+-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca2+-dependent interactions with specific target proteins. Using gel overlays, we identified a ∼210-kDa protein binding specifically and in a Ca2+-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human “unconventional” myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca2+-dependent. CLP interacted strongly with IQ motif 3 (Kd∼0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin Xin vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.

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Naydenov, Nayden, Susana Lechuga, Emina Huang, and Andrei Ivanov. "Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer." Cancers 13, no. 4 (February 11, 2021): 741. http://dx.doi.org/10.3390/cancers13040741.

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Colorectal cancer (CRC) remains the third most common cause of cancer and the second most common cause of cancer deaths worldwide. Clinicians are largely faced with advanced and metastatic disease for which few interventions are available. One poorly understood aspect of CRC involves altered organization of the actin cytoskeleton, especially at the metastatic stage of the disease. Myosin motors are crucial regulators of actin cytoskeletal architecture and remodeling. They act as mechanosensors of the tumor environments and control key cellular processes linked to oncogenesis, including cell division, extracellular matrix adhesion and tissue invasion. Different myosins play either oncogenic or tumor suppressor roles in breast, lung and prostate cancer; however, little is known about their functions in CRC. This review focuses on the functional roles of myosins in colon cancer development. We discuss the most studied class of myosins, class II (conventional) myosins, as well as several classes (I, V, VI, X and XVIII) of unconventional myosins that have been linked to CRC development. Altered expression and mutations of these motors in clinical tumor samples and their roles in CRC growth and metastasis are described. We also evaluate the potential of using small molecular modulators of myosin activity to develop novel anticancer therapies.

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Brown, Lisa D., and Marie E. Cantino. "Immunocytochemical Localization of Myosin Light Chains in the Abdominal Superficial Flexor Muscles of the American Lobster, Homarus Americanus." Microscopy and Microanalysis 4, S2 (July 1998): 1118–19. http://dx.doi.org/10.1017/s143192760002571x.

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Myosin is composed of two high-molecular weight heavy chains and four low-molecular weight hght chains. In both vertebrate and invertebrate skeletal muscle, each myosin heavy chain is associated with two myosin light chains. In skeletal muscle myosins studied by X-ray diffraction, each myosin heavy chain binds one of each of two distinct classes of hght chains. Thus, while isoform distributions may vary within and between fibers, the spatial distribution of each class of light chain should be uniform within the A band and between sarcomeres and fibers. Since no such study exists for crustacean myosin, we investigated the spatial distribution of the hght chains within the superficial flexor muscle (SFM) of the lobster, Homarus americanus, using immunoelectron microscopy. The SFM contains two classes of myosin hght chains, termed “alpha” (Mr = 21,000 to 23,500) and “beta” (Mr = 18,000 to 18,500). Immunocytochemical electron microscopic results suggest that the alpha light chains are not uniformly distributed at the subsarcomere level.

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6

Squire, John. "Special Issue: The Actin-Myosin Interaction in Muscle: Background and Overview." International Journal of Molecular Sciences 20, no. 22 (November 14, 2019): 5715. http://dx.doi.org/10.3390/ijms20225715.

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Muscular contraction is a fundamental phenomenon in all animals; without it life as we know it would be impossible. The basic mechanism in muscle, including heart muscle, involves the interaction of the protein filaments myosin and actin. Motility in all cells is also partly based on similar interactions of actin filaments with non-muscle myosins. Early studies of muscle contraction have informed later studies of these cellular actin-myosin systems. In muscles, projections on the myosin filaments, the so-called myosin heads or cross-bridges, interact with the nearby actin filaments and, in a mechanism powered by ATP-hydrolysis, they move the actin filaments past them in a kind of cyclic rowing action to produce the macroscopic muscular movements of which we are all aware. In this special issue the papers and reviews address different aspects of the actin-myosin interaction in muscle as studied by a plethora of complementary techniques. The present overview provides a brief and elementary introduction to muscle structure and function and the techniques used to study it. It goes on to give more detailed descriptions of what is known about muscle components and the cross-bridge cycle using structural biology techniques, particularly protein crystallography, electron microscopy and X-ray diffraction. It then has a quick look at muscle mechanics and it summarises what can be learnt about how muscle works based on the other studies covered in the different papers in the special issue. A picture emerges of the main molecular steps involved in the force-producing process; steps that are also likely to be seen in non-muscle myosin interactions with cellular actin filaments. Finally, the remarkable advances made in studying the effects of mutations in the contractile assembly in causing specific muscle diseases, particularly those in heart muscle, are outlined and discussed.

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Courson, David S., and Richard E. Cheney. "Myosin-X and disease." Experimental Cell Research 334, no. 1 (May 2015): 10–15. http://dx.doi.org/10.1016/j.yexcr.2015.03.014.

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8

Dillmann, W. H. "Methyl palmoxirate increases Ca2+-myosin ATPase activity and changes myosin isoenzyme distribution in the diabetic rat heart." American Journal of Physiology-Endocrinology and Metabolism 248, no. 5 (May 1, 1985): E602—E606. http://dx.doi.org/10.1152/ajpendo.1985.248.5.e602.

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Previous studies have shown that in rats diabetes mellitus leads to a decrease in cardiac ventricle myosin V1 and an increase in myosin V3 levels. Insulin administration reverts myosin isoenzyme distribution to normal levels. It is currently unclear whether the effects of insulin on myosin isoenzyme distribution are a direct effect of the hormone or are mediated through insulin-induced alterations in cardiac metabolism. To gain further insight into this question diabetic rats received methyl palmoxirate, a potent inhibitor of long-chain fatty acid oxidation. Administration of 25 mg methyl palmoxirate X kg body wt-1 X day-1 to diabetic rats for 4 wk leads to a partial reversal of the effects of diabetes. Myosin V1 predominance is re-established and Ca2+-activated myosin ATPase activity increases by 60% (Ca2+-myosin ATPase normal rats 1.067 +/- 0.13 mumol Pi X mg protein-1 X min-1, diabetic rats 0.609 +/- 0.05 mumol Pi X mg protein-1 X min-1, diabetic + methyl palmoxirate rats 0.912 +/- 0.06 mumol Pi X mg protein-1 X min-1). The methyl palmoxirate-induced increase in myosin V1 levels and Ca2+-activated myosin ATPase activity occurred in the absence of changes in insulin and thyroid hormone levels. Methyl palmoxirate may have acted through its known inhibitory effect on cardiac beta-oxidation and/or the resultant stimulatory effect on glycolytic flux. Our findings may indicate that changes in cardiac substrate consumption can influence myosin isoenzyme predominance.

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9

Almagro, Sébastien, Claire Durmort, Adeline Chervin-Pétinot, Stephanie Heyraud, Mathilde Dubois, Olivier Lambert, Camille Maillefaud, et al. "The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts." Molecular and Cellular Biology 30, no. 7 (February 1, 2010): 1703–17. http://dx.doi.org/10.1128/mcb.01226-09.

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ABSTRACT Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings.

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10

Ikebe, Mitsuo, Osamu Sato, and Tsuyoshi Sakai. "Myosin X and Cytoskeletal Reorganization." Applied Microscopy 48, no. 2 (June 30, 2018): 33–42. http://dx.doi.org/10.9729/am.2018.48.2.33.

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11

Vuillez, J. P., M. D. Brunet, E. Borrel, J. L. Bosson, M. J. Stasia, P. Eymerit, I. Perez, and D. Blin. "Place du dosage immunoradiométrique de la chaîne lourde de la myosine en chirurgie coronarienne." Immuno-analyse & Biologie Spécialisée 11, no. 1 (January 1996): 38–42. http://dx.doi.org/10.1016/0923-2532(96)88389-x.

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12

Iwano, Sayaka, Ayaka Satou, Shigeru Matsumura, Naoyuki Sugiyama, Yasushi Ishihama, and Fumiko Toyoshima. "PCTK1 Regulates Integrin-Dependent Spindle Orientation via Protein Kinase A Regulatory Subunit KAP0 and Myosin X." Molecular and Cellular Biology 35, no. 7 (January 20, 2015): 1197–208. http://dx.doi.org/10.1128/mcb.01017-14.

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Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with β1 integrin. This interaction between myosin X-FERM and β1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-β1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation.

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Berg, Jonathan S., and Richard E. Cheney. "Myosin-X is an unconventional myosin that undergoes intrafilopodial motility." Nature Cell Biology 4, no. 3 (February 18, 2002): 246–50. http://dx.doi.org/10.1038/ncb762.

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Knupp, Morris, and Squire. "The Interacting Head Motif Structure Does Not Explain the X-Ray Diffraction Patterns in Relaxed Vertebrate (Bony Fish) Skeletal Muscle and Insect (Lethocerus) Flight Muscle." Biology 8, no. 3 (September 14, 2019): 67. http://dx.doi.org/10.3390/biology8030067.

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Unlike electron microscopy, which can achieve very high resolution but to date can only be used to study static structures, time-resolved X-ray diffraction from contracting muscles can, in principle, be used to follow the molecular movements involved in force generation on a millisecond timescale, albeit at moderate resolution. However, previous X-ray diffraction studies of resting muscles have come up with structures for the head arrangements in resting myosin filaments that are different from the apparently ubiquitous interacting head motif (IHM) structures found by single particle analysis of electron micrographs of isolated myosin filaments from a variety of muscle types. This head organization is supposed to represent the super-relaxed state of the myosin filaments where adenosine triphosphate (ATP) usage is minimized. Here we have tested whether the interacting head motif structures will satisfactorily explain the observed low-angle X-ray diffraction patterns from resting vertebrate (bony fish) and invertebrate (insect flight) muscles. We find that the interacting head motif does not, in fact, explain what is observed. Previous X-ray models fit the observations much better. We conclude that the X-ray diffraction evidence has been well interpreted in the past and that there is more than one ordered myosin head state in resting muscle. There is, therefore, no reason to question some of the previous X-ray diffraction results on myosin filaments; time-resolved X-ray diffraction should be a reliable way to follow crossbridge action in active muscle and may be one of the few ways to visualise the molecular changes in myosin heads on a millisecond timescale as force is actually produced.

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Cheney, Richard E. "Dynamics of Myosin-X in Filopodia." Biophysical Journal 100, no. 3 (February 2011): 2a. http://dx.doi.org/10.1016/j.bpj.2010.11.064.

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Kerber, M. L., and R. E. Cheney. "Myosin-X: a MyTH-FERM myosin at the tips of filopodia." Journal of Cell Science 124, no. 22 (November 15, 2011): 3733–41. http://dx.doi.org/10.1242/jcs.023549.

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Holmes, K. C., D. R. Trentham, R. Simmons, Vincenzo Lombardi, Gabriella Piazzesi, Massimo Reconditi, Marco Linari, et al. "X-ray diffraction studies of the contractile mechanism in single muscle fibres." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1452 (December 29, 2004): 1883–93. http://dx.doi.org/10.1098/rstb.2004.1557.

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The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X–ray diffraction. Changes in the intensities of the axial X–ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X–ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X–ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.

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Prosser, Haydn M., Agnieszka K. Rzadzinska, Karen P. Steel, and Allan Bradley. "Mosaic Complementation Demonstrates a Regulatory Role for Myosin VIIa in Actin Dynamics of Stereocilia." Molecular and Cellular Biology 28, no. 5 (December 26, 2007): 1702–12. http://dx.doi.org/10.1128/mcb.01282-07.

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ABSTRACT We have developed a bacterial artificial chromosome transgenesis approach that allowed the expression of myosin VIIa from the mouse X chromosome. We demonstrated the complementation of the Myo7a null mutant phenotype producing a fine mosaic of two types of sensory hair cells within inner ear epithelia of hemizygous transgenic females due to X inactivation. Direct comparisons between neighboring auditory hair cells that were different only with respect to myosin VIIa expression revealed that mutant stereocilia are significantly longer than those of their complemented counterparts. Myosin VIIa-deficient hair cells showed an abnormally persistent tip localization of whirlin, a protein directly linked to elongation of stereocilia, in stereocilia. Furthermore, myosin VIIa localized at the tips of all abnormally short stereocilia of mice deficient for either myosin XVa or whirlin. Our results strongly suggest that myosin VIIa regulates the establishment of a setpoint for stereocilium heights, and this novel role may influence their normal staircase-like arrangement within a bundle.

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Hayden, S. M., J. S. Wolenski, and M. S. Mooseker. "Binding of brush border myosin I to phospholipid vesicles." Journal of Cell Biology 111, no. 2 (August 1, 1990): 443–51. http://dx.doi.org/10.1083/jcb.111.2.443.

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The actin filament core within each microvillus of the intestinal epithelial cell is attached laterally to the plasma membrane by brush border (BB) myosin I, a protein-calmodulin complex belonging to the myosin I class of actin-based mechanoenzymes. In this report, the binding of BB myosin I to pure phospholipid vesicles was examined and characterized. BB myosin I demonstrated saturable binding to liposomes composed of anionic phospholipids, but did not associate with liposomes composed of only neutral phospholipids. The binding of BB myosin I to phosphatidylserine and phosphatidylglycerol vesicles reached saturation at 4-5 x 10(-3) nmol protein/nmol phospholipid, while the apparent dissociation constant was determined to be 1-3 x 10(-7) M. Similar to the free protein, membrane-associated BB myosin I bound F-actin in an ATP-sensitive manner and demonstrated actin-activated Mg-ATPase activity. Immunoblot analysis of peptides generated from controlled proteolysis of vesicle-bound BB myosin I provided structural information concerning the site responsible for the membrane interaction. Immunoblot staining with domain-specific mAbs revealed a series of COOH-terminal, liposome-associated peptides that were protected from digestion, suggesting that the membrane-binding domain is within the carboxy-terminal "tail" of the BB myosin I heavy chain.

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de Lanerolle, P. "cAMP, myosin dephosphorylation, and isometric relaxation of airway smooth muscle." Journal of Applied Physiology 64, no. 2 (February 1, 1988): 705–9. http://dx.doi.org/10.1152/jappl.1988.64.2.705.

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The temporal relationships among increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels, myosin dephosphorylation, and relaxation were investigated to clarify the mechanisms of airway muscle relaxation. Canine tracheal muscles isometrically contracted (82% of maximum force) with 10(-6) M methacholine were relaxed by adding either 4 x 10(-7) M atropine or 4 x 10(-5) M forskolin. Atropine had no effect on cAMP levels; myosin phosphorylation and force, however, decayed at the same rates and these two parameters returned to their basal pre-methacholine levels within 5 min. Forskolin treatment results in about a 10-fold increase in cAMP levels; myosin phosphorylation and force decayed simultaneously to their respective steady-state levels by 10 min but neither parameter returned to its pre-methacholine level. The addition of forskolin to muscles maximally contracted with 10(-4) M methacholine leads to about a 30-fold increase in cAMP levels. However, there are minimal decreases in myosin phosphorylation and force in these muscles. Thus myosin dephosphorylation appears to be essential for airway muscle relaxation, whereas an increase in cAMP in the absence of myosin dephosphorylation is insufficient to cause relaxation. Moreover, myosin dephosphorylation appears to be a common step in the cAMP-independent and cAMP-dependent mechanisms for airway muscle relaxation.

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Liu, Katy C., Damon T. Jacobs, Brian D. Dunn, Alan S. Fanning, and Richard E. Cheney. "Myosin-X functions in polarized epithelial cells." Molecular Biology of the Cell 23, no. 9 (May 2012): 1675–87. http://dx.doi.org/10.1091/mbc.e11-04-0358.

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Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.

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Homma, Kazuaki, Junya Saito, Reiko Ikebe, and Mitsuo Ikebe. "Motor Function and Regulation of Myosin X." Journal of Biological Chemistry 276, no. 36 (July 16, 2001): 34348–54. http://dx.doi.org/10.1074/jbc.m104785200.

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Zepeda-Bastida, Armando, Natalia Chiquete-Felix, Juan Ocampo-López, Salvador Uribe-Carvajal, and Adela Mújica. "Possible participation of calmodulin in the decondensation of nuclei isolated from guinea pig spermatozoa." Zygote 18, no. 3 (November 26, 2009): 217–29. http://dx.doi.org/10.1017/s0967199409990220.

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SummaryThe guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

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Malhotra, A., P. Buttrick, and J. Scheuer. "Effects of sex hormones on development of physiological and pathological cardiac hypertrophy in male and female rats." American Journal of Physiology-Heart and Circulatory Physiology 259, no. 3 (September 1, 1990): H866—H871. http://dx.doi.org/10.1152/ajpheart.1990.259.3.h866.

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Previous studies have demonstrated a role for sex hormones in maintaining normal heart weight and myosin isoenzyme balance in the rat. To determine if sex hormones were necessary to elicit cardiac adaptations to the chronic loads of swimming or hypertension, female rats were gonadectomized (X) and then exposed either to a chronic swimming program (Sw) or to renal hypertension for 8-10 wk. Because gonadectomy in females increased heart and body weight, separate groups of food-restricted sedentary and Sw gonadectomized females (XFR) were included. Swimming resulted in significant increases in both heart weight and in the percent ventricular V1 isomyosin in female controls (C), X, and XFR. Hypertension was studied in C, X, and X with estrogen replacement. Cardiac hypertrophy developed in all groups, but estrogen therapy attenuated the decline in percent V1 isomyosin in both normotensive and hypertensive X animals. Swimming, which is generally not associated with cardiac hypertrophy in males, was also studied in that sex. Gonadectomy did not alter either the heart weight or the myosin isoenzyme response to Sw, although testosterone replacement in gonadectomized males restored ventricular V1 myosin levels to or above normal. Measures of serum thyroid levels and of myocardial catecholamines failed to demonstrate a causal relationship between these hormones and the various results. Therefore, although sex hormones are important for maintaining normal heart weights and myosin isoenzyme balance in rats, they do not appear to be important in the adaptations hearts exhibit when exposed to physiological or pathological loads.

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Skubiszak, Ludmila, and Leszek Kowalczyk. "The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data." Acta Biochimica Polonica 49, no. 4 (December 31, 2002): 841–53. http://dx.doi.org/10.18388/abp.2002_3744.

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Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.

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Sugi, Haruo, Maki Yamaguchi, Tetsuo Ohno, Hiroshi Okuyama, and Naoto Yagi. "X-ray Diffraction Studies on the Structural Origin of Dynamic Tension Recovery Following Ramp-Shaped Releases in High-Ca Rigor Muscle Fibers." International Journal of Molecular Sciences 21, no. 4 (February 13, 2020): 1244. http://dx.doi.org/10.3390/ijms21041244.

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It is generally believed that during muscle contraction, myosin heads (M) extending from myosin filament attaches to actin filaments (A) to perform power stroke, associated with the reaction, A-M-ADP-Pi → A-M + ADP + Pi, so that myosin heads pass through the state of A-M, i.e., rigor A-M complex. We have, however, recently found that: (1) an antibody to myosin head, completely covering actin-binding sites in myosin head, has no effect on Ca2+-activated tension in skinned muscle fibers; (2) skinned fibers exhibit distinct tension recovery following ramp-shaped releases (amplitude, 0.5% of Lo; complete in 5 ms); and (3) EDTA, chelating Mg ions, eliminate the tension recovery in low-Ca rigor fibers but not in high-Ca rigor fibers. These results suggest that A-M-ADP myosin heads in high-Ca rigor fibers have dynamic properties to produce the tension recovery following ramp-shaped releases, and that myosin heads do not pass through rigor A-M complex configuration during muscle contraction. To obtain information about the structural changes in A-M-ADP myosin heads during the tension recovery, we performed X-ray diffraction studies on high-Ca rigor skinned fibers subjected to ramp-shaped releases. X-ray diffraction patterns of the fibers were recorded before and after application of ramp-shaped releases. The results obtained indicate that during the initial drop in rigor tension coincident with the applied release, rigor myosin heads take up applied displacement by tilting from oblique to perpendicular configuration to myofilaments, and after the release myosin heads appear to rotate around the helical structure of actin filaments to produce the tension recovery.

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Ma, Weikang, Henry Gong, and Thomas Irving. "Myosin Head Configurations in Resting and Contracting Murine Skeletal Muscle." International Journal of Molecular Sciences 19, no. 9 (September 6, 2018): 2643. http://dx.doi.org/10.3390/ijms19092643.

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Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases, including those of skeletal muscle. We show that mouse skeletal muscle can produce high quality X-ray diffraction patterns establishing the mouse intact skeletal muscle X-ray preparation as a potentially powerful tool to test structural hypotheses in health and disease. A notable feature of the mouse model system is the presence of residual myosin layer line intensities in contracting mouse muscle patterns. This provides an additional tool, along with the I1,1/I1,0 intensity ratio, for estimating the proportions of active versus relaxed myosin heads under a given set of conditions that can be used to characterize a given physiological condition or mutant muscle type. We also show that analysis of the myosin layer line intensity distribution, including derivation of the myosin head radius, Rm, may be used to study the role of the super-relaxed state in myosin regulation. When the myosin inhibitor blebbistatin is used to inhibit force production, there is a shift towards a highly quasi-helically ordered configuration that is distinct from the normal resting state, indicating there are more than one helically ordered configuration for resting crossbridges.

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Milligan, R. A. "Structure and Action of Molecular Tracks and Motors." Microscopy and Microanalysis 4, S2 (July 1998): 456–57. http://dx.doi.org/10.1017/s1431927600022406.

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Molecular motors belonging to the myosin and kinesin superfamilies utilize ATP to move along their respective F-actin and microtubule tracks. The track-motor complexes have not been amenable to crystallization, so x-ray crystallographic investigations have focused on structure determinations of the individual proteins. Although providing detailed descriptions of the structure of each protein, this approach cannot reveal the geometry of interaction of the proteins nor the conformational changes which occur during the mechanochemical cycle. To obtain this information, we use cryoelectron microscopy and image analysis to calculate three dimensional maps of the track-motor complexes at moderate resolution (15-30A) and combine these data with the high resolution x-ray structures to provide near-atomic models of the working assemblies.We have so far built models of the rigor (nucleotide-free) and ADP actomyosin complexes. In smooth muscle myosin II (a collaboration with H.L. Sweeney, U. Perm.) and brush border myosin I (BBMI), the motor domain of the myosin head is similar in both biochemical states.

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29

Chatterjee, M., and M. Tejada. "Phorbol ester-induced contraction in chemically skinned vascular smooth muscle." American Journal of Physiology-Cell Physiology 251, no. 3 (September 1, 1986): C356—C361. http://dx.doi.org/10.1152/ajpcell.1986.251.3.c356.

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We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

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30

Whittaker, M., and R. Milligan. "Thin filament structure and activation." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 976–77. http://dx.doi.org/10.1017/s0424820100156869.

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Skeletal muscle contraction is triggered by the release of calcium ions from the sarcoplasmic reticulum of the myofibrils. Troponin and tropomyosin in the thin (actin-containing) filaments act together as the transducer of this signal. Evidence from reconstruction of electron images of negatively stained samples and from x-ray diffraction studies of intact myofibrils (e.g. 2) have led to the steric blocking model of regulation by the tropomyosin troponin complex. In this model, tropomyosin is believed to occupy a defined location on the myosin binding site in the absence of calcium. When calcium binds to the regulatory complex, tropomyosin is thought to change its location on actin, moving to occupy a second site adjacent to the myosin binding site. Time resolved x-ray diffraction studies on muscle during activation show that movement of tropomyosin precedes crossbridge attachment by 12-17ms, suggesting that it is a prerequisite for myosin binding. In contrast, biochemical experiments suggest that tropomyosin may not affect the ability of the myosin head to bind to actin but rather may interfere with a subsequent kinetic step in the crossbridge cycle.

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Homma, Kazuaki, and Mitsuo Ikebe. "Myosin X Is a High Duty Ratio Motor." Journal of Biological Chemistry 280, no. 32 (June 16, 2005): 29381–91. http://dx.doi.org/10.1074/jbc.m504779200.

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32

Lu, Q., F. Ye, Z. Wei, Z. Wen, and M. Zhang. "Antiparallel coiled-coil-mediated dimerization of myosin X." Proceedings of the National Academy of Sciences 109, no. 43 (September 10, 2012): 17388–93. http://dx.doi.org/10.1073/pnas.1208642109.

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FARUQI, A. R., R. A. CROSS, and J. KENDRICK-JONES. "Small angle X-ray scattering studies on myosin." Journal of Cell Science 1991, Supplement 14 (January 1, 1991): 23–26. http://dx.doi.org/10.1242/jcs.1991.supplement_14.5.

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34

Watanabe, Tomonobu M., Hiroshi Tokuo, Kohsuke Gonda, Hideo Higuchi, and Mitsuo Ikebe. "The Dynamics for Myosin-X Induced Filopodia Protrusion." Biophysical Journal 98, no. 3 (January 2010): 725a—726a. http://dx.doi.org/10.1016/j.bpj.2009.12.3976.

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Sato, Osamu, Satoshi Komatsu, Tomonobu M. Watanabe, Kazuaki Homma, and Mitsuo Ikebe. "Single Molecule Movement of Full-Length Myosin X." Biophysical Journal 102, no. 3 (January 2012): 570a. http://dx.doi.org/10.1016/j.bpj.2011.11.3103.

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Watanabe, Tomonobu M., Hiroshi Tokuo, Kohsuke Gonda, Hideo Higuchi, and Mitsuo Ikebe. "Myosin-X Induces Filopodia by Multiple Elongation Mechanism." Journal of Biological Chemistry 285, no. 25 (April 13, 2010): 19605–14. http://dx.doi.org/10.1074/jbc.m109.093864.

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Eakins, Felicity, Jeffrey J. Harford, Carlo Knupp, Manfred Roessle, and John M. Squire. "Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths." International Journal of Molecular Sciences 19, no. 7 (July 18, 2018): 2091. http://dx.doi.org/10.3390/ijms19072091.

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At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the ‘roll and lock’ mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.

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Flood, Veronica H., Tricia L. Slobodianuk, Daniel Keesler, Hannah K. Lohmeier, Scot Fahs, Liyun Zhang, Pippa Simpson, and Robert R. Montgomery. "von Willebrand factor binding to myosin assists in coagulation." Blood Advances 4, no. 1 (January 14, 2020): 174–80. http://dx.doi.org/10.1182/bloodadvances.2019000533.

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Abstract von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

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39

Verkhovsky, A. B., and G. G. Borisy. "Non-sarcomeric mode of myosin II organization in the fibroblast lamellum." Journal of Cell Biology 123, no. 3 (November 1, 1993): 637–52. http://dx.doi.org/10.1083/jcb.123.3.637.

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The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.

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40

Chang, Audrey N., Ning Gao, Zhenan Liu, Jian Huang, Angus C. Nairn, Kristine E. Kamm, and James T. Stull. "The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cβ, is essential for smooth muscle contraction." Journal of Biological Chemistry 293, no. 43 (September 5, 2018): 16677–86. http://dx.doi.org/10.1074/jbc.ra118.003083.

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Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo. In contrast, smooth muscle–specific conditional PP1cβ knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cβ to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cβ content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cβ forms in smooth muscle.

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41

Knupp and Squire. "Myosin Cross-Bridge Behaviour in Contracting Muscle—The T1 Curve of Huxley and Simmons (1971) Revisited." International Journal of Molecular Sciences 20, no. 19 (October 2, 2019): 4892. http://dx.doi.org/10.3390/ijms20194892.

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The stiffness of the myosin cross-bridges is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles. The seminal study of Huxley and Simmons (1971: Nature 233: 533) suggested that most of the observed half-sarcomere instantaneous compliance (=1/stiffness) resides in the myosin heads. They showed with a so-called T1 plot that, after a very fast release, the half-sarcomere tension reduced to zero after a step size of about 60Å (later with improved experiments reduced to 40Å). However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch slightly under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges. Here we have used a different approach, namely to model the compliances in a virtual half sarcomere structure in silico. We confirm that the T1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (probably greater than 0.4 pN/Å) is higher than previous studies have suggested. Our model demonstrates that the formulations produced by previous authors give very similar results to our model if the same starting parameters are used. However, we find that it is necessary to model the X-ray diffraction data as well as mechanics data to get a reliable estimate of the cross-bridge stiffness. In the light of the high cross-bridge stiffness found in the present study, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle. In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T1 response may come from weakly-bound myosin heads, not myosin heads in strongly attached states. The strongly attached heads would still contribute to the T1 curve, but only in a very minor way, with a stiffness that we postulate could be around 0.1 pN/Å, a value which would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule. The new model can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g., tension, T1 intercept, temperature, X-ray diffraction spacing results).

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Ma, Weikang, Marcus Henze, Robert L. Anderson, Henry Gong, Fiona L. Wong, Carlos L. del Rio, and Thomas Irving. "The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium." Circulation Research 129, no. 6 (September 3, 2021): 617–30. http://dx.doi.org/10.1161/circresaha.120.318647.

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Rationale: Myofilament length-dependent activation (LDA) is the key underlying mechanism of cardiac heterometric autoregulation, commonly referred as the Frank-Starling Law of the heart. Although alterations in LDA are common in cardiomyopathic states, the precise structural and biochemical mechanisms underlying LDA remain unknown. Objective: Here, we examine the role of structural changes in the thick filament during diastole, in particular changes in the availability of myosin heads, in determining both calcium sensitivity and maximum contractile force during systole in permeabilized porcine cardiac fibers. Methods and Results: Permeabilized porcine fibers from ventricular myocardium were studied under relaxing conditions at short and long sarcomere length using muscle mechanics, biochemical measurements, and X-ray diffraction. Upon stretch, the porcine myocardium showed the increased calcium sensitivity and maximum calcium-activated force characteristic of LDA. Stretch increased diastolic ATP turnover, recruiting reserve myosin heads from the super-relaxed state at longer sarcomere length. Structurally, X-ray diffraction studies in the relaxed-muscle confirmed a departure from the helical ordering of the thick filament upon stretch which occurred concomitantly with a displacement of myosin heads towards actin, facilitating cross-bridge formation upon systolic activation. Mavacamten, a selective myosin-motor inhibitor known to weaken the transition to actin-bound power-generating states and to enrich the ordered super-relaxed state myosin population, reversed the structural effects of stretch on the thick filament, blunting the mechanical consequences of stretch; mavacamten did not, however, prevent other structural changes associated with LDA in the sarcomere, such as decreased lattice spacing or troponin-displacement. Conclusions: Our findings strongly indicate that in ventricular muscle, LDA and its systolic consequences are dependent on the population of myosin heads competent to form cross bridges and involves the recruitment of myosin heads from the reserve super-relaxed state pool during diastole.

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Perz-Edwards, Robert J., Thomas C. Irving, Bruce A. J. Baumann, David Gore, Daniel C. Hutchinson, Uroš Kržič, Rebecca L. Porter, Andrew B. Ward, and Michael K. Reedy. "X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle." Proceedings of the National Academy of Sciences 108, no. 1 (December 9, 2010): 120–25. http://dx.doi.org/10.1073/pnas.1014599107.

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Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called “troponin bridges,” by analyzing real-time X-ray diffraction “movies” from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin’s steric blocking of myosin–actin binding. This enables subsequent force production, with cross-bridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.

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44

Niciforovic, Ana, Marija Radojcic, and Bratoljub Milosavljevic. "Gamma-radiation induced agglomeration of chicken muscle myosin and actine." Journal of the Serbian Chemical Society 69, no. 12 (2004): 999–1004. http://dx.doi.org/10.2298/jsc0412999n.

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Radiolytic behaviour of themajor vertebrate muscle proteins: fibrillarmyosin (molar mass, Mm = 520,000 g/mol) and filament forming actin (Mm = 42,050 g/mol) was studied using a SDS-polyacrylamide gel electrophoresis and quantified by high precision laser-densitometry. In order to study the OH radical contribution to the radiation damage, purified chicken myosin and actin (4 ?M) were prepared in N2O saturated solution and irradiated with 1?3 kGy at 60Co gamma source. With respect to changes in the molecular mass, the only observed myosin and actin damage was dose dependent agglomeration of proteins. The corresponding radiation chemical yields of 5 x?10-8 mol J-1 and 6.3 x?10-8 mol J-1 were obtained for myosin and actin, respectively. This result confirmed that only the radiation-induced agglomeration is initiated with the reaction of the OH radical even in the situation where the OH radical concentration produced exceeds the protein concentration 500 times thus enabling the multi-radical attack to occur.

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45

Baker, T. S., and D. A. Winkelmann. "Methodology for determining the three-dimensional crystal structure of myosin Sl from electron microscopy of orthogonal thin sections." Proceedings, annual meeting, Electron Microscopy Society of America 44 (August 1986): 26–29. http://dx.doi.org/10.1017/s0424820100141895.

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Detailed knowledge of the structure of myosin is essential for understanding the mechanism of muscle contraction. The discovery of conditions for crystallizing the head of myosin (Sl = subfragment 1) has led to systematic studies of the SI structure by x-ray crystallography and electron microscopy. We describe the method used to determine the structure of the myosin Sl molecule by electron microscopy (Fig. 3). This method involved independently reconstructing the three-dimensional density of several thin sections obtained from oriented crystals and then combining these reconstructions to produce a final, averaged density map of a single unit cell.

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Winkelmann, Donald A. "Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 156–57. http://dx.doi.org/10.1017/s0424820100102869.

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The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

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47

Taylor, Kenneth A., Hamidreza Rahmani, Robert J. Edwards, and Michael K. Reedy. "Insights into Actin-Myosin Interactions within Muscle from 3D Electron Microscopy." International Journal of Molecular Sciences 20, no. 7 (April 5, 2019): 1703. http://dx.doi.org/10.3390/ijms20071703.

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Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug Lethocerus sp. The Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states, as well as the fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures as determined by X-ray crystallography needed to explain the structure of the different actomyosin interactions observed in situ.

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48

Evans, L. L., A. J. Lee, P. C. Bridgman, and M. S. Mooseker. "Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport." Journal of Cell Science 111, no. 14 (July 30, 1998): 2055–66. http://dx.doi.org/10.1242/jcs.111.14.2055.

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Myosin-V has been linked to actin-based organelle transport by a variety of genetic, biochemical and localization studies. However, it has yet to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted a biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purification of brain myosin-V, we isolated a population of brain microsomes that is approx. fivefold enriched for myosin-V, and is similarly enriched for synaptic vesicle proteins. As demonstrated by immunoelectron microscopy, myosin-V associates with 30–40% of the vesicles in this population. Although a majority of myosin-V-associated vesicles also label with the synaptic vesicle marker protein, SV2, less than half of the total SV2-positive vesicles label with myosin-V. The average size of myosin-V/SV2 double-labeled vesicles (90+/−45 nm) is larger than vesicles that label only with SV2 antibodies (60+/−30 nm). To determine if these vesicles are capable of actin-based transport, we used an in vitro actin filament motility assay in which vesicles were adsorbed to motility assay substrates. As isolated, the myosin-V-associated vesicle fraction was nonmotile. However, vesicles pre-treated with ice-cold 0.1% Triton X-100 supported actin filament motility at rates comparable to those on purified myosin-V. This dilute detergent treatment did not disrupt vesicle integrity. Furthermore, while this treatment removed over 80% of the total vesicle proteins, myosin-V remained tightly vesicle-associated. Finally, function-blocking antibodies against the myosin-V motor domain completely inhibited motility on these substrates. These studies provide direct evidence that vesicle-associated myosin-V is capable of actin transport, and suggest that the activity of myosin-V may be regulated by proteins or lipids on the vesicle surface.

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Melo, Hugo Christiano Soares, and Milton Vieira Coelho. "A new method to precipitate myosin V from rat brain soluble fraction." Acta Biochimica Polonica 54, no. 3 (September 17, 2007): 575–81. http://dx.doi.org/10.18388/abp.2007_3231.

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Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.

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Wagner, M. C., and B. A. Molitoris. "ATP depletion alters myosin I beta cellular location in LLC-PK1 cells." American Journal of Physiology-Cell Physiology 272, no. 5 (May 1, 1997): C1680—C1690. http://dx.doi.org/10.1152/ajpcell.1997.272.5.c1680.

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The brush border (BB) of the proximal tubule cell (PTC) requires dynamic membrane events for function. The actin cytoskeleton is necessary for structure and function in this region. ATP depletion disrupts both structure and function. In this report, myosin 1 beta location in LLC-PK1 cells was followed during ATP depletion and repletion using immunofluorescence and Western blot techniques. Myosin I beta colocalized with F-actin in the microvilli and cell periphery, but no colocalization was observed with stress fibers. ATP depletion increased the apical F-actin, and myosin I beta was colocalized there. In addition, after ATP depletion, myosin I beta was extracted less by Triton X-100. These changes were reversed after ATP repletion. Finally, immunofluorescence of kidney sections shows myosin I beta in the BB. These results place this motor in a dynamic region of the PTC where its actin and membrane binding domains can contribute to PTC function.

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Journal articles on the topic 'Myosine-X'

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