Chimera-users] Chimera: Myosin thick filament movie
Elaine pointed out a few more movie command script examples: Hi Tom, Just thought I'd mention that the 'movies' page in the User's Guide, the bottom URL you gave, has a 'Movie Command File Examples' section that links to a few files and says there are more in the Animation Gallery. http://www.cgl.ucsf.edu/chimera/current/docs/UsersGuide/movies.html#examples Elaine , Hi Rogelio, , , The myosin thick filament movie in the Chimera animation gallery was , made in 2006 using a 700 line python program I wrote. Fortunately , Chimera animation capabilities are much better now, so I've made a , Chimera command script that makes the same movie in Chimera 1.5.2 (Jan , 2011), about 150 commands. I've attached the script. It of course , requires the myosin data files to work. So I've put the script and data , files in myomovie.zip and put a link to that in the animation gallery , with the myosin animation (will appear after web site is rebuilt tonight). , , ...http://www.cgl.ucsf.edu/pipermail/chimera-users/2011-April/006310.html
Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain | JCB
Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC had no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduced velocity by approximately 30%. Very slow movement was observed once 26 residues were deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins were monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A ...http://jcb.rupress.org/content/124/6/963
Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain |...
Conventional myosin ('myosin II') is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested ...http://jcs.biologists.org/content/107/10/2875
Shaking the myosin family tree: Biochemical kinetics defines four types of myosin motor - Kent Academic Repository
Although all myosin motors follow the same basic cross-bridge cycle, they display a large variety in the rates of transition between different states in the cycle, allowing each myosin to be finely tuned for a specific task. Traditionally, myosins have been classified by sequence analysis into a large number of sub-families (∼35). Here we use a different method to classify the myosin family members which is based on biochemical and mechanical properties. The key properties that define the type of mechanical activity of the motor are duty ratio (defined as the fraction of the time myosin remains attached to actin during each cycle), thermodynamic coupling of actin and nucleotide binding to myosin and the degree of strain-sensitivity of the ADP release step. Based on these properties we propose to classify myosins into four different groups: (I) fast movers, (II) slow/efficient force holders, ...https://kar.kent.ac.uk/29008/
Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly | JCB
This work has also provided discrete evidence of abnormal structure in unc-45 mutant thick filaments. Comparison of thick filaments from the CB286 strain grown at the restrictive versus the permissive temperature has demonstrated that this ts mutation, located within the CRO1/ She4p-like domain, affects the accumulation of assembled thick filaments, as well as their myosin isoform distribution and in vitro stability.. The results presented here suggest that UNC-45 functions as a thick filament assemblase through its CRO1/ She4p-like domain. Assemblase activity may be defined as a catalyst or chaperone that mediates the incorporation of myosin and related proteins into thick filaments (Liu et al., 1997). Several mechanisms may be involved, including catalysis of myosin folding at the ribosome, intracellular myosin transport, posttranslational modification of myosin and associated proteins, catalysis of myosin ...http://jcb.rupress.org/content/143/5/1215
"A unique ATP hydrolysis mechanism of single-headed processive myosin, " by Taketoshi Kambara and Mitsuo Ikebe
Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. ...http://escholarship.umassmed.edu/oapubs/684/
Local diversity of myosin expression in mammalian atrial muscles. Variations depending on age and thyroid state in the rat and...
Rat, rabbit, pig, and bovine atrial myocardia were investigated with anti-alpha and anti-beta myosin heavy chain monoclonal antibodies. Analysis of atrial fibers by indirect immunofluorescence and assay of myosin heavy chains in tissue micro samples by immunoaffinity chromatography revealed both heterogeneity and plasticity in the atrial myosin heavy chains, undetected by electrophoresis of native atrial myosins under nondenaturing conditions. We found both alpha- and beta-like myosin heavy chains to be expressed in rat and rabbit, as they are in pig and bovine, atrial myocardia. They were regionally distributed within atrial muscles. The beta-like myosin heavy chains were present at much lower levels in rat and rabbit atria than in pig and bovine atria. Young rat atrial myosin was composed of only alpha-like heavy chains. In the rat and the rabbit, hyperthyroidism induced a beta- to ...http://circres.ahajournals.org/content/57/5/767
Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human skeletal muscles...
Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, ...https://iris.unipv.it/handle/11571/15881
Myosin phospho S19/phospho S20 Antibody 600-401-416
Myosin is the major component of thick muscle filaments, and is a long asymmetric molecule containing a globular head and a long tail. The molecule consists of two heavy chains each ~200,000 daltons, and four light chains each ~16,000 - 21,000 daltons. Activation of smooth and cardiac muscle primarily involves pathways that increase calcium levels and myosin phosphorylation, resulting in contraction. Myosin light chain phosphatase acts to regulate muscle contraction by dephosphorylating activated myosin light chain. This antibody is specific for the phosphorylated form of myosin light chain. The selected peptide sequence used to generate the polyclonal antibody is located near the amino terminal end of the polypeptide corresponding to the smooth/non-muscle form of myosin regulatory light chain found in cardiac myocytes in addition to smooth and non-muscle cells. This ...https://rockland-inc.com/store/Enzyme-Antibodies-600-401-416-O4L_18319.aspx
Force-generating capacity of human myosin isoforms extracted from single muscle fibre segments
Muscle, motor unit and muscle fibre type-specific differences in force-generating capacity have been investigated for many years, but there is still no consensus regarding specific differences between slow- and fast-twitch muscles, motor units or muscle fibres. This is probably related to a number of different confounding factors disguising the function of the molecular motor protein myosin. We have therefore studied the force-generating capacity of specific myosin isoforms or combination of isoforms extracted from short single human muscle fibre segments in a modified single fibre myosin in vitro motility assay, in which an internal load (actin-binding protein) was added in different concentrations to evaluate the force-generating capacity. The force indices were the x-axis intercept and the slope of the relationship between the fraction of moving filaments and the α-actinin concentration. The force-generating capacity of the β/slow myosin ...http://uu.diva-portal.org/smash/record.jsf?pid=diva2:401619
A myosin family tree | Journal of Cell Science
This unrooted phylogenetic tree of the myosin superfamily (see insert) is derived from an alignment of 139 members of the myosin superfamily. The alignment compared the core motor domains (equivalent to residues 88-780 of chicken skeletal myosin II) of each myosin, using distance matrix analysis performed with the Clustal-W package. The exceptions, shown with a dotted line, are SsVIIa, which is a partial sequence as reported in the databases, and Hs MysPDZ. The latter is reported as a complete coding sequence but has a truncated N terminus starting some 52 residues into the core motor region (i.e. residue 140 of chicken skeletal myosin II). These shorter sequences have no significant effect on the branching order of the rest of the tree.. Optimally, when one produces a phylogenetic tree from such an alignment, any positions in the alignment with gaps are excluded. Such a strategy would have excluded a large proportion of the ...http://jcs.biologists.org/content/113/19/3353
Location and functional characterization of myosin contact sites in smooth- muscle caldesmon | Biochemical Journal
Caldesmon interaction with smooth muscle myosin and its ability to cross-link actin filaments to myosin were investigated by the use of several bacterially expressed myosin-binding fragments of caldesmon. We have confirmed the presence of two functionally different myosin-binding sites located in domains 1 and 3/4a of caldesmon. The binding of the C-terminal site is highly sensitive to ionic strength and hardly participates in acto-myosin cross-linking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains two separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involved in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosin filaments, without affecting the affinity of ...http://www.biochemj.org/content/328/1/211
Contributions of intracellular and extracellular Ca2+ pools to activation of myosin phosphorylation and stress in swine carotid...
Contractile agonists can mobilize Ca2+ from both intracellular and extracellular stores in smooth muscle. This study addresses the role of Ca2+ mobilization as it relates to the complex manner by which Ca2+ regulates the contractile system in smooth muscle. In swine carotid media, both histamine and phenylephrine produced initial rapid increases in myosin phosphorylation and stress. Stress was sustained for the duration of the stimulus while myosin phosphorylation slowly declined to steady-state levels. Removal of extracellular Ca2+ or elimination of cellular Ca2+ influx did not dramatically reduce the initial rapid increase in myosin phosphorylation produced by either agonist but reduced steady-state levels of myosin phosphorylation to basal values. Initial rapid increases in stress were seen, but stress was not maintained. Following depletion of Ca2+ from sarcoplasmic reticulum, muscle activation by Ca2+ influx in the presence of ...http://circres.ahajournals.org/content/60/3/410
The myosin superfamily at a glance | Journal of Cell Science
The diversity of the myosin superfamily becomes particularly evident in the variety of domains that are found in the C-terminal tail of these proteins. Whereas the catalytic heads share a number of conserved elements, the tail regions of the various myosin classes are highly divergent (Thompson and Langford, 2002). The unconventional myosins possess specific domains in their tail regions that are thought to enable their individual cellular functions through association with adaptors and other binding proteins (Akhmanova and Hammer, 2010).. Myosins localize to a number of intracellular compartments and participate in many trafficking and anchoring events. The recruitment of motor proteins to an organelle or molecular complex is generally the result of the tail binding to a specific adaptor protein. A number of myosin-binding proteins have been discovered, and their identities have often provided information that is essential ...http://jcs.biologists.org/content/125/7/1627.long
Regulatory Light Chains of Striated Muscle Myosin. Structure, Function and Malfunction | BenthamScience
Title: Regulatory Light Chains of Striated Muscle Myosin. Structure, Function and Malfunction. VOLUME: 3 ISSUE: 2. Author(s):Danuta Szczesna-Cordary. Affiliation:Department of Molecular&Cellular Pharmacology, University of Miami School of MedicineRosenstiel Medical Sciences Building R-189, Room 6113, USA. Keywords:regulatory light chains of myosin (rlc), Phosphorylation, skinned fibers, familial hypertrophic, cardiomyopathy (fhc) mutations.. Abstract: Striated (skeletal and cardiac) muscle is activated by the binding of Ca2+ to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a Ca2+-Mg2+-binding site and, analogous to that of smooth muscle myosin, also ...http://www.eurekaselect.com/91509/article
"Shortening Velocity and Myosin Heavy- and Light-Chain Isoform mRNA in " by Jennifer J. Sherwood and Thomas J. Eddinger
In smooth muscle cells (SMCs) isolated from rabbit carotid, femoral, and saphenous arteries, relative myosin isoform mRNA levels were measured in RT-PCR to test for correlations between myosin isoform expression and unloaded shortening velocity. Unloaded shortening velocity and percent smooth muscle myosin heavy chain 2 (SM2) and myosin light chain 17b (MLC17b) mRNA levels were not significantly different in single SMCs isolated from the luminal and adluminal regions of the carotid media. Saphenous artery SMCs shortened significantly faster (P | 0.05) than femoral SMCs and had more SM2 mRNA (P | 0.05) than carotid SMCs and less MLC17b mRNA (P | 0.001) and higher tissue levels of SMB mRNA (P | 0.05) than carotid and femoral SMCs. No correlations were found between percent SM2 and percent MLC17b mRNA levels and unloaded shortening velocity in SMCs from these arteries. We have previously shown that myosin heavy chain (MHC) ...http://epublications.marquette.edu/bio_fac/60/
Increases in phosphorylation of the myosin II heavy chain, but not regulatory light chains, correlate with insulin secretion in...
Although cytoskeletal proteins such as myosin II are implicated in the control of insulin secretion, their precise role is poorly understood. In other secretory cells, myosin II is predominantly regulated via the phosphorylation of the regulatory light chains (RLC). The current study was aimed at investigating RLC phosphorylation in beta-cells. In both the insulin-secreting cell line RINm5F and rat pancreatic islets, the RLC was basally phosphorylated on the myosin light chain kinase sites (Ser19/Thr18). Phosphorylation at these sites was not consistently increased by either metabolic stimuli (glyceraldehyde/glucose) or the depolarizing agent KCl. The RLC sites phosphorylated by protein kinase C (PKC) (Ser1/Ser2) were unphosphorylated in the basal state, not affected by nutrients or KCl, and only slightly increased by the PKC activator phorbol 12-myristate 13-acetate (PMA). Like the other insulin secretagogues, however, PMA did promote serine phosphorylation ...https://www.garvan.org.au/research/publications/1316
Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells | JCB
Cultured cells used include REF-52 cells (rat embryo derived fibroblastic cells), REF-2A cells (SV-40 transformed REF52 cells), gerbil fibroma cells (IMR-33; CCL146; American Type Culture Collection, Rockville, MD), and epithelial type NRK cells (NRK-52E, CRL1571, American Type Culture Collection). Cells were maintained in DME containing 10% new born calf serum.. Double-label immunofluorescence was performed as described before (34). Briefly, cells grown on glass coverslips were fixed with 3.7% formaldehyde solution in PBS, permeabilized with acetone at −20°C, and then incubated with primary antibodies of pp2b (60 μg/ml) and a mouse monoclonal antibody against heavy chain of myosin II (called monoclonal myosin antibody; Immunotech, Westbrook, ME). After extensive washing, cells were labeled with affinity-purified, rhodamine-conjugated goat anti- mouse, and FITC-conjugated goat anti-rabbit antibodies. The monoclonal myosin antibody was originally reported to ...http://jcb.rupress.org/content/140/1/119
Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force...
Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca2+ sensitivity of force (pCa50), PKA treatment has been shown to decrease pCa50, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca2+-independent force and maximum Ca2+-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d1,0) to assess the inter-thick filament ...http://onlinelibrary.wiley.com/doi/10.1113/jphysiol.2009.183897/abstract
Myosin phosphorylation and cyclic adenosine 3',5'-monophosphate in relaxation of arterial smooth muscle by vasodilators. |...
Recent evidence indicates that contraction of vascular smooth muscle may be regulated by two calcium-dependent mechanisms: activation of myosin kinase, and calcium binding to a second, unknown regulatory site. This hypothesis implies that vasodilators could modify vascular tone by several mechanisms, including inactivation of myosin kinase. Since relaxation of the carotid artery following agonist removal may occur when myosin phosphorylation is at resting levels, we could determine whether dephosphorylation of myosin is necessarily involved in the molecular mechanisms mediating relaxation in response to vasodilators. The relaxant effects of adenosine, 3-isobutyl-1-methylxanthine, forskolin, sodium nitroprusside, and 8-bromo-cGMP were tested under conditions where myosin phosphorylation was at basal levels (0.08 +/- 0.02 mol Pi/mol light chain). All of these agents increased the rate of relaxation in nonsteady state ...http://circres.ahajournals.org/content/54/1/83
Polyclonal Antibody to Myosin Light Chain 3, Alkali, Ventricular, Slow Skeletal (MYL3), Polyclonal antibody, CLOUD-CLONE CORP....
PAD425Hu01, Polyclonal Antibody to Myosin Light Chain 3, Alkali, Ventricular, Slow Skeletal (MYL3), Homo sapiens (Human), Polyclonal antibody, CMH8, MLC1V, VLC1, MLC1SB, Cardiac myosin light chain 1, Ventricular/slow twitch myosin alkali light chain, Designed by Cloud-Clone Corp.http://www.cloud-clone.us/antibody/Polyclonal-Antibody-to-Myosin-Light-Chain-3--Alkali--Ventricular--Slow-Skeletal-
A mechanical function of myosin II in cell motility | Journal of Cell Science
Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell's rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a ...http://jcs.biologists.org/content/108/1/387
Studies of the interaction between titin and myosin. | JCB
The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal ...http://jcb.rupress.org/content/131/6/1471
Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular...
Arterial vascular smooth muscle cells (VSMCs) play an important role in the function of many organ systems. Abnormality in the contractile and/or regulatory apparatus of smooth muscle is implicated in the pathogenesis of a variety of disease conditions such as hypertension, coronary and cerebral vasospasm, miscarriage, and erectile dysfunction. VSMCs in vivo show remarkable plasticity once they need to adapt to changes in environments, such as new development of vasculature and remodeling after vascular injury or during vascular diseases like arteriosclerosis (Owens, 1995). These arterial cells undergo rapid changes in shape and functional property from non-proliferative and contractile to proliferative and mobile phenotype.. Agonist stimulation of VSMCs induces phosphorylation of the 20 kDa regulatory light chain of myosin (MLC), which increases actin-activated myosin ATPase activity and contraction (Hartshorne, 1987; Somlyo and Somlyo, 2003). MLC phosphorylation is ...http://jcs.biologists.org/content/119/9/1769
Fiber types and myosin types in human atrial and ventricular myocardium. An anatomical description. | Circulation Research
Hybridomas were prepared from mice immunized with myosin from the enlarged left ventricle of a 53-year-old female with an obstructive cardiomyopathy. The specificity of 15 monoclonal antibodies to myosin heavy chains was assessed by the reactivity of muscle extracts and of chymotryptic myosin fragments of different sizes with these antibodies, as determined by the immune replicate technique; some of the monoclonal antibodies cross-reacted only with the ventricular V3-type myosin from hypothyroid rats, whereas the other antibodies cross-reacted both with the latter and with the ventricular V1-type myosins from normal young rats. Immunological heterogeneity of the fibers from human atrial muscles and from human ventricular muscles was detected by some of the antimyosin antibodies by means of indirect immunofluorescence. Histochemical fiber heterogeneity was also detected by adenosine triphosphatase staining of ...http://circres.ahajournals.org/content/55/6/794