Science.gov

Sample records for skeletal myosin light

  1. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    PubMed

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  2. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  3. Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

    PubMed

    Borejdo, Julian; Ushakov, Dmitry S; Akopova, Irina

    2002-06-01

    Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body.

  4. In vivo myosin step-size from zebrafish skeletal muscle

    PubMed Central

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

    2016-01-01

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

  5. In vivo myosin step-size from zebrafish skeletal muscle.

    PubMed

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

    2016-05-01

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

  6. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

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

  8. Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

    PubMed Central

    Moreno-Domínguez, Alejandro; Colinas, Olaia; El-Yazbi, Ahmed; Walsh, Emma J; Hill, Michael A; Walsh, Michael P; Cole, William C

    2013-01-01

    The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca2+ entry via voltage-gated Ca2+ channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca2+ sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate

  9. Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries.

    PubMed

    Moreno-Domínguez, Alejandro; Colinas, Olaia; El-Yazbi, Ahmed; Walsh, Emma J; Hill, Michael A; Walsh, Michael P; Cole, William C

    2013-03-01

    Abstract  The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our

  10. Slow myosin in developing rat skeletal muscle

    PubMed Central

    1987-01-01

    Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC

  11. Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

    PubMed Central

    Nabeshima, Y; Fujii-Kuriyama, Y; Muramatsu, M; Ogata, K

    1982-01-01

    We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA. PMID:6128725

  12. The expression of myosin genes in developing skeletal muscle in the mouse embryo

    SciTech Connect

    Lyons, G.E.; Ontell, M.; Cox, R.; Sassoon, D.; Buckingham, M. )

    1990-10-01

    Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and acculumation.

  13. Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

    PubMed Central

    Findlay, W. A.; Gradwell, M. J.; Bayley, P. M.

    1995-01-01

    The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM. PMID:8563635

  14. In vivo orientation of single myosin lever arms in zebrafish skeletal muscle.

    PubMed

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

    2014-09-16

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

  15. All-atom molecular dynamics simulations of actin-myosin interactions: a comparative study of cardiac α myosin, β myosin, and fast skeletal muscle myosin.

    PubMed

    Li, Minghui; Zheng, Wenjun

    2013-11-26

    Myosins are a superfamily of actin-binding motor proteins with significant variations in kinetic properties (such as actin binding affinity) between different isoforms. It remains unknown how such kinetic variations arise from the structural and dynamic tuning of the actin-myosin interface at the amino acid residue level. To address this key issue, we have employed molecular modeling and simulations to investigate, with atomistic details, the isoform dependence of actin-myosin interactions in the rigor state. By combining electron microscopy-based docking with homology modeling, we have constructed three all-atom models for human cardiac α and β and rabbit fast skeletal muscle myosin in complex with three actin subunits in the rigor state. Starting from these models, we have performed extensive all-atom molecular dynamics (MD) simulations (total of 100 ns per system) and then used the MD trajectories to calculate actin-myosin binding free energies with contributions from both electrostatic and nonpolar forces. Our binding calculations are in good agreement with the experimental finding of isoform-dependent differences in actin binding affinity between these myosin isoforms. Such differences are traced to changes in actin-myosin electrostatic interactions (i.e., hydrogen bonds and salt bridges) that are highly dynamic and involve several flexible actin-binding loops. By partitioning the actin-myosin binding free energy to individual myosin residues, we have also identified key myosin residues involved in the actin-myosin interactions, some of which were previously validated experimentally or implicated in cardiomyopathy mutations, and the rest make promising targets for future mutational experiments. PMID:24224850

  16. Myosin light chain genes in the turkey (Meleagris gallopavo).

    PubMed

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  17. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

    PubMed

    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F

    1998-04-21

    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

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

    PubMed

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

    2014-06-15

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

  19. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis. PMID:27548633

  20. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  1. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

    PubMed

    Patrinou-Georgoulas, M; John, H A

    1977-10-01

    A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

  2. Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle.

    PubMed Central

    Saez, L; Leinwand, L A

    1986-01-01

    In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions. Images PMID:2421254

  3. A comparison of rat myosin from fast and slow skeletal muscle and the effect of disuse

    NASA Technical Reports Server (NTRS)

    Unsworth, B. R.; Witzmann, F. A.; Fitts, R. H.

    1981-01-01

    Certain enzymatic and structural features of myosin, purified from rat skeletal muscles representative of the fast twitch glycolytic (type IIb), the fast twitch oxidative (type IIa), and the slow twitch oxidative (type I) fiber, were determined and the results were compared with the measured contractile properties. Good correlation was found between the shortening velocities and Ca(2+)-activated ATPase activity for each fiber type. Short term hind limb immobilization caused prolongation of contraction time and one-half relaxation time in the fast twitch muscles and a reduction of these contractile properties in slow twitch soleus. Furthermore, the increased maximum shortening velocity in the immobilized soleus could be correlated with increased Ca(2+)-ATPase, but no change was observed in the enzymatic activity of the fast twitch muscles. No alteration in light chain distribution with disuse was observed in any of the fiber types. The myosin from slow twitch soleus could be distinguished from fast twitch myosins on the basis of the pattern of peptides generated by proteolysis of the heavy chains. Six weeks of hind limb immobilization resulted in both an increased ATPase activity and an altered heavy chain primary structure in the slow twitch soleus muscle.

  4. Improving human skeletal muscle myosin heavy chain fiber typing efficiency.

    PubMed

    Murach, Kevin A; Bagley, James R; McLeland, Kathryn A; Arevalo, Jose A; Ciccone, Anthony B; Malyszek, Kylie K; Wen, Yuan; Galpin, Andrew J

    2016-04-01

    Single muscle fiber sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE) is a sensitive technique for determining skeletal muscle myosin heavy chain (MHC) composition of human biopsy samples. However, the number of fibers suitable to represent fiber type distribution via this method is undefined. Muscle biopsies were obtained from the vastus lateralis (VL) of nine resistance-trained males (25 ± 1 year, height = 179 ± 5 cm, mass = 82 ± 8 kg). Single fiber MHC composition was determined via SDS-PAGE. VL fiber type distribution [percent MHC I, I/IIa, IIa, IIa/IIx, and total "hybrids" (i.e. I/IIa + IIa/IIx)] was evaluated according to number of fibers analyzed per person (25 vs. 125). VL fiber type distribution did not differ according to number of fibers analyzed (P > 0.05). VL biopsy fiber type distribution of nine subjects is represented by analyzing 25 fibers per person. These data may help minimize cost, personnel-time, and materials associated with this technique, thereby improving fiber typing efficiency in humans. PMID:26842420

  5. Myosin rods are a source of second harmonic generation signals in skeletal muscle

    NASA Astrophysics Data System (ADS)

    Schürmann, Sebastian; Weber, Cornelia; Fink, Rainer H. A.; Vogel, Martin

    2007-02-01

    Intrinsic second harmonic generation (SHG) signals can be used to visualize the three-dimensional structure of cardiac and skeletal muscle with high spatial resolution. Fluorescence labeling of complementary sarcomeric proteins, e.g. actin, indicates that the observed SHG signals arise from the myosin filaments. Recently, the myosin rod domain or LMM - light meromyosin - has been reported to be the dominant source of this SHG signal. However, to date, mostly negative and indirect evidence has been presented to support this assumption. Here, we show, to our knowledge, the first direct evidences that strong SHG signals can be obtained from synthetic paracrystals. These rod shaped filaments are formed from purified LMM. SDS-PAGE protein analysis confirmed that the LMM crystals lack myosin head domains. Some regions of the LMM paracrystals produce a strong SHG signal whereas others did not. The SHG signals were recorded with a laser-scanning microscope (Leica SP2). A ps laser tuned to 880 nm was used to excite the sample through an 63x objective of 1.2 NA. In order to visualize the synthetic filaments - in addition to SHG imaging -, the LMM was labeled with the fluorescent marker 5-IAF. We were able to observe filaments of 1 to 50 μm in length and of up to 5 μm in diameter. In conclusion, we can show that the myosin rod domain (LMM) is a dominant source for intrinsic SHG signals. There seems, however, a signal dependence on the paracrystals' morphology. This dependence is being investigated.

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2015-08-15

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

  8. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    PubMed

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS.

  9. Effect of spaceflight on skeletal muscle: Mechanical properties and myosin isoform content of a slow muscle

    NASA Technical Reports Server (NTRS)

    Caiozzo, Vincent J.; Baker, Michael J.; Herrick, Robert E.; Tao, Ming; Baldwin, Kenneth M.

    1994-01-01

    This study examined changes in contractile, biochemical, and histochemical properties of slow antigravity skeletal muscle after a 6-day spaceflight mission. Twelve male Sprague-Dawley rats were randomly divided into two groups: flight and ground-based control. Approximately 3 h after the landing, in situ contractile measurements were made on the soleus muscles of the flight animals. The control animals were studied 24 h later. The contractile measurements included force-velocity relationship, force-frequency relationship, and fatigability. Biochemical measurements focused on the myosin heavy chain (MHC) and myosin light chain profiles. Adenosinetriphosphatase histochemistry was performed to identify cross-sectional area of slow and fast muscle fibers and to determine the percent fiber type distribution. The force-velocity relationships of the flight muscles were altered such that maximal isometric tension P(sub o) was decreased by 24% and maximal shortening velocity was increased by 14% (P less than 0.05). The force-frequency relationship of the flight muscles was shifted to the right of the control muscles. At the end of the 2-min fatigue test, the flight muscles generated only 34% of P(sub o), whereas the control muscles generated 64% of P(sub o). The flight muscles exhibited de novo expression of the type IIx MHC isoform as well as a slight decrease in the slow type I and fast type IIa MHC isoforms. Histochemical analyses of flight muscles demonstrated a small increase in the percentage of fast type II fibers and a greater atrophy of the slow type I fibers. The results demonstrate that contractile properties of slow antigravity skeletal muscle are sensitive to the microgravity environment and that changes begin to occur within the 1st wk. These changes were at least, in part, associated with changes in the amount and type of contractile protein expressed.

  10. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2011-01-01

    Summary To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin. PMID:19361417

  11. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  12. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

    PubMed

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-10-10

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

  13. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    PubMed

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  14. Cardiac Myosin Binding Protein-C Plays No Regulatory Role in Skeletal Muscle Structure and Function

    PubMed Central

    Lin, Brian; Govindan, Suresh; Lee, Kyounghwan; Zhao, Piming; Han, Renzhi; Runte, K. Elisabeth; Craig, Roger; Palmer, Bradley M.; Sadayappan, Sakthivel

    2013-01-01

    Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C(t/t)). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C(t/t) mice. Slow and fast skeletal muscles in cMyBP-C(t/t) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C(t/t) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C. PMID:23936073

  15. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

    PubMed

    Linari, Marco; Brunello, Elisabetta; Reconditi, Massimo; Fusi, Luca; Caremani, Marco; Narayanan, Theyencheri; Piazzesi, Gabriella; Lombardi, Vincenzo; Irving, Malcolm

    2015-12-10

    Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart. PMID:26560032

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

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

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

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

    PubMed

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

    2015-04-24

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

  18. Characteristics of light chains of Chara myosin revealed by immunological investigation.

    PubMed

    Kakei, Toshihito; Sumiyoshi, Hiroki; Higashi-Fujime, Sugie

    2012-01-01

    Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca⁺⁺-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively.

  19. Electric birefringence study of rabbit skeletal myosin subfragments HMM, LMM, and rod in solution.

    PubMed Central

    Cardinaud, R; Bernengo, J C

    1985-01-01

    Electric birefringence measurements and depolarized light scattering experiments were performed with HMM, LMM, and rod, the three fragments of myosin, under conditions (0.3 M KCl, 0.02 M PO4, pH 7.3) the medium currently used for biochemical assays of myosin in its native state as well as of its subfragments. The comparison of myosin and rod relaxation times (17.2 and 22.8 microseconds, respectively) suggests that the average bend angle in the tail is sharper in intact myosin (90 degrees) whereas rod, when detached from the heads, is a more elongated species with an average bend angle of 120-135 degrees. The LMM relaxation time (6.4 microseconds) is consistent with a rigid linear stick model of length 78 nm. Flexibility in myosin tail is thus confirmed as located in the HMM-LMM hinge. LMM and rod did not exhibit any significant variation of their apparent relaxation times with concentration and the decay curves were best fitted by a single exponential, evidence that the concentration of parallel staggered dimers was negligible in the concentration range studied here (0-7 g/l). This observation lends support to previous results obtained with myosin. Respective HMM, LMM, and rod molecular weights and homogeneity as evaluated by SDS-PAGE analysis were correlated to the Kerr constants of their solutions. Large variations in LMM Kerr constants could be related to the loss of a COOH-terminal peptide on prolonged chymotryptic digestion. Electric birefringence combined with depolarized light scattering is presented as a potential method for net charge distribution studies. Images FIGURE 1 PMID:4074835

  20. Functions of Myosin Light Chain-2 (MYL2) In Cardiac Muscle and Disease

    PubMed Central

    Sheikh, Farah; Lyon, Robert C.; Chen, Ju

    2015-01-01

    Myosin light chain-2 (MYL2, also called MLC-2) is an ∼19 kDa sarcomeric protein that belongs to the EF-hand calcium binding protein superfamily and exists as three major isoforms encoded by three distinct genes in mammalian striated muscle. Each of the three different MLC-2 genes (MLC-2f; fast twitch skeletal isoform, MLC-2v; cardiac ventricular and slow twitch skeletal isoform, MLC-2a; cardiac atrial isoform) has a distinct developmental expression pattern in mammals. Genetic loss-of-function studies in mice demonstrated an essential role for cardiac isoforms of MLC-2, MLC-2v and MLC-2a, in cardiac contractile function during early embryogenesis. In the adult heart, MLC-2v function is regulated by phosphorylation, which displays a specific expression pattern (high in epicardium and low in endocardium) across the heart. These data along with new data from computational models, genetic mouse models, and human studies have revealed a direct role for MLC-2v phosphorylation in cross-bridge cycling kinetics, calcium-dependent cardiac muscle contraction, cardiac torsion, cardiac function and various cardiac diseases. This review focuses on the regulatory functions of MLC-2 in the embryonic and adult heart, with an emphasis on phosphorylation-driven actions of MLC-2v in adult cardiac muscle, which provide new insights into mechanisms regulating myosin cycling kinetics and human cardiac diseases. PMID:26074085

  1. Slow skeletal muscle myosin-binding protein-C (MyBPC1) mediates recruitment of muscle-type creatine kinase (CK) to myosin.

    PubMed

    Chen, Zhe; Zhao, Tong-Jin; Li, Jie; Gao, Yan-Song; Meng, Fan-Guo; Yan, Yong-Bin; Zhou, Hai-Meng

    2011-06-01

    Muscle contraction requires high energy fluxes, which are supplied by MM-CK (muscle-type creatine kinase) which couples to the myofibril. However, little is known about the detailed molecular mechanisms of how MM-CK participates in and is regulated during muscle contraction. In the present study, MM-CK is found to physically interact with the slow skeletal muscle-type MyBPC1 (myosin-binding protein C1). The interaction between MyBPC1 and MM-CK depended on the creatine concentration in a dose-dependent manner, but not on ATP, ADP or phosphocreatine. The MyBPC1-CK interaction favoured acidic conditions, and the two molecules dissociated at above pH 7.5. Domain-mapping experiments indicated that MM-CK binds to the C-terminal domains of MyBPC1, which is also the binding site of myosin. The functional coupling of myosin, MyBPC1 and MM-CK is further corroborated using an ATPase activity assay in which ATP expenditure accelerates upon the association of the three proteins, and the apparent K(m) value of myosin is therefore reduced. The results of the present study suggest that MyBPC1 acts as an adaptor to connect the ATP consumer (myosin) and the regenerator (MM-CK) for efficient energy metabolism and homoeostasis. PMID:21426302

  2. Thirteen is enough: the myosins of Dictyostelium discoideum and their light chains

    PubMed Central

    Kollmar, Martin

    2006-01-01

    Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree

  3. Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.

    PubMed

    Chantler, P D; Tao, T

    1986-11-01

    Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified either with 1,5-IAEDANS (N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine), as energy transfer donor, or with IAF (5-(iodoacetamido)fluorescein) or DABMI (4-dimethylaminophenylazophenyl-4'-maleimide), as energy transfer acceptor. The sulfhydryl groups on these light chains are located at different positions within the regulatory light-chain primary sequence; this enables one to probe a variety of locations, with respect to regulatory light-chain topology, on each myosin head. These independently modified regulatory light chains were added back to desensitized scallop myosin under a variety of conditions, including biphasic re-addition, the aim being to maximize the number of interhead energy transfer couples present. The efficiency of energy transfer was determined on the same samples by both steady-state and time-decay techniques. Results obtained by these two techniques were in good agreement with each other and indicated that the efficiency of energy transfer did not exceed 20% in any of the hybrids studied. Transfer efficiencies were invariant, irrespective of the presence or absence of MgATP, calcium or actin, either separately or in combination. Results using heavy meromyosin at low ionic strength were identical. It is shown that these results, in conjunction with the results of recent crosslinking studies performed on comparable myosin hybrids, may place certain restrictions on the configurations of the two heads of myosin.

  4. The importance of subfragment 2 and C-terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells.

    PubMed

    Ojima, Koichi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Muroya, Susumu; Chikuni, Koichi; Hattori, Akihito; Nishimura, Takanori

    2015-04-01

    In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.

  5. [The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin].

    PubMed

    Vorovikov, Iu S; Szczesna, D; Kakol, I

    1989-06-01

    The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.

  6. A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

    PubMed Central

    Wu, X; Blank, P S; Carlson, F D

    1992-01-01

    We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments. PMID:1420864

  7. Indirect myosin immunocytochemistry for the identification of fibre types in equine skeletal muscle

    NASA Technical Reports Server (NTRS)

    Sinha, A. K.; Rose, R. J.; Pozgaj, I.; Hoh, J. F.

    1992-01-01

    The histochemical ATPase method for muscle fibre typing was first described by Brooke and Kaiser in 1970. However, problems have been found with the subdivision of type II fibres using this technique. To determine whether indirect myosin immunocytochemistry using anti-slow (5-4D), anti-fast (1A10) and anti-fast red (5-2B) monoclonal antibodies with cross reactivity for type I, II and IIa fibres, respectively, in a number of species, could identify three fibre types in equine skeletal muscle, data on fibre type composition and fibre size obtained using the two different techniques were compared. Results indicate that different myosin heavy chains can coexist in single equine muscle fibres. Type I and type II fibres were identified by immunocytochemistry, but subdivision of type II fibres was not possible. Although the percentage of type I and type II fibres was not significantly different for the two techniques, a few fibres reacted with both the 1A10 and 5-4D antibodies.

  8. Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation.

    PubMed

    Dhawan, Jyotsna; Helfman, David M

    2004-08-01

    Cell-substratum interactions trigger key signaling pathways that modulate growth control and tissue-specific gene expression. We have previously shown that abolishing adhesive interactions by suspension culture results in G(0) arrest of myoblasts. We report that blocking intracellular transmission of adhesion-dependent signals in adherent cells mimics the absence of adhesive contacts. We investigated the effects of pharmacological inhibitors of acto-myosin contractility on growth and differentiation of C2C12 myogenic cells. ML7 (5-iodonaphthalene-1-sulfonyl homopiperazine) and BDM (2,3, butanedione monoxime) are specific inhibitors of myosin light chain kinase, and myosin heavy chain ATPase, respectively. ML7 and BDM affected cell shape by reducing focal adhesions and stress fibers. Both inhibitors rapidly blocked DNA synthesis in a dose-dependent, reversible fashion. Furthermore, both ML7 and BDM suppressed expression of MyoD and myogenin, induced p27(kip1) but not p21(cip1), and inhibited differentiation. Thus, as with suspension-arrest, inhibition of acto-myosin contractility in adherent cells led to arrest uncoupled from differentiation. Over-expression of inhibitors of the small GTPase RhoA (dominant negative RhoA and C3 transferase) mimicked the effects of myosin inhibitors. By contrast, wild-type RhoA induced arrest, maintained MyoD and activated myogenin and p21 expression. The Rho effector kinase ROCK did not appear to mediate Rho's effects on MyoD. Thus, ROCK and MLCK play different roles in the myogenic program. Signals regulated by MLCK are critical, since inhibition of MLCK suppressed MyoD expression but inhibition of ROCK did not. Inhibition of contractility suppressed MyoD but did not reduce actin polymer levels. However, actin depolymerization with latrunculin B inhibited MyoD expression. Taken together, our observations indicate that actin polymer status and contractility regulate MyoD expression. We suggest that in myoblasts, the Rho pathway and

  9. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  10. Dictyostelium discoideum myosin: Isolation and characterization of cDNAs encoding the essential light chain

    SciTech Connect

    Chisholm, R.L.; Rushforth, A.M.; Pollenz, R.S.; Kuczmarski, E.R.; Tafuri, S.R.

    1988-02-01

    The authors used an antibody specific for Dictyostelium discoideum myosin to screen a lambdagt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca/sup 2 +/-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

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

    PubMed Central

    1992-01-01

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

  12. Expression of developmental myosin and morphological characteristics in adult rat skeletal muscle following exercise-induced injury.

    PubMed

    Smith, H K; Plyley, M J; Rodgers, C D; McKee, N H

    1999-07-01

    The extent and stability of the expression of developmental isoforms of myosin heavy chain (MHCd), and their association with cellular morphology, were determined in adult rat skeletal muscle fibres following injury induced by eccentrically-biased exercise. Adult female Wistar rats [274 (10) g] were either assigned as non-exercised controls or subjected to 30 min of treadmill exercise (grade, -16 degrees; speed, 15 m x min(-1)), and then sacrificed following 1, 2, 4, 7, or 12 days of recovery (n = 5-6 per group). Histologically and immunohistologically stained serial, transverse cryosections of the soleus (S), vastus intermedius (VI), and tibialis anterior (TA) muscles were examined using light microscopy and digital imaging. Fibres staining positively for MHCd (MHCd+) were seldom detected in the TA. In the VI and S, higher proportions of MHCd+ fibres (0.8% and 2.5%, respectively) were observed in rats at 4 and 7 days post-exercise, in comparison to all other groups combined (0.2%, 1.2%; P < or = 0.01). In S, MHCd+ fibres were observed less frequently by 12 days (0.7%) than at 7 days (2.6%) following exercise. The majority (85.1%) of the MHCd+ fibres had morphological characteristics indicative of either damage, degeneration, repair or regeneration. Most of the MHCd+ fibres also expressed adult slow, and/or fast myosin heavy chain. Quantitatively, the MHCd+ fibres were smaller (< 2500 microm2) and more angular than fibres not expressing MHCd. Thus, there was a transient increase in a small, but distinct population of MHCd+ fibres following unaccustomed, functional exercise in adult rat S and VI muscles. The observed close coupling of MHCd expression with morphological changes within muscle fibres suggests that these characteristics have a common, initial exercise-induced injury-related stimulus.

  13. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

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

  14. Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Adams, Gregory R.; Baldwin, Kenneth M.

    1995-01-01

    This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid (creatine depletion juvenile (CDJ) group). Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens (normal diet adult and creatine depletion adult (CDA) groups). After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC ditribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC tranitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of the animals to be used.

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

    NASA Astrophysics Data System (ADS)

    Bandman, Everett

    1985-02-01

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

  16. Regulatory Light Chain Mutations Associated with Cardiomyopathy Affect Myosin Mechanics and Kinetics

    PubMed Central

    Greenberg, Michael J.; Watt, James D.; Jones, Michelle; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2009-01-01

    The myosin regulatory light chain (RLC) wraps around the alpha helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K showed reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation. PMID:18929571

  17. Regulatory and structural motifs of chicken gizzard myosin light chain kinase.

    PubMed Central

    Olson, N J; Pearson, R B; Needleman, D S; Hurwitz, M Y; Kemp, B E; Means, A R

    1990-01-01

    The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed definition of both the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain. PMID:2315320

  18. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

    PubMed

    Hong, Feng; Brizendine, Richard K; Carter, Michael S; Alcala, Diego B; Brown, Avery E; Chattin, Amy M; Haldeman, Brian D; Walsh, Michael P; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2015-10-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

  19. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

    PubMed Central

    Hong, Feng; Brizendine, Richard K.; Carter, Michael S.; Alcala, Diego B.; Brown, Avery E.; Chattin, Amy M.; Haldeman, Brian D.; Walsh, Michael P.; Facemyer, Kevin C.; Baker, Josh E.

    2015-01-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. PMID:26415568

  20. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts.

  1. Structure of the light chain-binding domain of myosin V

    PubMed Central

    Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C.; Grabarek, Zenon; Dominguez, Roberto

    2005-01-01

    Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM–IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein–protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. PMID:16120677

  2. Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.

    1993-01-01

    This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

  3. Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

    PubMed Central

    Štrbenc, Malan; Smerdu, Vika; Pogačnik, Azra; Fazarinc, Gregor

    2006-01-01

    To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (α-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4–6 weeks of age, which coincides with the increased physical activity of puppies. PMID:16879596

  4. Absence of the functional Myosin heavy chain 2b isoform in equine skeletal muscles.

    PubMed

    Chikuni, Koichi; Muroya, Susumu; Nakajima, Ikuyo

    2004-05-01

    Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3' untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.

  5. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  6. An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

    PubMed Central

    Elangovan, R; Capitanio, M; Melli, L; Pavone, F S; Lombardi, V; Piazzesi, G

    2012-01-01

    A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4–30°C) and substrate concentration. VF was 8.88 ± 0.26 μm s−1 at 30.6°C and decreased to 1.60 ± 0.09 μm s−1 at 4.5°C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40–50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5°C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads. PMID:22199170

  7. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    SciTech Connect

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony; Bernstein, Sanford I.

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  8. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  9. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-18

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

  12. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

    PubMed Central

    Bottinelli, R; Canepari, M; Pellegrino, M A; Reggiani, C

    1996-01-01

    1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C. Images Figure 1 Figure 3 Figure 6 PMID:8887767

  13. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

    PubMed Central

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D.

    2015-01-01

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  14. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  15. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    PubMed

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

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

    PubMed Central

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

    1989-01-01

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

  17. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  18. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  19. Cardiac and skeletal myopathy in beta myosin heavy-chain simian virus 40 tsA58 transgenic mice.

    PubMed Central

    De Leon, J R; Federoff, H J; Dickson, D W; Vikstrom, K L; Fishman, G I

    1994-01-01

    The mechanisms regulating cardiac muscle differentiation and development are incompletely understood. To examine the relationships between cardiocyte proliferation and differentiation, we tested the ability of a fragment from the rat beta myosin heavy-chain (MHC beta) gene to correctly target expression of a thermolabile simian virus 40 large tumor antigen allele (tsA58) in the developing mouse. Transgene expression in the heart was observed as early as 10 days postconception and was developmentally regulated in parallel with the endogenous MHC beta gene. Expression was also detected in developing skeletal muscle, although at low levels. Despite the temperature sensitivity of the mutant large tumor antigen protein, a subset of transgenic mice in several lineages developed marked cardiac and skeletal myopathies. Images Fig. 2 Fig. 3 Fig. 4 PMID:8290557

  20. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

    PubMed

    Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I; Geeves, Michael A

    2016-01-22

    The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

  1. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    PubMed Central

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2009-01-01

    SUMMARY The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ~9 nm distance between the myosin lever arm and the thin filament. In order to localize the N-terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle. PMID:17597155

  2. Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn

    PubMed Central

    Griffin, J.; St-Pierre, N.; Lilburn, M. S.; Wick, M.

    2016-01-01

    There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development. PMID:26908894

  3. Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states.

    PubMed

    Squire, J M; Harford, J J

    1988-08-01

    The structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200A) and azimuthally (total range less than 60 degrees) from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 A) or an azimuthal range of actin binding sites of about 100 degrees around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a 'weak binding' state, then the predicted X-ray diffraction pattern would comprise layer lines at the same axial spacings (orders of 429 A) as those seen in patterns from resting muscle. It is shown that actin target areas in vertebrate skeletal muscles are probably arranged on an approximate 62 (right-handed) helix of pitch (P) of about 720 A, subunit translation P/6 and near repeat P/2. Troponin position need not be considered in defining the labelling pattern of cross-bridges on this 62 helix of target areas; the target areas appear to be defined solely by the azimuthal position of the actin binding sites. The distribution of actin filament labelling patterns could be regular in fish muscle which has a 'crystalline' A-band, but will be irregular in higher vertebrate muscles such as frog sartorius muscle.

  4. [Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber].

    PubMed

    Levistkiĭ, D I; Borovikov, Iu S; Nikolaeva, O P; Golitsyna, N L; Poglazov, B F

    1990-09-01

    At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).

  5. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  6. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.

    PubMed

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

    2010-01-15

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

  7. Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

  8. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  9. Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

    PubMed

    Trus, B L; Steven, A C; McDowall, A W; Unser, M; Dubochet, J; Podolsky, R J

    1989-04-01

    For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

  10. Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

    PubMed Central

    Oshima, Kanji; Sugimoto, Yasunobu; Irving, Thomas C.; Wakabayashi, Katsuzo

    2012-01-01

    The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions

  11. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-01

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

  12. Conformational changes of myosin by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Lee, Ju.-Woon; Yook, Hong.-Sun; Lee, Kyong.-Haeng; Kim, Jae.-Hun; Kim, Woo.-Jung; Byun, Myung.-Woo

    2000-05-01

    Conformational and decompositional changes of bovine skeletal muscle myosin caused by gamma irradiation were studied for understanding the effects of irradiation treatment on myofibrillar proteins. Myosin solution and beef cuts were irradiated 0, 1, 3, 5 and 10 kGy. Competitive indirect enzyme linked immunosorbent assay (Ci-ELISA) showed that subunits of myosin were structurally modified with different patterns. Binding abilities of anti-myosin whole molecule and anti heavy meromyosin S-1 IgG, which were produced from rabbits, with irradiated myosin decreased in the same tendency depending upon the dose. Anti-light meromyosin IgG appeared to have the highest binding ability at 3 kGy. Irradiated beef cuts (≥5 kGy) could be identified by Ci-ELISA. Myosin solution became increasingly turbid with increasing dose. Hydrophobicity of myosin solution also increased by irradiation. Electrophoretic patterns showed that the myosin heavy chain disappeared and new bands were generated at higher molecular weight ranges.

  13. Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

    PubMed

    Burghardt, Thomas P; Sikkink, Laura A

    2013-02-19

    Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

  14. Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Chu, Wuying; Fu, Guihong; Bing, Shiyu; Meng, Tao; Zhou, Ruixue; Cheng, Jia; Zhao, Falan; Zhang, Hongfang; Zhang, Jianshe

    2010-03-01

    The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%-76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%-80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

  15. PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization.

    PubMed

    El-Yazbi, Ahmed F; Abd-Elrahman, Khaled S; Moreno-Dominguez, Alejandro

    2015-06-15

    Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries. PMID:25931148

  16. Impact of resistance exercise during bed rest on skeletal muscle sarcopenia and myosin isoform distribution

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Feeback, D. L.; Talmadge, R. J.; Stevens, B. R.; Lieberman, S. A.; Greenisen, M. C.

    1998-01-01

    Because resistance exercise (REx) and bed-rest unloading (BRU) are associated with opposing adaptations, our purpose was to test the efficacy of REx against the effects of 14 days of BRU on the knee-extensor muscle group. Sixteen healthy men were randomly assigned to no exercise (NoEx; n = 8) or REx (n = 8). REx performed five sets of leg press exercise with 80-85% of one repetition maximum (1 RM) every other day during BRU. Muscle samples were removed from the vastus lateralis muscle by percutaneous needle biopsy. Myofiber distribution was determined immunohistochemically with three monoclonal antibodies against myosin heavy chain (MHC) isoforms (I, IIa, IIx). MHC distribution was further assessed by quantitative gel electrophoresis. Dynamic 1-RM leg press and unilateral maximum voluntary isometric contraction (MVC) were determined. Maximal neural activation (root mean squared electromyogram) and rate of torque development (RTD) were measured during MVC. Reductions (P < 0.05) in type I (15%) and type II (17%) myofiber cross-sectional areas were found in NoEx but not in REx. Electrophoresis revealed no changes in MHC isoform distribution. The percentage of type IIx myofibers decreased (P < 0.05) in REx from 9 to 2% and did not change in NoEx. 1 RM was reduced (P < 0.05) by 9% in NoEx but was unchanged in REx. MVC fell by 15 and 13% in NoEx and REx, respectively. The agonist-to-antagonist root mean squared electromyogram ratio decreased (P < 0.05) 19% in REx. RTD slowed (P < 0.05) by 54% in NoEx only. Results indicate that REx prevented BRU-induced myofiber atrophy and also maintained training-specific strength. Unlike spaceflight, BRU did not induce shifts in myosin phenotype. The reported benefits of REx may prove useful in prescribing exercise for astronauts in microgravity.

  17. Identification of myosin heavy chain I, IIa and IIx in canine skeletal muscles by an electrophoretic and immunoblotting study.

    PubMed

    Smerdu, V; Strbenc, M; Meznaric-Petrusa, M; Fazarinc, G

    2005-01-01

    To determine which myosin heavy chain (MHC) isoforms are expressed in canine skeletal muscles, different muscle samples of five mixed-breed dogs were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated MHC isoforms were identified by immunoblotting technique using a set of specific monoclonal antibodies. To compare the results of the electrophoretic and immunoblotting study, the pattern of MHC isoform expression and histochemical profiles of canine fibres were additionally demonstrated on serial muscle sections by immunohistochemistry and myofibrillar adenosine triphosphatase (mATPase) histochemistry. Not more than three MHC isoforms were demonstrated by SDS-PAGE in the analysed canine muscles. By the immunoblotting technique, the fastest migrating MHC band was identified as slow or MHC-I, the intermediate one as MHC-IIx and the slowest migrating band as MHC-IIa isoform. Since none of the three MHC bands and none of the analysed fibres were recognized by the antibody specific to MHC-IIb of rats, we concluded that MHC-IIb is not expressed in large skeletal muscles of dogs. Similarly, only three major fibre types, i.e. I, IIA and IIX, were revealed according to the pattern of MHC immunohistochemistry and mATPase reaction. Type IIA fibres were more alkali- and acid-stable than type IIX fibres after mATPase histochemistry; hence, the latter corresponded to type IIDog fibres. However, beside the three major fibre types, scarce hybrid fibres co-expressing two MHC isoforms (I/IIA and IIA/IIX) were demonstrated by immunohistochemistry.

  18. Dynamic nature of fibre-type specific expression of myosin heavy chain transcripts in 14 different human skeletal muscles.

    PubMed

    Smerdu, V; Erzen, I

    2001-01-01

    The main goal of this study was to find out, whether the appearance of fibres without evident myosin heavy chain (MyHC) transcript expression (negative fibres) implies the existence of additional MyHC transcripts in human muscle fibres. Fourteen different skeletal muscles were analysed also to verify how MyHC transcript expression matches histochemical phenotypes of fibres. For this purpose, the expression of beta-slow, 2a and 2x MyHC transcripts, demonstrated by in situ hybridisation technique, was analysed within type I, IIC, IIA, IIAX and IIX fibres, determined according to the activity of myofibrillar ATPase. Additionally, MyHC isoform expression was immunohistochemically demonstrated and metabolic profiles of negative fibres were estimated. From a total of 4444 muscle fibres analysed, only 0.8% of fibres were negative, among them type I prevailed, the remainder were type IIA and IIX fibres. The majority of fibres expressed only beta, 2a and 2x MyHC transcripts and they mostly matched type I, IIA and IIX fibres respectively, but two minor hybrid fibre groups (beta/2a and 2ax) exhibited variable histochemical phenotype. The infrequency, the prevailing oxidative-glycolytic metabolic profile of negative type I fibres and frequent co-appearance with transitional type IIC fibres imply that the negative fibres rather result from fibre type transition than express an additional slow or even 2b MyHC transcripts. The appearance of hybrid and mismatched fibres additionally indicates that fibre type transition occurs also in presumably normal skeletal muscles, what enables the muscles to tune even with minimal changes in mechanical demands.

  19. Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis

    PubMed Central

    Jeong, Jin-Yeon; Jung, Eun-Young; Jeong, Tae-Chul; Yang, Han-Sul; Kim, Gap-Don

    2016-01-01

    The purpose of this study was to analyze oxidized methionines in the myosin isoforms of porcine longissimus thoracis, psoas major, and semimembranosus muscles by liquid chromatography (LC) and mass spectrometry (MS). A total of 836 queries matched to four myosin isoforms (myosin-1, -2, -4, and -7) were analyzed and each myosin isoform was identified by its unique peptides (7.3-13.3). Forty-four peptides were observed from all three muscles. Seventeen peptides were unique to the myosin isoform and the others were common peptides expressed in two or more myosin isoforms. Five were identified as oxidized peptides with one or two methionine sulfoxides with 16 amu of mass modification. Methionines on residues 215 (215), 438 (438), 853 (851), 856 (854), 1071 (1069), and 1106 (1104) of myosin-1 (myosin-4) were oxidized by the addition of oxygen. Myosin-2 had two oxidized methionines on residues 215 and 438. No queries matched to myosin-7 were observed as oxidized peptides. LC-MS/MS allows analysis of the oxidation of specific amino acids on specific residue sites, as well as in specific proteins in the food system. PMID:27194935

  20. Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

    PubMed

    Kuang, Liangde; Xie, Xiaohong; Zhang, Xiangyu; Lei, Min; Li, Congyan; Ren, Yongjun; Zheng, Jie; Guo, Zhiqiang; Zhang, Cuixia; Yang, Chao; Zheng, Yucai

    2014-01-01

    The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

  1. Myosin light chain kinase-dependent microvascular hyperpermeability in thermal injury.

    PubMed

    Huang, Qiaobing; Xu, Wenjuan; Ustinova, Elena; Wu, Mack; Childs, Ed; Hunter, Felicia; Yuan, Sarah

    2003-10-01

    Although the critical role of systemic inflammatory edema in the development of multiple organ failure in patients with massive burns has been fully recognized, the precise mechanisms responsible for the accumulation of blood fluid and proteins in tissues remote from the burn wound are poorly understood. The aim of this study was to test the hypothesis that circulating factors released during thermal injury cause microvascular leakage by triggering endothelial cell contraction and barrier dysfunction. A third-degree scald burn was induced in rats on the dorsal skin covering 25% total body surface area. The microcirculation and transvascular flux of albumin were observed in the rat mesentery using intravital fluorescence microscopy. The direct effect of circulating factors on microvascular barrier function was assessed by measuring the apparent permeability coefficient of albumin in isolated rat mesenteric venules during perfusion of plasma freshly withdrawn from burned rats. The in vivo study showed that the transvenular flux of albumin was significantly increased over a 6-h period with a maximal response seen at 3 h postburn. Importantly, perfusion of noninjured venules with burn plasma induced a time-dependent increase in albumin permeability. Pharmacological inhibition of protein kinase C, Src tyrosine kinases, or mast cell activation did not significantly affect the hyperpermeability response; however, blockage of myosin light chain phosphorylation with the myosin light chain kinase inhibitor ML-7 greatly attenuated the burn-induced increase in venular permeability in a dose-related pattern. The results support a role for endogenous circulating factors in microvascular leakage during burns. Myosin light chain phosphorylation-dependent endothelial contractile response may serve as an end-point effector leading to microvascular barrier dysfunction. PMID:14501951

  2. EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

    PubMed

    Huang, W; Wilson, G J; Brown, L J; Lam, H; Hambly, B D

    1998-10-15

    The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

  3. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  4. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

  5. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction.

    PubMed

    Stroka, Kimberly M; Aranda-Espinoza, Helim

    2011-08-11

    A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness. PMID:21652678

  6. 3-D structural analysis of the crucial intermediate of skeletal muscle myosin and its role in revised actomyosin cross-bridge cycle

    PubMed Central

    Katayama, Eisaku

    2014-01-01

    Skeletal myosin S1 consists of two functional segments, a catalytic-domain and a lever-arm. Since the crystal structure of ADP/Vi-bound S1 exhibits a strong intramolecular flexure between two segments, inter-conversion between bent and extended forms; i.e. “tilting of the lever-arm” has been accepted as the established molecular mechanism of skeletal muscle contraction. We utilized quick-freeze deep-etch replica electron microscopy to directly visualize the structure of in vitro actin-sliding myosin, and found the existence of a novel oppositely-bent configuration, instead of the expected ADP/Vi-bound form. We also noticed that SH1–SH2 cross-linked myosin gives an aberrant appearance similar to the above structure. Since SH1–SH2-cross-linked myosin is a well-studied analogue of the transient intermediate of the actomyosin cross-bridge cycle, we devised a new image-processing procedure to define the relative view-angles between the catalytic-domain and the lever-arm from those averaged images, and built a 3-D model of the new conformer. The lever-arm in that model was bent oppositely to the ADP/Vi-bound form, in accordance with observed actin-sliding cross-bridge structure. Introducing this conformer as the crucial intermediate that transiently appears during sliding, we propose a revised scheme of the cross-bridge cycle. In the scenario, the novel conformer keeps actin-binding in two different modes until it forms a primed configuration. The final extension of the lever-arm back to the original rigor-state constitutes the “power-stroke”. Various images observed during sliding could be easily interpreted by the new conformer. Even the enigmatic behavior of the cross-bridges reported as “loose chemo-mechanical coupling” might be adequately explained under some assumptions. PMID:27493503

  7. Coordinated expression of myosin heavy chains, metabolic enzymes, and morphological features of porcine skeletal muscle fiber types.

    PubMed

    Quiroz-Rothe, Eugenio; Rivero, José-Luis L

    2004-09-01

    Combined methodologies of electrophoresis, immunoblots, immunohistochemistry, histochemistry, and photometric image analysis were applied to characterize porcine skeletal muscle fibers according to their myosin heavy chain (MyHC) composition, and to determine on a fiber-to-fiber basis the correlation between contractile [MyHC (s), myofibrillar ATPase (mATPase), and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [succinate dehydrogenase (SDH), and glycerol-3-phosphate dehydrogenase (GPDH) activities, glycogen, and phospholamban (PLB) contents], and morphological [cross-sectional area (CSA), capillary, and nuclear densities] features of individual myofibers. An accurate delineation of MyHC-based fiber types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibers with overwhelming dominance of one MyHC isoform. The phenotypic differences in contractile, metabolic, and morphological properties seen between fiber types were related with MyHC content. Slow fibers had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPDH activity (glycolytic metabolism), and glycogen content, the smallest CSA, the greatest capillary, and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IIB fibers, whereas type IIA and IIX fibers had intermediate properties. Hybrid fibers had mean values intermediate in-between their respective pure phenotypes. Discrimination of myofibers according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafiber interrelationships suggest that myofibers of control pigs exhibit a high degree of co-ordination in their physiological, biochemical, and anatomical features. This study may well be a useful baseline for future work on the pig meat

  8. Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

    PubMed

    Sellers, James R; Wang, Fei; Chantler, Peter D

    2003-01-01

    Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

  9. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  10. Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

    PubMed

    Zhang, Yong-Xia; Chen, Heng-Li; Maleki, Soheila J; Cao, Min-Jie; Zhang, Ling-Jing; Su, Wen-Jin; Liu, Guang-Ming

    2015-07-15

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

  11. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

    PubMed Central

    1988-01-01

    The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges. PMID:3216188

  12. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  13. An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle.

    PubMed

    Smith, D A; Stephenson, D G

    2011-06-01

    The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl(-) binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small. PMID:21641314

  14. Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis.

    PubMed

    Kondo, Tomo; Isoda, Rieko; Uchimura, Takashi; Sugiyama, Mutsumi; Hamao, Kozue; Hosoya, Hiroshi

    2012-01-13

    Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II. PMID:22166199

  15. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  16. Nemaline myopathy-related skeletal muscle α-actin (ACTA1) mutation, Asp286Gly, prevents proper strong myosin binding and triggers muscle weakness.

    PubMed

    Ochala, Julien; Ravenscroft, Gianina; Laing, Nigel G; Nowak, Kristen J

    2012-01-01

    Many mutations in the skeletal muscle α-actin gene (ACTA1) lead to muscle weakness and nemaline myopathy. Despite increasing clinical and scientific interest, the molecular and cellular pathogenesis of weakness remains unclear. Therefore, in the present study, we aimed at unraveling these mechanisms using muscles from a transgenic mouse model of nemaline myopathy expressing the ACTA1 Asp286Gly mutation. We recorded and analyzed the mechanics of membrane-permeabilized single muscle fibers. We also performed molecular energy state computations in the presence or absence of Asp286Gly. Results demonstrated that during contraction, the Asp286Gly acts as a "poison-protein" and according to the computational analysis it modifies the actin-actin interface. This phenomenon is likely to prevent proper myosin cross-bridge binding, limiting the fraction of actomyosin interactions in the strong binding state. At the cell level, this decreases the force-generating capacity, and, overall, induces muscle weakness. To counterbalance such negative events, future potential therapeutic strategies may focus on the inappropriate actin-actin interface or myosin binding. PMID:23029319

  17. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  18. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

  19. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

    PubMed Central

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-01-01

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  20. Effects of a Fluorescent Myosin Light Chain Phosphatase Inhibitor on Prostate Cancer Cells

    PubMed Central

    Grindrod, Scott; Suy, Simeng; Fallen, Shannon; Eto, Masumi; Toretsky, Jeffrey; Brown, Milton L.

    2011-01-01

    Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/32P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI50 values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. β-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype. PMID:22655237

  1. Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

    PubMed

    Hoar, P E; Kerrick, W G

    1988-08-01

    Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions. PMID:3186428

  2. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    PubMed

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  3. [The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent].

    PubMed

    Borovikov, Iu S; Szczesna, D; Khoroshev, M I; Kakol, I

    1990-01-01

    Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.

  4. Myosin: A Link between Streptococci and Heart

    NASA Astrophysics Data System (ADS)

    Krisher, Karen; Cunningham, Madeleine W.

    1985-01-01

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

  5. New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

    PubMed Central

    Taniguchi, Masaya; Okamoto, Ryuji; Ito, Masaaki; Goto, Itaru; Fujita, Satoshi; Konishi, Katsuhisa; Mizutani, Hideo; Dohi, Kaoru; Hartshorne, David J.; Itoh, Takeo

    2015-01-01

    Background & Aims Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated. Methods and Results By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%. Conclusions We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. PMID:26512720

  6. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  7. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    PubMed Central

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  8. Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles

    PubMed Central

    Ackermann, Maegen A.; Ward, Christopher W.; Gurnett, Christina; Kontrogianni-Konstantopoulos, Aikaterini

    2015-01-01

    Myosin Binding Protein-C slow (sMyBP-C), encoded by MYBPC1, comprises a family of regulatory proteins of skeletal muscles that are phosphorylated by PKA and PKC. MYBPC1 missense mutations are linked to the development of Distal Arthrogryposis-1 (DA-1). Although structure-function details for this myopathy are evolving, function is undoubtedly driven by sequence variations and post-translational modifications in sMyBP-C. Herein, we examined the phosphorylation profile of sMyBP-C in mouse and human fast-twitch skeletal muscles. We used Flexor Digitorum Brevis (FDB) isolated from young (~2-months old) and old (~14-months old) wild type and mdx mice, and human Abductor Hallucis (AH) and gastrocnemious muscles carrying the DA-1 mutations. Our results indicate both constitutive and differential phosphorylation of sMyBP-C in aged and diseased muscles. We report a 7–35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx FDB mouse muscles, compared to young wild type tissue. Similarly, we observe a 30–70% decrease in the phosphorylation levels of all PKA and PKC phospho-sites in the DA-1 AH, but not gastrocnemius, muscle. Overall, our studies show that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age and disease, suggesting that phosphorylation plays important roles in these processes. PMID:26287277

  9. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    SciTech Connect

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  10. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  11. [Role of phosphatidylinositol 3-kinase and myosin light chain kinase during the activation of thrombin receptors].

    PubMed

    Han, Yue; Gao, Hai-Li; Zhang, Wei; Bai, Xia; Dai, Lan; Sheng, Wen-Hong; Sun, Ai-Ning; Wu, De-Pei; Wang, Zhao-Yue; Ruan, Chang-Geng

    2009-06-01

    The objective of study was to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptor activation, and to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptor activation. Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet, and the changes of platelet aggregation and GPIb were analyzed with 100 nmol/L wortmannin (inhibitor of PI3-K) and 10 micromol/L wortmannin (inhibitor of MLCK). The results indicated that the platelet activation was influenced by either concentration of wortmannin in response to PAR stimulation. Platelet aggregation was apparently inhibited by 10 micromol/L wortmannin through both PAR peptides, and was slightly inhibited by 100 nmol/L wortmannin only under PAR1-AP activation. In addition, GPIbalpha internalization was partly inhibited by 100 nmol/L wortmannin in response to PAR1 (p < 0.05 at 1, 2, 5 min) and PAR4 (p < 0.05 at 2, 5, 10 min) activation. Meanwhile, 10 micromol/L wortmannin induced little change for GPIbalpha centralisation in the course of PAR activation, with a delayed restoration of surface GPIbalpha observed under PAR1-AP activation, and no change of GPIbalpha redistribution existed under PAR4-AP activation. It is concluded that the different roles of PI3-K and MLCK exist in the course of thrombin receptor activation. PI3-K accelerates the short course of GPIb centralisation for two PAR signal pathways, while MLCK inhibits the restoration of GPIbalpha in PAR1 pathway. PMID:19549383

  12. Myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle.

    PubMed

    Martinsen, A; Schakman, O; Yerna, X; Dessy, C; Morel, N

    2014-07-01

    The Ca(2+)-dependent kinase myosin light chain kinase (MLCK) is the activator of smooth muscle contraction. In addition, it has been reported to be involved in Ca(2+) channel regulation in cultured cells, and we previously showed that the MLCK inhibitor ML-7 decreases arginine vasopressin (AVP)-induced Ca(2+) influx in rat aorta. This study was designed to investigate whether MLCK is involved in Ca(2+) regulation in resistance artery smooth muscle cell, which plays a major role in the control of blood pressure. As ML compounds were shown to have off-target effects, MLCK was downregulated by transfection with a small interfering RNA targeting MLCK (MLCK-siRNA) in rat small resistance mesenteric artery (RMA) and in the rat embryonic aortic cell line A7r5. Noradrenaline-induced contraction and Ca(2+) signal were significantly depressed in MLCK-siRNA compared to scramble-siRNA-transfected RMA. Contraction and Ca(2+) signal induced by high KCl and voltage-activated Ca(2+) current were also significantly decreased in MLCK-siRNA-transfected RMA, suggesting that MLCK depletion modifies voltage-operated Ca(2+) channels. KCl- and AVP-induced Ca(2+) signals and voltage-activated Ca(2+) current were decreased in MLCK-depleted A7r5 cells. Eventually, real-time quantitative PCR analysis indicated that in A7r5, MLCK controlled mRNA expression of CaV1.2 (L-type) and CaV3.1 (T-type) voltage-dependent Ca(2+) channels. Our results suggest that MLCK controls the transcription of voltage-dependent Ca(2+) channels in vascular smooth muscle cells. PMID:24162233

  13. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  14. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  15. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  16. Tarantula Myosin Free Head Regulatory Light Chain Phosphorylation Stiffens N-terminal Extension Releasing it and Blocking its Docking Back

    PubMed Central

    Alamo, Lorenzo; Li, Xiaochuan (Edward); Espinoza-Fonseca, L. Michel; Pinto, Antonio; Thomas, David D.; Lehman, William; Padrón, Raúl

    2015-01-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence lengths analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft in the relaxed state. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  17. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    PubMed

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  18. Skeletal and cardiac α-actin isoforms differently modulate myosin cross-bridge formation and myofibre force production.

    PubMed

    Ochala, Julien; Iwamoto, Hiroyuki; Ravenscroft, Gianina; Laing, Nigel G; Nowak, Kristen J

    2013-11-01

    Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies. PMID:23784376

  19. Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase.

    PubMed

    Mamar-Bachi, A; Cox, J A

    1987-12-01

    Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.

  20. β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

    PubMed Central

    Simard, Elie; Kovacs, Jeffrey J.; Miller, William E.; Kim, Jihee; Grandbois, Michel; Lefkowitz, Robert J.

    2013-01-01

    Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility. PMID:24255721

  1. A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

    PubMed

    Facemyer, K C; Cremo, C R

    1992-01-01

    We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.

  2. Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding

    PubMed Central

    1987-01-01

    Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing. PMID:2826495

  3. Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation.

    PubMed

    Taverner, Alistair; Dondi, Ruggero; Almansour, Khaled; Laurent, Floriane; Owens, Siân-Eleri; Eggleston, Ian M; Fotaki, Nikoletta; Mrsny, Randall J

    2015-07-28

    The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein-protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3-4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal

  4. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    PubMed

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  5. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  6. Various Themes of Myosin Regulation.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2016-05-01

    Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

  7. 5'-flanking sequences of zebrafish fast myosin heavy chain genes regulate unique expression in the anterior, medial subsection and posterior tail somites of the skeletal muscle.

    PubMed

    Asaduzzaman, M; Shakur Ahammad, A K; Asakawa, S; Kinoshita, S; Watabe, S

    2016-01-01

    In zebrafish, fast muscle-specific myosin heavy chain genes have their unique expression patterns in a well-defined and restricted region of the skeletal muscle. However, the transcriptional regulatory mechanisms involved have remained unclear. Here, we examined the regulation of spatio-temporal expression patterns of myhz1 (myhz1.1, myhz1.2 and myhz1.3) and myhz2 during their development by using transient gene and stable transgenic techniques. Embryos microinjected with different length 5'-flanking sequences of myhz1 conjugated with the enhanced green fluorescent protein (EGFP) gene showed EGFP expression in the anterior and medial subsections of somites, but not in the tail somite region. In contrast, embryos microinjected with different length 5'-flanking sequences of myhz2 showed EGFP expression exclusively at the posterior tail somite domain. Promoter deletion analyses demonstrated that reduced EGFP fluorescence typically is correlated with smaller 5'-flanking sequences. The immunohistochemical observation revealed that zebrafish larvae provided with the transient gene and those from stable transgenic lines consistently expressed EGFP in the fast muscle fibers. r-VISTA plot identified one common conserved region of about 140°bp among myhz1.1, myhz1.2 and myhz1.3. Deletion of this conserved region from the 5'-flanking sequence of each myhz1 markedly reduced EGFP expression in its unique spatial somite region. Deletion mutation analysis demonstrated that myhz2 expression in the tail somite region might be mediated by Tbx (family of transcription factors having a common DNA-binding sequence known as T-box) binding elements. In summary, 5'-flanking sequences of myhz1 and myhz2 regulate their unique expression patterns in a well-defined and restricted somite region of the skeletal muscle in zebrafish.

  8. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway.

  9. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway. PMID:26634902

  10. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    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 Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-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 (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  11. Effects of creatine supplementation during resistance training on myosin heavy chain (MHC) expression in rat skeletal muscle fibers.

    PubMed

    Aguiar, Andreo F; Aguiar, Danilo H; Felisberto, Alan D S; Carani, Fernanda R; Milanezi, Rachel C; Padovani, Carlos R; Dal-Pai-Silva, Maeli

    2010-01-01

    The purpose of this study was to utilize a rodent model to test the hypothesis that creatine (Cr) supplementation during resistance training would influence the pattern of slow-twitch muscle myosin heavy chain (MHC) isoforms expression. Male Wistar rats (2-3 months old, 250-300 g) were divided into 4 groups: Nontrained without creatine supplementation (CO), nontrained with creatine supplementation (CR), trained without creatine supplementation (TR), and trained with creatine supplementation (TRCR). TR and TRCR groups were submitted to a resistance training program for 5 weeks (5 days/week) for morphological and biochemical analysis of the soleus muscle. Weightlifting exercise involved jump sessions into water, carrying progressive overload equivalent to percentage of body weight. CR and TRCR groups were given creatine at 0.5 g/kg(-1)/d(-1). Both Cr supplementation and resistance training alone or associated did not result in significant alterations (p > 0.05) in body weight gain, food intake, and muscle weight in the CR, TR and TRCR groups compared to the CO group. Also compared to the CO group, the CR group showed a significant (p < 0.02) increase in MHCI content and a reduction in MHCII; inversely, the TR group increased the MHCII content and reduced MHCI (p < 0.02). When combined, both creatine and resistance training did not promote significant (p > 0.05) changes in MHC content of the TRCR group compared to the CO group. The data show that Cr supplementation provides a potential action to abolish the exercise-induced MHC isoform transitions from slow to fast in slow-twitch muscle. Thus, Cr supplementation might be a suitable strategy to maintaining a slow phenotype in slow muscle during resistance training, which may be favorable to maintenance of muscle oxidative capacity of endurance athletes.

  12. Regulatory and Catalytic Domain Dynamics of Smooth Muscle Myosin Filaments†

    PubMed Central

    Li, Hui-Chun; Song, Likai; Salzameda, Bridget; Cremo, Christine R.; Fajer, Piotr G.

    2016-01-01

    Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, τr, were 24 ± 6 μs and 441 ± 79 μs for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 ± 4° and 24 ± 9° as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643–9647. Brown et al. (2001) Biochemistry 40, 8283–8291), the latter displayed higher regulatory domain mobility, τr = 40 ± 3 μs, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (~4 μs) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 ± 4° to 36 ± 7°) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 μs) or the catalytic domain (24 to 17 μs). These data are not consistent with a stable interaction between the two catalytic domains in

  13. Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

    PubMed

    Wu, Limin; Ramirez, Servio H; Andrews, Allison M; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. We propose the following events for neuregulin-1-mediated effects on Interleukin-1 β (IL-1β)-induced endothelial hyperpermeability: IL-1β leads to RhoA activation, resulting in an increase in phosphorylation of myosin light chain (MLC). Phosphorylation of MLC is known to result in actin contraction and alterations in the f-actin cytoskeletal structure. These changes are associated with increased endothelial permeability

  14. High-intensity resistance training with insufficient recovery time between bouts induce atrophy and alterations in myosin heavy chain content in rat skeletal muscle.

    PubMed

    De Souza, Rodrigo Wagner Alves; Aguiar, Andreo Fernando; Carani, Fernanda Regina; Campos, Gerson Eduardo Rocha; Padovani, Carlos Roberto; Silva, Maeli Dal Pai

    2011-08-01

    The aim of this study was to test whether high-intensity resistance training with insufficient recovery time between bouts, could result in a decrease of muscle fiber cross-sectional area (CSA), alter fiber-type frequencies and myosin heavy chain (MHC) isoform content in rat skeletal muscle. Wistar rats were divided into two groups: trained (Tr) and control (Co). Tr group were subjected to a high-intensity resistance training program (5 days/week) for 12 weeks, involving jump bouts into water, carrying progressive overloads based on percentage body weight. At the end of experiment, animals were sacrificed, superficial white (SW) and deep red (DR) portions of the plantaris muscle were removed and submitted to mATPase histochemical reaction and SDS-PAGE analysis. Throughout the experiment, both groups increased body weight, but Tr was lower than Co. There was a significant reduction in IIA and IID muscle fiber CSA in the DR portion of Tr compared to Co. Muscle fiber-type frequencies showed a reduction in Types I and IIA in the DR portion and IID in the SW portion of Tr compared to Co; there was an increase in Types IIBD frequency in the DR portion. Change in muscle fiber-type frequency was supported by a significant decrease in MHCI and MHCIIa isoforms accompanied by a significant increase in MHCIIb isoform content. MHCIId showed no significant differences between groups. These data show that high-intensity resistance training with insufficient recovery time between bouts promoted muscle atrophy and a transition from slow-to-fast contractile activity in rat plantaris muscle.

  15. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    SciTech Connect

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  16. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  17. Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

    PubMed Central

    1990-01-01

    The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation. PMID:2202734

  18. SLOW MYOSIN ATP TURNOVER IN THE SUPER-RELAXED STATE IN TARANTULA MUSCLE

    PubMed Central

    Naber, Nariman; Cooke, Roger

    2011-01-01

    We measured the nucleotide turnover rate of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ~30 minutes. This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations. PMID:21763701

  19. EPR and fluorescence depolarization studies on bovine cardiac myosin.

    PubMed

    Stone, D B; Mendelson, R A; Botts, J; Cheung, P H

    1981-09-01

    To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.

  20. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  1. Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

    PubMed

    Kengyel, András; Bécsi, Bálint; Kónya, Zoltán; Sellers, James R; Erdődi, Ferenc; Nyitrai, Miklós

    2015-05-01

    The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

  2. The importance of complete tissue homogenization for accurate stoichiometric measurement of myosin light chain phosphorylation in airway smooth muscle.

    PubMed

    Wang, Lu; Paré, Peter D; Seow, Chun Y

    2015-02-01

    The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10(-6) mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate.

  3. Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

    PubMed Central

    Kondo, Tomo; Isoda, Rieko; Ookusa, Takayuki; Kamijo, Keiju; Hamao, Kozue; Hosoya, Hiroshi

    2013-01-01

    Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells. PMID:23951055

  4. Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages

    SciTech Connect

    Mykles, D.L.

    1985-01-01

    Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrilla proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed alpha and beta. There were three ..cap alpha..-LC variants and two ..beta..-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles. 31 references, 6 figures, 2 tables.

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    1995-04-01

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

  7. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

    PubMed

    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  8. Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes.

    PubMed Central

    Boxer, L A; Stossel, T P

    1976-01-01

    Actin, myosin, and a high molecular weight actin-binding protein were purified from chronic myelogenous leukemia (CML) leukocytes. CML leukocyte actin resembled skeletal muscle and other cytoplasmic actins by its subunit molecular weight, by its ability to polymerize in the presence of salts, and to activate the Mg2+-ATPase activity of rabbit skeletal muscle myosin. CML leukocyte myosin was similar to other vertebrate cytoplasmic myosins in having heavy chains and two light subunits. However, its apparent heavy-chain molecular weight and Stokes radius suggested that it was variably degraded during purification. Purified CML leukocyte myosin had average specific EDTA- AND Ca2+-activated ATPase activities of 125 and 151 nmol Pi released/mg protein per min, respectively and low specific Mg2+-ATPase activity. The Mg2+-ATPase activity of CML myosin was increased 200-fold by rabbit skeletal muscle F-actin, but the specific activity relative to that of actin-activated rabbit skeletal muscle myosin was low. CML leukocyte myosin, like other vertebrate cytoplasmic myosins, formed filaments in 0.1 M KCl solutions. Reduced and denatured CML leukocyte-actin-binding protein had a single high molecular weight subunit like a recently described actin-binding protein of rabbit pulmonary macrophages which promotes the polymerization and gelation of actin. Cytoplasmic extracts of CML leukocytes prepared with ice-cold 0.34-M sucrose solutions containing Mg2+-ATP, dithiothreitol, and EDTA at pH 7.0 underwent rapid gelation when warmed to 25 degrees C. Initially, the gel could be liquified by cooling to ice-bath temperature. With time, warmed cytoplasmic extract gels shrunk ("contracted") into aggregates. The following findings indicated that CML leukocyte actin-binding protein promoted the temperature-dependent gelation of actin in the cytoplasmic extracts and that CML leukocyte myosin was involved in the contraction of the actin gels: (a) Cytoplasmic extract gels initially contained

  9. A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability*

    PubMed Central

    Clayburgh, Daniel R.; Rosen, Shari; Witkowski, Edwina D.; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G. N.; Alverdy, John C.; Turner, Jerrold R.

    2005-01-01

    Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes. PMID:15507455

  10. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.

    PubMed

    Clayburgh, Daniel R; Rosen, Shari; Witkowski, Edwina D; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G N; Alverdy, John C; Turner, Jerrold R

    2004-12-31

    Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.

  11. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation

    PubMed Central

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J.; Spears, Danna A.; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S.; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R.; Stainier, Didier Y. R.; Gollob, Michael H.

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836

  12. Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

    PubMed Central

    Farahbakhsh, Nasser A.; Narins, Peter M.

    2008-01-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca2+/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca2+/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter the phase 1 response. However, they appear to counter effects of the inhibitors of Ca2+/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells. PMID:18534795

  13. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    PubMed

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

  14. [Seasonal changes in phosphorylation of myosin regulatory light chains and C-protein in myocardium of hibernating ground squirrel Citellus undulatus].

    PubMed

    Malyshev, S L; Osipova, D A; Vikhliantsev, I M; Podlubnaia, Z A

    2006-01-01

    A comparative study concerning the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of hibernating ground squirrel Citellus undulatus during the periods of hibernation and activity was carried out. During hibernation, regulatory light chains of ground squirrel were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averages 40-45% of their total amount. The extent of phosphorylation of the cardiac C-protein during hibernation is about two times higher than that in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.

  15. Pressure-jump studies on the length-regulation kinetics of the self-assembly of myosin from vertebrate skeletal muscle into thick filament.

    PubMed Central

    Davis, J S

    1981-01-01

    The self-assembly of myosin monomer into thick filament occurs via a two-step mechanism. At first a pair of myosin monomers reacts to form a parallel dimer; the dimer in turn adds to the filament ends at a rate that is independent of filament length. The rate of the dissociation reaction on the other hand is length-dependent. The 'off' rate constant has been shown to increase exponentially by a factor of 500 as the filament grows from the bare-zone out to its full length. The length of the filament is thus kinetically controlled; myosin is added to the filament at a fixed rate, whereas the dissociation rate increases to a point where equilibrium is established and the filament ceases to grow. The structural implications implicit in the mechanism are discussed. PMID:7198910

  16. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

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

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  17. Myosins in protists.

    PubMed

    Gavin, R H

    2001-01-01

    This review focuses on selected papers that illustrate an historical perspective and the current knowledge of myosin structure and function in protists. The review contains a general description of myosin structure, a phylogenetic tree of the myosin classes, and descriptions of myosin isoforms identified in protists. Each myosin is discussed within the context of the taxonomic group of the organism in which the myosin has been identified. Domain structure, cellular location, function, and regulation are described for each myosin.

  18. Mechanosensing in Myosin Filament Solves a 60 Years Old Conflict in Skeletal Muscle Modeling between High Power Output and Slow Rise in Tension

    PubMed Central

    Marcucci, Lorenzo; Reggiani, Carlo

    2016-01-01

    Almost 60 years ago Andrew Huxley with his seminal paper (Huxley, 1957) laid the foundation of modern muscle modeling, linking chemical to mechanical events. He described mechanics and energetics of muscle contraction through the cyclical attachment and detachment of myosin motors to the actin filament with ad-hoc assumptions on the dependence of the rate constants on the strain of the myosin motors. That relatively simple hypothesis is still present in recent models, even though with several modifications to adapt the model to the different experimental constraints which became subsequently available. However, already in that paper, one controversial aspect of the model became clear. Relatively high attachment and detachment rates of myosin to the actin filament were needed to simulate the high power output at intermediate velocity of shortening. However, these rates were incompatible with the relatively slow rise in tension upon activation, despite the rise should be generated by the same rate functions. This discrepancy has not been fully solved till today, despite several hypotheses have been forwarded to reconcile the two aspects. Here, using a conventional muscle model, we show that the recently revealed mechanosensing mechanism of recruitment of myosin motors (Linari et al., 2015) can solve this long standing problem without any further ad-hoc hypotheses. PMID:27721796

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    PubMed Central

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  3. Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of occludin, without altering epithelial permeability.

    PubMed

    Lapointe, Tamia K; Buret, Andre G

    2012-02-01

    Compromised epithelial barrier function and tight junction alterations are hallmarks of a number of gastrointestinal disorders, including inflammatory bowel disease (IBD). Increased levels of IL-18 have been observed in mucosal samples from Crohn's disease and ulcerative colitis patients. Remarkably, several reports have demonstrated that immunological or genetic blockage of IL-18 ameliorates the severity of colitis in multiple in vivo models of IBD. Nevertheless, the effects of IL-18 on intestinal epithelial barrier function remain unclear. We hypothesized that IL-18 could disrupt intestinal epithelial barrier structure and function, thus contributing to tissue damage in the context of IBD. The aims of the present study were to determine the effects of IL-18 on epithelial barrier structure and function and to characterize the mechanisms involved in these modulatory properties. Human colonic epithelial Caco-2 monolayers were coincubated with IL-18 for 24 h and processed for immunocytochemistry, immunoblotting, quantitative PCR, and permeability measurements (transepithelial resistance, FITC-dextran fluxes, and bacterial translocation). Our findings indicate that IL-18 selectively disrupts tight junctional occludin, without affecting the distribution pattern of claudin-4, claudin-5, zonula occludens-1, or E-cadherin. This effect coincided with a significant increase in myosin light chain kinase (MLCK) protein levels and activity. Pharmacological inhibition of MLCK and NF-κB prevented IL-18-induced loss of occludin. Although too subtle to alter paracellular permeability, these fine changes correlated with an MLCK-dependent increase in neutrophil transepithelial migration. In conclusion, our data suggest that IL-18 may potentiate inflammation in the context of IBD by facilitating neutrophil transepithelial migration via MLCK-dependent disruption of tight junctional occludin.

  4. Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury

    PubMed Central

    Mirzapoiazova, Tamara; Moitra, Jaideep; Moreno-Vinasco, Liliana; Sammani, Saad; Turner, Jerry R.; Chiang, Eddie T.; Evenoski, Carrie; Wang, Ting; Singleton, Patrick A.; Huang, Yong; Lussier, Yves A.; Watterson, D. Martin; Dudek, Steven M.; Garcia, Joe G. N.

    2011-01-01

    Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation. PMID:20139351

  5. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    PubMed

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene. PMID:25911560

  6. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm.

    PubMed

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  7. Berberine ameliorates severe acute pancreatitis‑induced intestinal barrier dysfunction via a myosin light chain phosphorylation‑dependent pathway.

    PubMed

    Liang, Hong-Yin; Chen, Tao; Yan, Hong-Tao; Huang, Zhu; Tang, Li-Jun

    2014-05-01

    Berberine is a traditional drug used to treat gastrointestinal disorders in China and has been demonstrated to attenuate intestinal barrier dysfunction in certain animal models. However, the effects of berberine on pancreatitis-induced intestinal barrier dysfunction are yet to be fully elucidated. This study aimed to investigate the effect of berberine pretreatment on the attenuation of intestinal barrier dysfunction induced by severe acute pancreatitis (SAP). A total of 36 rats were randomly divided into Sham, SAP and SAP plus berberine groups. Pancreatitis was induced using retrograde injection of 3% Na-taurocholate into the pancreatic duct. Histological examinations of the pancreas were performed and intestinal barrier dysfunction was characterized by histological measurements and the assessment of serum diamine oxidase activity and endotoxin levels. Zonula occludens-1 and occludin mRNA and protein expression, as well as myosin light chain (MLC) phosphorylation, were assessed. SAP rat models were successfully established. Berberine treatment was found to have no significant effect on the histological changes in the pancreas, but was observed to ameliorate the intestinal mucosal barrier damage and membrane permeability associated with SAP. Although berberine exerted minimal effects on tight junction proteins in the ilea of SAP rats, it was observed to significantly inhibit SAP-induced MLC phosphorylation. To the best of our knowledge, this is the first study to demonstrate that berberine attenuates SAP‑induced intestinal barrier dysfunction in vivo. In addition, this study shows that the effect of berberine on intestinal barrier function may be associated with the inhibition of SAP‑induced upregulation of MLC phosphorylation.

  8. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  9. Kinetic and Motor Functions Mediated by Distinct Regions of the Regulatory Light Chain of Smooth Muscle Myosin1,2

    PubMed Central

    Ni, Shaowei; Hong, Feng; Brewer, Paul D.; Ikebe, Mitsuo; Onishi, Hirofumi; Baker, Jonathan E.; Facemyer, Kevin C.; Cremo, Christine R.

    2009-01-01

    To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP95-98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg2+-ATPase (Vmax and Katpase) and in vitro actin sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the upstate (0.007-0.030 s-1) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (Katpase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the Vmax was 64% and actin sliding velocity was ∼50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation. PMID:19635597

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    1995-06-01

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

  12. Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.

    PubMed

    Smyczynski, C; Kasprzak, A A

    1997-10-28

    The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models

  13. Effect of chronic excess of tumour necrosis factor-alpha on contractile proteins in rat skeletal muscle.

    PubMed

    Cheema, I R; Hermann, C; Postell, S; Barnes, P

    2000-01-01

    The effect of chronic tumour necrosis factor-alpha (TNF-alpha) treatment on the synthesis of specific myofibrillar proteins such as heavy chain myosin, light chain myosin and G-actin in rat diaphragm were evaluated. Muscles (diaphragm) from control and experimental groups (TNF-alpha i.v. at 50 microg/kg body wt for 5 days) were incubated in the presence of 35S-methionine for 2 h. Myofibrillar protein extracts were prepared and protein was electrophoresed on sodium dodecyl sulphate-polyacrylamide gels. Heavy chain myosin, light chain myosin and G-actin were identified by Western blot analysis using specific monoclonal antibodies. Polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis revealed two types of heavy chain myosin (206 and 212 kD), all four types of light chain myosin (15, 16.5, 18 and 20 kD) and a single type of G-actin (42 kD). Chronic TNF-alpha treatment produced a significant decline in the synthesis of all types of myofibrillar proteins, namely heavy chain myosin, light chain myosin and G-actin. TNF-alpha impaired peptide-chain initiation in diaphragm muscle which was reversed by the branched-chain amino acids (BCAA) therapy of TNF-alpha treated rats. These findings indicate a significant role for TNF-alpha in the translational regulation of protein synthesis in skeletal muscle.

  14. Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

    PubMed

    Takemoto, Kenji; Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-01

    Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

  15. Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

    PubMed

    Di Ciano-Oliveira, Caterina; Lodyga, Monika; Fan, Lingzhi; Szászi, Katalin; Hosoya, Hiroshi; Rotstein, Ori D; Kapus, András

    2005-07-01

    Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl- cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl- depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl- depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. PMID:15728707

  16. Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

    PubMed

    Van Lierop, Jacquelyn E; Wilson, David P; Davis, Jonathan P; Tikunova, Svetlana; Sutherland, Cindy; Walsh, Michael P; Johnson, J David

    2002-02-22

    Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.

  17. Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins.

    PubMed

    Seki, Masaya; Kashiyama, Taku; Hachikubo, You; Ito, Kohji; Yamamoto, Keiichi

    2004-11-19

    A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.

  18. [Effect of hypothyreosis on actin subdomain-1 movement induced by myosin subfragment 1-binding in fast and slow rat skeletal muscles].

    PubMed

    Kirillina, V P; Jakubiec-Puka, A; Borovikov, Iu S

    2009-01-01

    Orientation and mobility of fluorescent probe N-((iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine)(1.5-IAEDANS)) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of myosin subfragment-1 (S1) in intact rats and in the animals with gradual (during 2-5 weeks) reduction of thyroid hormones synthesis (hypothyreosis development). S1 binding to F-actin of ghost muscle fibers was shown to induce changes in orientation of the dipoles of the fluorescent probe 1.5-IAEDANS and in the relative amount of the randomly oriented fluorophores that indicated changes in actin subdomain-1 orientation and mobility resulting from the formation of its strong binding with S1. This effect is markedly inhibited by hypothyreosis development. The maximal effect of hypothyreosis is observed after 34 days of disease development. It is suggested that the change of thyroid status in the muscle inhibits the ability of F-actin to form strong binding with myosin which is essential for force generation.

  19. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    SciTech Connect

    Zhang, Ying; Li, Jianguo

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  20. A Millennial Myosin Census

    PubMed Central

    Berg, Jonathan S.; Powell, Bradford C.; Cheney, Richard E.

    2001-01-01

    The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms. PMID:11294886

  1. Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

    PubMed

    Bertrand, R; Capony, J P; Derancourt, J; Kassab, R

    1999-09-14

    We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could

  2. Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.

    PubMed

    Sutton, T A; Haeberle, J R

    1990-02-15

    In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase

  3. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs

    PubMed Central

    Sharanek, Ahmad; Burban, Audrey; Burbank, Matthew; Le Guevel, Rémy; Li, Ruoya; Guillouzo, André; Guguen-Guillouzo, Christiane

    2016-01-01

    Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis. PMID:27169750

  6. Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

    PubMed Central

    Jalagadugula, Gauthami; Mao, Guangfen; Kaur, Gurpreet; Goldfinger, Lawrence E.; Dhanasekaran, Danny N.

    2010-01-01

    Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region −729/−542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations. PMID:20876458

  7. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    PubMed

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  8. Myosin filament 3D structure in mammalian cardiac muscle☆

    PubMed Central

    AL-Khayat, Hind A.; Morris, Edward P.; Kensler, Robert W.; Squire, John M.

    2008-01-01

    A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac). PMID:18472277

  9. UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification

    SciTech Connect

    Grammer, J.C.; Cremo, C.R.; Yount, R.G.

    1988-11-01

    Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (V/sub i/)-myosin subfragment 1(S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and V/sub i/. This photomodified S1 had Ca/sup 2 +/ATPase activity 4-5-fold higher than that of the nonirradiated control S1, while the K/sup +/EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca/sup 2 +/ATPase and the release of both ADP and V/sub i/ with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiols reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-V/sub i/ complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of V/sub i/ from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg/sup 2 +/ with either Co/sup 2 +/, Mn/sup 2 +/, or Ni/sup 2 +/. Unlike previous irradiation studies of V/sub i/-dynein complexes, no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-V/sub i/ at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-V/sub i/ complex, MgADP and V/sub i/ were again released from the active site, resulting in heavy chain cleavage to form NH/sub 2/-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.

  10. Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Wuethrich, Andrew J.; Hancock, Deana L.

    2002-01-01

    Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were

  11. Myosins and cell dynamics in cellular slime molds.

    PubMed

    Yumura, Shigehiko; Uyeda, Taro Q P

    2003-01-01

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

  12. Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants

    PubMed Central

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R.; Gokhale, Vijay; Brown, Mary E.; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M.; Wang, Ting; Garcia, Joe G. N.

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities. PMID:26111161

  13. Translation termination factors function outside of translation: yeast eRF1 interacts with myosin light chain, Mlc1p, to effect cytokinesis.

    PubMed

    Valouev, I A; Urakov, V N; Kochneva-Pervukhova, N V; Smirnov, V N; Ter-Avanesyan, M D

    2004-07-01

    The translation termination factor eRF1 recognizes stop codons at the A site of the ribosome and induces peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Recent data show that, besides translation, yeast eRF1 is also involved in cell cycle regulation. To clarify the mechanisms of non-translational functions of eRF1, we performed a genetic screen for its novel partner proteins. This screen revealed the gene for myosin light chain, Mlc1p, acting as a dosage suppressor of a temperature-sensitive mutation in the SUP45 gene encoding eRF1. eRF1 and Mlc1p are able to interact with each other and, similarly to depletion of Mlc1p, mutations in the SUP45 gene may affect cytokinesis. Immunofluorescent staining performed to determine localization of Mlc1p has shown that the sup45 mutation, which arrests cytokinesis, redistributed Mlc1p, causing its disappearance from the bud tip and the bud neck. The data obtained demonstrate that yeast eRF1 has an important non-translational function effecting cytokinesis via interaction with Mlc1p.

  14. All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

    PubMed

    Zuo, Li; Yang, Xiaoping; Lu, Man; Hu, Ruolei; Zhu, Huaqing; Zhang, Sumei; Zhou, Qing; Chen, Feihu; Gui, Shuyu; Wang, Yuan

    2016-10-01

    All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway. PMID:27564600

  15. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    PubMed

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. PMID:26175189

  16. Activation of Endothelial Pro-resolving Anti-Inflammatory Pathways by Circulating Microvesicles from Non-muscular Myosin Light Chain Kinase-Deficient Mice

    PubMed Central

    Gaceb, Abderahim; Vergori, Luisa; Martinez, M. C.; Andriantsitohaina, Ramaroson

    2016-01-01

    Microvesicles, small membrane vesicles released from cells, have beneficial and/or deleterious effects in sepsis. We previously reported that non-muscle myosin light chain kinase (nmMLCK) deletion protects mice against endotoxic shock by reducing inflammation. Here, we have evaluated the consequences of nmMLCK deletion on microvesicle phenotypes and their effects on mouse aortic endothelial cells in association with vascular inflammation and endothelial dysfunction during endotoxic shock induced by lipopolysaccharide in mice. Treatment with lipopolysaccharide induced an increase in levels of circulating microvesicles in wild type but not in nmMLCK-deficient mice. Microvesicles from nmMLCK-deficient mice (MVsnmMLCK-/-) prevented the inflammatory effects of lipopolysaccharide with concomitant increase of anti- inflammatory and reduction of pro-inflammatory secretome in mouse aortic endothelial cells. In addition, MVsnmMLCK-/- reduced the efficacy of lipopolysaccharide to increase aortic oxidative and nitrosative stresses as well as macrophage infiltration in the aorta. Moreover, MVsnmMLCK-/- prevented ex vivo endothelial dysfunction, vascular hyporeactivity, and in vivo overproduction of nitric oxide in heart and liver in response to lipopolysaccharide. Altogether, these findings provide evidence that nmMLCK deletion generates circulating microvesicles displaying protective effects by activating endothelial pro-resolving anti-inflammatory pathways allowing the effective down-regulation of oxidative and nitrative stresses associated with endotoxic shock. Thus, nmMLCK plays a pivotal role in susceptibility to sepsis via the control of cellular activation and release of circulating microvesicles. PMID:27708581

  17. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    PubMed

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation.

  18. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    PubMed

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  19. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

    PubMed

    McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

    2015-01-01

    Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation. PMID:25689628

  20. Photoaffinity labelling of smooth-muscle myosin by methylanthraniloyl-8-azido-ATP.

    PubMed

    Maruta, S; Ikebe, M

    1993-06-01

    Methylanthraniloyl-8-azido-ATP (Mant-8-N3-ATP), which binds to the 20 kDa C-terminal tryptic fragment of skeletal-muscle myosin subfragment-1 [Maruta, Miyanishi and Matsuda (1989) Eur. J. Biochem. 184, 213-221], was synthesized and used as a probe of the conformational change of smooth-muscle myosin. Mant-8-N3-ATP, like ATP, induced the formation of the 10 S conformation at low ionic strength. In the presence of vanadate, smooth-muscle myosin formed a stable complex with Mant-8-N3-ADP, and this complex showed the 10 S-->6 S transition of myosin. ATP-binding sites for 6 S (extended state) and 10 S (folded state) myosin were studied by photolabelling of myosin with Mant-8-N3-ADP. For both 6 S and 10 S myosin, Mant-8-N3-ATP was incorporated into the 29 kDa N-terminal tryptic fragment of myosin heavy chain. This is unlike the labelling of skeletal-muscle myosin, in which the 20 kDa C-terminal fragment is labelled. The labelling of 29 kDa fragment was diminished significantly by addition of ATP. These results suggest that the conformation of the ATP-binding site of smooth-muscle myosin is different from that of skeletal-muscle myosin. To examine further the possible differences in the labelling site between 6 S and 10 S myosin, the affinity-labelled 29 kDa fragment was subjected to complete proteolysis by lysylendo-peptidase. The fluorescent-labelled-peptide map suggested that the Mant-8-N3-ADP-binding sites for 6 S and 10 S myosin were identical.

  1. Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms

    PubMed Central

    Zhu, Jingui; Sun, Yongqiao; Zhao, Fa-Qing; Yu, Jun; Craig, Roger; Hu, Songnian

    2009-01-01

    Background Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking. Results We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins). Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types. Conclusion The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species. PMID:19298669

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

    SciTech Connect

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.

    2009-07-01

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

  3. Effect of light and brine shrimp on skeletal δ 13C in the Hawaiian coral Porites compressa: a tank experiment

    NASA Astrophysics Data System (ADS)

    Grottoli, Andréa G.

    2002-06-01

    Previous experimental fieldwork showed that coral skeletal δ 13C values decreased when solar intensity was reduced, and increased in the absence of zooplankton. However, actual seasonal changes in solar irradiance levels are typically less pronounced than those used in the previous experiment and the effect of increases in the consumption of zooplankton in the coral diet on skeletal δ 13C remains relatively unknown. In the present study, the effects of four different light and heterotrophy regimes on coral skeletal δ 13C values were measured. Porites compressa corals were grown in outdoor flow-through tanks under 112%, 100%, 75%, and 50% light conditions at the Hawaii Institute of Marine Biology, Hawaii. In addition, corals were fed either zero, low, medium, or high concentrations of brine shrimp. Decreases in light from 100% resulted in significant decreases in δ 13C that is most likely due to a corresponding decrease in photosynthesis. Increases in light to 112% also resulted in a decrease in δ 13C values. This latter response may be a consequence of photoinhibition. The overall curved response in δ 13C values was described by a significant quadratic function. Increases in brine shrimp concentrations resulted in increased skeletal δ 13C levels. This unexpected outcome appears to be attributable to enhanced nitrogen supply associated with the brine shrimp diet which led to increased zooxanthellae concentrations, increased photosynthesis rates, and thus increased δ 13C values. This result highlights the potential influence of nutrients from heterotrophically acquired carbon in maintaining the zooxanthellae-host symbiosis in balance. In addition, evidence is presented that suggests that coral skeletal growth and δ 13C are decoupled. These results increase our knowledge of how light and heterotrophy affects the δ 13C of coral skeletons.

  4. TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism.

    PubMed

    Wu, Xiaoyan; Guo, Rongqing; Chen, Peili; Wang, Quan; Cunningham, Patrick N

    2009-08-01

    The pathogenesis of LPS-induced acute kidney injury (AKI) requires signaling through tumor necrosis factor-alpha (TNF) receptor 1 (TNFR1), which within the kidney is primarily located in the endothelium. We showed previously that caspase inhibition protected mice against LPS-induced AKI and in parallel significantly inhibited LPS-induced renal inflammation. Therefore we hypothesized that caspase activation amplifies TNF-induced inflammation in renal endothelial cells (ECs). In cultured renal ECs, TNF induced apoptosis through a caspase-8-dependent pathway. TNF caused translocation of the p65 subunit of NF-kappaB to the nucleus, resulting in upregulation of inflammatory markers such as adhesion molecules ICAM-1 and VCAM-1. However, the broad-spectrum caspase inhibitor Boc-d-fmk reduced NF-kB activation as assessed by gel shift assay, reduced phosphorylation of subunit IkappaBalpha, and significantly inhibited TNF-induced expression of ICAM-1 and VCAM-1 as assessed by both real-time PCR and flow cytometry. Broad-spectrum caspase inhibition markedly inhibited neutrophil adherence to the TNF-activated endothelial monolayer, supporting the functional significance of this effect. Specific inhibitors of caspases-8 and -3, but not of caspase-1, reduced TNF-induced NF-kappaB activation. Caspase inhibition also reduced TNF-induced myosin light chain (MLC)-2 phosphorylation, and activation of upstream regulator RhoA. Consistent with this, MLC kinase (MLCK) inhibitor ML-7 reduced TNF-induced NF-kappaB activation. Thus caspase activation influences NF-kappaB signaling via its affect on cytoskeletal changes occurring through RhoA and MLCK pathways. These cell culture experiments support a role for caspase activation in TNF-induced inflammation in the renal endothelium, a key event in LPS-induced AKI. PMID:19420112

  5. Myosin VI: a multifunctional motor.

    PubMed

    Lister, I; Roberts, R; Schmitz, S; Walker, M; Trinick, J; Veigel, C; Buss, F; Kendrick-Jones, J

    2004-11-01

    Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed. PMID:15493988

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-01-01

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

  8. Still and rotating myosin clusters determine cytokinetic ring constriction

    PubMed Central

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

    2016-01-01

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

  9. The Conformation of Myosin Head Domains in Rigor Muscle Determined by X-Ray Interference

    PubMed Central

    Reconditi, M.; Koubassova, N.; Linari, M.; Dobbie, I.; Narayanan, T.; Diat, O.; Piazzesi, G.; Lombardi, V.; Irving, M.

    2003-01-01

    In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T0), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T0), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T0, the heads' center of mass moved back by 1.1–1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys707–Lys843 vector and the filament axis increases by ∼36° between isometric contraction and low-force rigor, and decreases by 7–10° when the rigor fiber is stretched to 0.55 T0. PMID:12885655

  10. Light-emitting diode therapy increases collagen deposition during the repair process of skeletal muscle.

    PubMed

    de Melo, Claudia Aparecida Viana; Alves, Agnelo Neves; Terena, Stella Maris Lins; Fernandes, Kristianne Porta Santos; Nunes, Fábio Daumas; da Silva, Daniela de Fátima Teixeira; Bussadori, Sandra Kalil; Deana, Alessandro Melo; Mesquita-Ferrari, Raquel Agnelli

    2016-04-01

    This study analyzed the effects of light-emitting diode (LED) therapy on the morphology of muscle tissue as well as collagen remodeling and matrix metalloproteinase 2 (MMP-2) activity in the skeletal muscle of rats following acute injury. Wistar rats were divided into four groups: (1) control, (2) sham, (3) untreated cryoinjury, and (4) cryoinjury treated with LED. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior muscle. For treatment, the LED equipment (wavelength 850 nm, output power 30 mW, and total energy 3.2 J) was used daily. The study periods were 1, 3, and 7 days after cryoinjury. Morphological aspects were evaluated through hematoxylin-eosin staining. The amount of collagen fibers was evaluated using Picro Sirius Red staining under polarized light. The gelatinase activity of MMP-2 was evaluated using zymography. The results showed significant reductions in inflammatory infiltrate after 3 days and an increased number of immature muscle fibers after 7 days. Furthermore, treatment induced a reduction in the gelatinolytic activity of MMP-2 after 1, 3, and 7 days in comparison to the untreated injury groups and increased the collagen deposition after 3 and 7 days in the treated groups. LED therapy at 850 nm induced a significant reduction in inflammation, decreased MMP-2 activity, and increased the amount of immature muscle and collagen fibers during the muscle repair process following acute injury.

  11. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    PubMed Central

    1987-01-01

    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg- ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin. PMID:2958483

  12. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system.

    PubMed

    Caldwell, James T; Melkani, Girish C; Huxford, Tom; Bernstein, Sanford I

    2012-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

  13. Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes.

    PubMed

    Werneck-de-Castro, Joao P; Fonseca, Tatiana L; Ignacio, Daniele L; Fernandes, Gustavo W; Andrade-Feraud, Cristina M; Lartey, Lattoya J; Ribeiro, Marcelo B; Ribeiro, Miriam O; Gereben, Balazs; Bianco, Antonio C

    2015-10-01

    The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers. PMID:26214036

  14. Myosin V motor proteins

    PubMed Central

    Vale, Ronald D.

    2003-01-01

    Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how “dynamic” single molecule measurements can synergize with “static” protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine. PMID:14610051

  15. Myosin flexibility: structural domains and collective vibrations.

    PubMed

    Navizet, Isabelle; Lavery, Richard; Jernigan, Robert L

    2004-02-15

    The movement of the myosin motor along an actin filament involves a directed conformational change within the cross-bridge formed between the protein and the filament. Despite the structural data that has been obtained on this system, little is known of the mechanics of this conformational change. We have used existing crystallographic structures of three conformations of the myosin head, containing the motor domain and the lever arm, for structural comparisons and mechanical studies with a coarse-grained elastic network model. The results enable us to define structurally conserved domains within the protein and to better understand myosin flexibility. Notably they point to the role of the light chains in rigidifying the lever arm and to changes in flexibility as a consequence of nucleotide binding.

  16. Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction via tight junction regulation in a rabbit model of atherosclerosis.

    PubMed

    Cheng, Xiaowen; Wang, Xiaobian; Wan, Yufeng; Zhou, Qing; Zhu, Huaqing; Wang, Yuan

    2015-09-01

    Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet‑induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)‑1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high‑fat diet‑fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high‑fat diet (AS group) or a high‑fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium‑dependent relaxation and endothelium‑independent relaxation were measured using high-frequency ultrasound. Administration of a high‑fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high‑fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high‑fat diet. A similar change was observed for the TJ proteins ZO‑1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ

  17. Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction via tight junction regulation in a rabbit model of atherosclerosis

    PubMed Central

    CHENG, XIAOWEN; WANG, XIAOBIAN; WAN, YUFENG; ZHOU, QING; ZHU, HUAQING; WANG, YUAN

    2015-01-01

    Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet-induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)-1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high-fat diet-fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high-fat diet (AS group) or a high-fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium-dependent relaxation and endothelium-independent relaxation were measured using high-frequency ultrasound. Administration of a high-fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high-fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high-fat diet. A similar change was observed for the TJ proteins ZO-1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ, is associated with

  18. Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

    PubMed Central

    Aguilar, Hector N.; Zielnik, Barbara; Tracey, Curtis N.; Mitchell, Bryan F.

    2010-01-01

    Background Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the “in-cell western” (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC20) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses. Methodology/Principal Findings ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey®) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC20 phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT. Conclusion/Significance ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an

  19. Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-α-induced regulation of myosin light chain kinase gene activity

    PubMed Central

    Ye, Dongmei; Ma, Thomas Y

    2008-01-01

    The patients with Crohn's disease (CD) have a ‘leaky gut’ manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-α (TNF-α) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-α is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-α increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-α-induced increase in MLCK gene activity. By progressive 5′ deletion, minimal MLCK promoter was localized between −313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-α-induced increase in MLCK promoter activity was mediated by NF-κB activation. There were eight κB binding sites on MLCK promoter. The NF-κB1 site at +48 to +57 mediated TNF-α-induced increase in MLCK promoter activity. The NF-κB2 site at −325 to −316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-κB dimer type binding to the κB sites. p50/p65 dimer preferentially binds to the NF-κB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-κB2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-α-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate

  20. Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

    PubMed

    Zhang, Ying; Kawamichi, Hozumi; Tanaka, Hideyuki; Yoshiyama, Shinji; Kohama, Kazuhiro; Nakamura, Akio

    2012-08-01

    We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

  1. Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement

    PubMed Central

    Fabian, Lacramioara; Troscianczuk, Joanna; Forer, Arthur

    2007-01-01

    Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. PMID:17381845

  2. Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.

    PubMed

    Fabian, Lacramioara; Troscianczuk, Joanna; Forer, Arthur

    2007-01-01

    Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. PMID:17381845

  3. Phosphorylation of lymphocyte myosin catalyzed in vitro and in intact cells

    PubMed Central

    1982-01-01

    Myosin has been isolated from guinea pig B-lymphocytic leukemia cells (L2C). The myosin has been enzymatically phosphorylated and dephosphorylated in vitro using both heterologous and lymphocyte- derived enzymes. Both the heavy chain and 20,000-dalton light chain of lymphocyte myosin are phosphorylated in vitro. Phosphorylation of myosin enhances actin-activated ATPase activity. Phosphorylation of myosin in murine lymphocytes was analyzed by use of a novel technique for rapid immunoprecipitation of myosin from cell extracts. Both the heavy chain and 20,000-dalton light chain of myosin are phosphorylated in intact cells. Addition of antibody reactive with cell-surface immunoglobulin to lymphocyte populations enriched for B cells stimulates locomotion of these cells and also increases the quantity of 32P isolated in association with the 20,000-dalton light chain of lymphocyte myosin, when 32Pi was present in the medium. In addition, an unidentified, phosphorylated polypeptides with a molecular mass of 22,000 daltons is co-isolated with myosin from cells by rapid immunoprecipitation. These results are consistent with the hypothesis that phosphorylation of myosin may contribute to regulation of movements performed by lymphocytes which are related to their participation in immunologic reactions. PMID:6212588

  4. Locking regulatory myosin in the off-state with trifluoperazine.

    PubMed

    Patel, H; Margossian, S S; Chantler, P D

    2000-02-18

    Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.

  5. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine modification of myosin from chicken gizzard.

    PubMed

    Onishi, H

    1985-07-01

    Previously, we (Suzuki et al. (1978) J. Biochem. 84, 1529) reported that the sedimentation constant of chicken gizzard myosin in the presence of ATP was approximately 10S in 0.15 M or 0.2 M KCl and approximately 6S in 0.3 M or higher concentrations of KCl. The 10S-myosin and 6S-myosin were considerably different in conformation from each other. I now report the finding that the transformation of 6S-myosin to the 10S conformation results in a drastic change in the reactivity of thiol groups of gizzard myosin with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (abbreviated as IAEDANS). The so-called SH1-type thiol groups (Sekine et al. (1962) J. Biol. Chem. 237, 2769) were present on 68 kilodalton fragments (produced by tryptic digestion) of gizzard myosin. The reactivity of the thiol groups with IAEDANS was greatly decreased by the 6S to 10S transformation of gizzard myosin molecules. Two other findings were obtained. Blocking the SH1-type thiol groups made the Mg-ATPase activities (in the presence of gizzard native tropomyosin) of gizzard myosin and of acto-gizzard myosin insensitive to calcium and to phosphorylation of regulatory light chains, although calcium-dependent phosphorylation of the IAEDANS-modified myosin could still occur. It also made gizzard myosin filaments resistant to the disassembly action of ATP.

  6. The role of myosin phosphorylation in anaphase chromosome movement.

    PubMed

    Sheykhani, Rozhan; Shirodkar, Purnata V; Forer, Arthur

    2013-01-01

    This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production. Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin. PMID:23566798

  7. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2010-08-01

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

  9. Influence of the Cardiac Myosin Hinge Region on Contractile Activity

    NASA Astrophysics Data System (ADS)

    Margossian, Sarkis S.; Krueger, John W.; Sellers, James R.; Cuda, Giovanni; Caulfield, James B.; Norton, Paul; Slayter, Henry S.

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the antihinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

  10. Influence of the cardiac myosin hinge region on contractile activity.

    PubMed

    Margossian, S S; Krueger, J W; Sellers, J R; Cuda, G; Caulfield, J B; Norton, P; Slayter, H S

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

  11. Plasmodium falciparum myosins: transcription and translation during asexual parasite development.

    PubMed

    Chaparro-Olaya, Jacqueline; Margos, Gabriele; Coles, Deborah J; Dluzewski, Anton R; Mitchell, Graham H; Wasserman, Moisés M; Pinder, Jennifer C

    2005-04-01

    Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically; accordingly, we refer to the two latest additions as Pfmyo-E and Pfmyo-F. Both new myosins contain regions characteristic of the functional motor domain of "true" myosins and, unusually for P. falciparum myosins, Pfmyo-F encodes two consensus IQ light chain-binding motifs. Phylogenetic analysis of the 17 currently known apicomplexan myosins together with one representative of each myosin class clusters all but one of the apicomplexan sequences together in Class XIV. This refines the earlier definition of the Class XIV Subclasses XIVa and XIVb. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Thus: Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation. It is known that Pfmyo-A and Pfmyo-B are synthesised during late schizogony and we now show that Pfmyo-D expression is also temporally regulated to late trophozoites and schizonts where it distributes close to segregating nuclei. Thus, in asexual stages myosin synthesis does not always parallel transcript accumulation, showing that translation is also regulated. The implication is that the mRNAs are either subjected to turnover, synthesised and degraded, or that they are sequestered in an inactivate form until required for protein synthesis.

  12. Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division.

    PubMed

    Silverman-Gavrila, Rosalind V; Forer, Arthur

    2003-06-01

    We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the

  13. Molecular engineering of myosin.

    PubMed Central

    Manstein, Dietmar J

    2004-01-01

    Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins. PMID:15647166

  14. The Effect of Time on Bone Fluorescence: Implications for Using Alternate Light Sources to Search for Skeletal Remains.

    PubMed

    Swaraldahab, Mohamed A H; Christensen, Angi M

    2016-03-01

    Bones fluoresce when exposed to certain wavelengths of shortwave light, and this property can be useful in locating and sorting skeletal remains in forensic contexts. The proteins in bone collagen are largely responsible for its fluorescent properties, but these proteins degrade and denature over time. This study examined the fluorescence of bones from four temporal groups (recent, semi-recent, ancient, and historic) ranging from 0 to 1064 years before present. Specimens were photographed under 490 nm wavelength light, and fluorescence was quantified by converting intensity to a gray scale value based on the RGB color model using ImageJ(®) software. Significant (p < 0.05) differences were found in mean fluorescence between all four temporal groups, and a 0.324 coefficient of correlation indicates a significant (inverse) relationship between fluorescence and time. Bone fluorescence decreases with time, but some fluorescence is retained even in older samples. Fluorescence can therefore be reliably used in many modern skeletal remains searches. PMID:27404617

  15. Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle.

    PubMed

    Nogara, Leonardo; Naber, Nariman; Pate, Edward; Canton, Marcella; Reggiani, Carlo; Cooke, Roger

    2016-01-01

    In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity. PMID:27479128

  16. Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle

    PubMed Central

    Naber, Nariman; Pate, Edward; Canton, Marcella; Reggiani, Carlo; Cooke, Roger

    2016-01-01

    In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity. PMID:27479128

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

    SciTech Connect

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-02-25

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

  18. Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.

    PubMed

    Alamo, Lorenzo; Qi, Dan; Wriggers, Willy; Pinto, Antonio; Zhu, Jingui; Bilbao, Aivett; Gillilan, Richard E; Hu, Songnian; Padrón, Raúl

    2016-03-27

    Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles. PMID:26851071

  19. Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

    PubMed Central

    Shu, Shi; Liu, Xiong; Korn, Edward D.

    2005-01-01

    Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II. PMID:15671182

  20. Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.

    PubMed

    Clow, P A; Chen, T; Chisholm, R L; McNally, J G

    2000-06-01

    During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

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

    PubMed Central

    Spudich, James A.; Sivaramakrishnan, Sivaraj

    2010-01-01

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

  2. Myosin II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

    PubMed Central

    Song, Feifei; Sang, Helen M.; Martin, René; Knölker, Hans-Joachim; MacDonald, Michael P; Weijer, Cornelis J

    2016-01-01

    Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables. PMID:25812521

  3. Regulation of myosin II during cytokinesis in higher eukaryotes.

    PubMed

    Matsumura, Fumio

    2005-07-01

    Cellular myosin II is the principal motor responsible for cytokinesis. In higher eukaryotes, phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Because signals transmitted by the mitotic spindle coordinate key spatial and temporal aspects of cytokinesis, such signals should ultimately function to activate myosin II. Thus, it follows that identification of regulatory factors involved in MLC phosphorylation should elucidate the nature of spindle-derived regulatory signals and lead to a model for how they control cytokinesis. However, the identity of these upstream molecules remains elusive. This review (which is part of the Cytokinesis series) summarizes current views of the regulatory pathway controlling MLC phosphorylation and features four candidate molecules that are likely immediate upstream myosin regulators. I discuss proposed functions for MLCK, ROCK, citron kinase and myosin phosphatase during cytokinesis and consider the possibility of a link between these molecules and the signals transmitted by the mitotic spindle.

  4. Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

    PubMed Central

    Johnson, Terezina M.; Rajfur, Zenon; Jacobson, Ken

    2007-01-01

    The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes. PMID:17538016

  5. Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick.

    PubMed Central

    Hoh, J Y; McGrath, P A; White, R I

    1976-01-01

    1. A method is described for the electrophoretic analysis of intact myosin in polyacrylamide gel in a buffer system containing 0.02 M-pyrophosphate and 10% (v/v) glycerol, pH 8.8. 2. In this system chicken skeletal-muscle myosins reveal five distinct electrophoretic components, three components from the fast-twitch posterior latissimus dorsi muscle and two slower-migrating components from the slow-twitch anterior latissimus dorsi muscle. 3. The Ca2+-activated ATPase (adenosine triphosphatase) activity of myosin components was measured by densitometric scanning of the gel for the Ca3(PO4)2 precipitate formed during the ATPase reaction and subsequently for stained protein. Each component from the same muscle appears to have identical ATPase activity, but components from the fast-twitch muscle had an activity 2.2 times higher than those from the slow-twitch muscle. 4. On re-electrophoresis in the same buffer system, individual fractions of fast-twitch myosin did not reproduce the three-band pattern of the original myosin, but migrated at rates consistent with their original mobility. 5. Analysis of the mobility of the three fast-twitch myosin components in gels of different concentrations suggests that they are not stable oligomers of each other. 6. It is suggested that these components of fast-twitch myosin and slow-twitch myosin are isoenzymes of myosin. PMID:183747

  6. Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle.

    PubMed

    O'Connell, Kathleen; Gannon, Joan; Doran, Philip; Ohlendieck, Kay

    2007-08-01

    Extended longevity is often accompanied by frailty and increased susceptibility to a variety of crippling disorders. One of the most striking features of human aging is sarcopenia, which is defined as the age-related decline in skeletal muscle mass and strength. Although various metabolic and functional defects in aging muscle fibres have been described over the last decade, it is not known whether a pathophysiological hierarchy exists within degenerative pathways leading to muscle wasting. Hence, in order to identify novel biomarkers of age-dependent skeletal muscle degeneration, we have here applied mass spectrometry-based proteomics for studying global muscle protein expression patterns. As a model system of sarcopenia, we have employed crude extracts from senescent rat gastrocnemius muscle, as compared to young adult tissue preparations. Using the highly sensitive protein dye Deep Purple for the analysis of the 2-D separated muscle proteome and peptide mass fingerprinting for the identification of individual protein spots, a differential expression pattern was observed for contractile proteins, metabolic factors, regulatory components and heat shock elements. A drastic increase was shown for alpha B-crystallin, myosin light chain MLC-1, phosphoglycerate kinase, adenylate kinase, triosephosphate isomerase, albumin, aconitase and nucleoside-diphosphate kinase in aged fibres. In contrast, the expression of pyruvate kinase, aldolase, creatine kinase, transferrin, alpha-tropomyosin and myosin light chain MLC-3 was decreased in old skeletal muscle. Comparative 2-D immunoblotting of selected candidate proteins has confirmed the effect of aging on the skeletal muscle proteome. These findings demonstrate a severely perturbed protein expression pattern in aged skeletal muscle, which reflects the underlying molecular alterations causing a drastic decline of muscle strength in the senescent organism. In the long-term, the systematic deduction of abnormal protein expression

  7. The energetics of allosteric regulation of ADP release from myosin heads.

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  8. The energetics of allosteric regulation of ADP release from myosin heads.

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  9. Redox-sensitive residue in the actin-binding interface of myosin.

    PubMed

    Moen, Rebecca J; Cornea, Sinziana; Oseid, Daniel E; Binder, Benjamin P; Klein, Jennifer C; Thomas, David D

    2014-10-24

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  10. Redox-sensitive residue in the actin-binding interface of myosin

    PubMed Central

    Moen, Rebecca J.; Cornea, Sinziana; Oseid, Daniel E.; Binder, Benjamin P.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  11. Phosphoproteomic analysis of aged skeletal muscle.

    PubMed

    Gannon, Joan; Staunton, Lisa; O'Connell, Kathleen; Doran, Philip; Ohlendieck, Kay

    2008-07-01

    One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phospho-specific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month- and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin alpha, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins. PMID:18575773

  12. Neurotensinergic Augmentation of Glutamate Release at the Perforant Path-Granule Cell Synapse in Rat Dentate Gyrus: Roles of L-Type Ca2+ Channels, Calmodulin and Myosin Light-Chain Kinase

    PubMed Central

    Zhang, Haopeng; Dong, Hailong; Lei, Saobo

    2015-01-01

    Neurotensin (NT) serves as a neuromodulator in the brain where it is involved in modulating a variety of physiological functions including nociception, temperature, blood pressure and cognition, and many neurological diseases such as Alzheimer’s disease, schizophrenia and Parkinson’s disease. Whereas there is compelling evidence demonstrating that NT facilitates cognitive processes, the underlying cellular and molecular mechanisms have not been fully determined. Because the dentate gyrus expresses high densities of NT and NT receptors, we examined the effects of NT on the synaptic transmission at the synapse formed between the perforant path (PP) and granule cells (GC) in the rats. Our results demonstrate that NT persistently increased the amplitude of the AMPA receptor-mediated EPSCs at the PP-GC synapse. NT-induced increases in AMPA EPSCs were mediated by presynaptic NTS1 receptors. NT reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that NT facilitates presynaptic glutamate release. NT increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. NT-mediated augmentation of glutamate release required the influx of Ca2+ via L-type Ca2+ channels and the functions of calmodulin and myosin light chain kinase. Our results provide a cellular and molecular mechanism to explain the roles of NT in the hippocampus. PMID:25842242

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2014-05-01

    Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

  16. Space flight effects on skeletal bones of rats (light and electron microscopic examination).

    PubMed

    Yagodovsky, V S; Triftanidi, L A; Gorokhova, G P

    1976-07-01

    In the light and electron microscopes, long tubular bones of Wistar rats that were flown for 22 d onboard the Cosmos-605 biosatellite and were exposed to a ground-based simulation experiment were examined. About half of the flight rats showed osteoporosis of metaphyses which was usually combined with a decrease of the mass of the primary spongiosa in the vicinity of the epiphyseal cartilaginous plate. This gives evidence that the growth of the bones could have been inhibited in flight. The light and electron microscopy of bones of flight rats revealed wide osteocyte lacunae which could have been produced by perilacunar osteolysis. In the simulation experiment, reduction in the metaphyseal spongiosa occurred only in one-third of the rats and was less pronounced than in flight rats; no decrease of the mass of the primary spongiosa near the cartilaginous plate was noted. Histological investigation of bones 27 d postflight demonstrated that that time period was not enough to eliminate all the changes in the bones tested.

  17. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad

    PubMed Central

    Ono, Kanako; Ono, Shoichiro

    2016-01-01

    The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle–like contractile apparatuses, it has a striated muscle–like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  18. Metal cation controls myosin and actomyosin kinetics

    PubMed Central

    Tkachev, Yaroslav V; Ge, Jinghua; Negrashov, Igor V; Nesmelov, Yuri E

    2013-01-01

    We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time-resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number. PMID:24115140

  19. Directional Mechanosensing in Myosin VI

    NASA Astrophysics Data System (ADS)

    Yang, Yubo; Tehver, Riina

    2013-03-01

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

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

    SciTech Connect

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

    1987-10-01

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

  1. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD. PMID:27440420

  2. Recombinant bovine somatotropin (rbST) administration to creep-fed beef calves increases muscle mass but does not affect satellite cell number or concentration of myosin light chain-1f mRNA.

    PubMed

    Vann, R C; Althen, T G; Smith, W K; Veenhuizen, J J; Smith, S B

    1998-05-01

    Our objective in this study was to determine the effect of recombinant bovine somatotropin (rbST) on indices of muscle development in creep-fed beef calves. Crossbred steer calves were assigned to one of two treatment groups: control (sham-injected; n = 12) or rbST-treated (.09 mg x kg(-1) x d(-1); n = 12). Calves were injected every 14 d starting at d 28 of age and were weaned at 205 d of age. Supplemental creep feed was supplied free access to all calves to compensate for an expected increased protein and energy requirement in calves given rbST. Biopsy (d 100) and slaughter (d 206) samples of semitendinosus muscle were evaluated for satellite cell, myofiber nuclei numbers, and myosin light chain (MLC-1f) mRNA quantification. Myofiber nuclei and satellite cell numbers per 100 myofibers and MLC-1f mRNA:rRNA ratios at 100 and 206 d of age were not different (P > .10) between control and rbST-treated calves. Total gain, ADG, quality grade, femur length, percentage kidney, pelvic, and heart fat, dressing percentage, plasma IGF-I, and plasma urea nitrogen concentrations did not differ (P > .10) between control and rbST-treated calves. However, rbST-treated calves had larger longissimus muscle areas (P < .03), less marbling (P < .001), higher carcass conformation scores (P < .04), greater mass of separated muscle (P < .03), more ground meat (P < .01), and heavier carcass weights (P < .05) than control calves. Thus, rbST treatment increased muscle characteristics while nuclei number and MLC-1f mRNA concentrations remained the same, implying that the additional muscle growth was in a normal fashion. PMID:9621943

  3. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

  4. Species differences in the effects of prostaglandins on inositol trisphosphate accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cyclic AMP system.

    PubMed

    Yousufzai, S Y; Chen, A L; Abdel-Latif, A A

    1988-12-01

    Comparative studies on the effects of prostaglandins (PGs) on 1,2-diacylglycerol, measured as phosphatidic acid (PA), and inositol trisphosphate (IP3) production, cyclic AMP (cAMP) formation, myosin light chain (MLC) phosphorylation and contraction in the iris sphincter smooth muscle of rabbit, bovine and other mammalian species were undertaken and functional and biochemical relationships between the IP3-Ca++ and cAMP second messenger systems were demonstrated. The findings obtained from these studies can be summarized as follows: 1) all PGs investigated, including PGE2, PGF2 alpha, PGF2 alpha-ester, PGE1 and PGA2 increased IP3 accumulation and PA formation, and the extent of stimulation was dependent on the animal species. Thus, PGF2 alpha-ester (1 microM), the most potent of the PGs, increased IP3 accumulation in rabbit and bovine sphincters by 33 and 58%, respectively, and increased PA formation by 67 and 56%, respectively. The PG increased IP3 accumulation in both rabbit and bovine sphincters very rapidly (T1/2 values about 26 sec) and in a dose-dependent manner. 2) The PG had no effect on MLC phosphorylation in the rabbit sphincter, but it increased that of the bovine by 36%. 3) The PG increased cAMP formation by 75% in the rabbit sphincter but it had no effect on that of the bovine. 4) The PG induced a maximal contractile response in the bovine sphincter but it had no effect on that of the rabbit. 5) In the bovine, PGA2 induced IP3 accumulation and contraction, without an effect on cAMP formation; however, in the rabbit, cat and dog it increased cAMP formation and had no effect on IP3 accumulation and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Proper expression of myosin genes in transgenic nematodes.

    PubMed Central

    Fire, A; Waterston, R H

    1989-01-01

    Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product. Images PMID:2583105

  6. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

    PubMed Central

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-01-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

  7. Small-molecule inhibitors of myosin proteins

    PubMed Central

    Bond, Lisa M; Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2014-01-01

    Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design. PMID:23256812

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

    SciTech Connect

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

    2009-01-01

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

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

    SciTech Connect

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

    2008-04-25

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

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

    NASA Astrophysics Data System (ADS)

    Nie, Wei

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

  11. Dynamics of myosin II organization into cortical contractile networks and fibers

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  12. Myosin VI: cellular functions and motor properties.

    PubMed Central

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

    2004-01-01

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

  13. Myosin VI: cellular functions and motor properties.

    PubMed

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

    2004-12-29

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

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

    PubMed Central

    Root, D D; Reisler, E

    1992-01-01

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

  15. Proteomic analysis of skeletal deformity in diploid and triploid rainbow trout (Oncorhynchus mykiss) larvae.

    PubMed

    Babaheydari, Samad Bahrami; Keyvanshokooh, Saeed; Dorafshan, Salar; Johari, Seyed Ali

    2016-09-01

    A proteomic screening approach was employed to achieve a better understanding of the changes that occur in protein expression patterns associated with skeletal deformities in both diploid and triploid rainbow trout larvae. Triploidy was induced through the application of heat shock of 28°C for 10min to eggs 10-min post fertilization in an aquarium equipped with a heater. Percentage of skeletal deformity in heat-shocked larvae (2.88±0.30, mean±S.E.) was significantly (P<0.05) greater than that of the diploids (0.55±0.24). At five days after hatching, proteins of normal and deformed specimens of deyolked larvae were subjected to proteomic analysis using two-dimensional electrophoresis and mass spectrometry. Among the identified protein spots from diploids, creatine kinase was found to be increased in larvae with skeletal deformities, while apolipoprotein A-I-2, apolipoprotein A-II and calmodulin were found to be decreased in deformed fish. Among the five protein spots that were identified in heat-shocked fish, apolipoprotein A-I-2, apolipoprotein A-II, parvalbumin, myosin light chain 1-1 and nucleoside diphosphate kinase were found to be decreased in deformed larvae. The identification of nine protein spots showing altered expression in deformed fish allows us to reach a preliminary view of the molecular mechanisms that are involved in the development of skeletal malformations in diploid and triploid fish.

  16. Proteomic analysis of skeletal deformity in diploid and triploid rainbow trout (Oncorhynchus mykiss) larvae.

    PubMed

    Babaheydari, Samad Bahrami; Keyvanshokooh, Saeed; Dorafshan, Salar; Johari, Seyed Ali

    2016-09-01

    A proteomic screening approach was employed to achieve a better understanding of the changes that occur in protein expression patterns associated with skeletal deformities in both diploid and triploid rainbow trout larvae. Triploidy was induced through the application of heat shock of 28°C for 10min to eggs 10-min post fertilization in an aquarium equipped with a heater. Percentage of skeletal deformity in heat-shocked larvae (2.88±0.30, mean±S.E.) was significantly (P<0.05) greater than that of the diploids (0.55±0.24). At five days after hatching, proteins of normal and deformed specimens of deyolked larvae were subjected to proteomic analysis using two-dimensional electrophoresis and mass spectrometry. Among the identified protein spots from diploids, creatine kinase was found to be increased in larvae with skeletal deformities, while apolipoprotein A-I-2, apolipoprotein A-II and calmodulin were found to be decreased in deformed fish. Among the five protein spots that were identified in heat-shocked fish, apolipoprotein A-I-2, apolipoprotein A-II, parvalbumin, myosin light chain 1-1 and nucleoside diphosphate kinase were found to be decreased in deformed larvae. The identification of nine protein spots showing altered expression in deformed fish allows us to reach a preliminary view of the molecular mechanisms that are involved in the development of skeletal malformations in diploid and triploid fish. PMID:27219664

  17. Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

    PubMed Central

    Kron, S J; Drubin, D G; Botstein, D; Spudich, J A

    1992-01-01

    The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin. Images PMID:1533933

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

    PubMed Central

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

    2014-01-01

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

  19. Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

    NASA Technical Reports Server (NTRS)

    Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

    1992-01-01

    This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

  20. Myosin heavy chain expression in rabbit masseter muscle during postnatal development.

    PubMed Central

    Bredman, J J; Weijs, W A; Korfage, H A; Brugman, P; Moorman, A F

    1992-01-01

    The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' alpha-MHC. During postnatal growth myosin transitions take place from neonatal and fast (IIA, IIA/IIB--referring to a fibre containing both IIA and IIB MHCs) MHC to adult 'cardiac' alpha-MHC and I/alpha-MHC. Since there is a temporary population of fibres containing IIA/alpha-MHC during the first 4 wk of development with a peak in the 3rd to 4th wk, the transition from IIA-MHC to alpha-MHC may occur in these IIA/alpha-MHC-containing fibres. The appearance of 'cardiac' alpha-MHC coincides with the timing of weaning, suggesting that the changes in MHC content, that probably result in a transition to a lower speed of contraction, have functional significance related to weaning. The finding of neonatal MHC in adult rabbits indicates that the masseter develops at a rate and in a way that is distinct from most other skeletal muscles. A spatiotemporal variation in expression of myosin isozymes within the masseter was observed, with many fibres containing more than one myosin type, indicating developmentally regulated spatial differences in function. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:1387129

  1. Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells

    PubMed Central

    Bhat, Purnima

    2009-01-01

    Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (−)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (−)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening. PMID:19158378

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

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

  4. The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion.

    PubMed

    Pinto, Antonio; Sánchez, Fredi; Alamo, Lorenzo; Padrón, Raúl

    2012-12-01

    Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures. PMID:22982253

  5. Myosin lever arm directs collective motion on cellular actin network

    PubMed Central

    Hariadi, Rizal F.; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-01-01

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions. PMID:24591646

  6. Myosin lever arm directs collective motion on cellular actin network.

    PubMed

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2009-04-01

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

  9. Evaluation of Acanthamoeba myosin-IC as a potential therapeutic target.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

    PubMed Central

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

    2014-01-01

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

  13. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

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

  14. Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii.

    PubMed

    Kuznicki, J; Atkinson, M A; Korn, E D

    1984-07-25

    Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed. PMID:6235225

  15. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles.

    PubMed

    Gally, Christelle; Wissler, Frédéric; Zahreddine, Hala; Quintin, Sophie; Landmann, Frédéric; Labouesse, Michel

    2009-09-01

    Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4. PMID:19675126

  16. Myosin II Dynamics during Embryo Morphogenesis

    NASA Astrophysics Data System (ADS)

    Kasza, Karen

    2013-03-01

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

  17. New insights into myosin evolution and classification.

    PubMed

    Foth, Bernardo J; Goedecke, Marc C; Soldati, Dominique

    2006-03-01

    Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

  18. Sex-Based Differences in Skeletal Muscle Kinetics and Fiber-Type Composition

    PubMed Central

    Haizlip, K. M.; Harrison, B. C.

    2015-01-01

    Previous studies have identified over 3,000 genes that are differentially expressed in male and female skeletal muscle. Here, we review the sex-based differences in skeletal muscle fiber composition, myosin heavy chain expression, contractile function, and the regulation of these physiological differences by thyroid hormone, estrogen, and testosterone. The findings presented lay the basis for the continued work needed to fully understand the skeletal muscle differences between males and females. PMID:25559153

  19. Supervillin binding to myosin II and synergism with anillin are required for cytokinesis.

    PubMed

    Smith, Tara C; Fridy, Peter C; Li, Yinyin; Basil, Shruti; Arjun, Sneha; Friesen, Ryan M; Leszyk, John; Chait, Brian T; Rout, Michael P; Luna, Elizabeth J

    2013-12-01

    Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II-binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831-1281, which bind central spindle proteins, and residues 1-170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis. PMID:24088567

  20. Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

    PubMed Central

    Giuliano, K A; Kolega, J; DeBiasio, R L; Taylor, D L

    1992-01-01

    The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors. Images PMID:1421576

  1. Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

    PubMed

    Al-Haddad, A; Shonn, M A; Redlich, B; Blocker, A; Burkhardt, J K; Yu, H; Hammer, J A; Weiss, D G; Steffen, W; Griffiths, G; Kuznetsov, S A

    2001-09-01

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

  2. The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

    PubMed

    He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

    2015-01-01

    Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

  3. The non-linear elasticity of the muscle sarcomere and the compliance of myosin motors

    PubMed Central

    Fusi, Luca; Brunello, Elisabetta; Reconditi, Massimo; Piazzesi, Gabriella; Lombardi, Vincenzo

    2014-01-01

    Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre–microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4°C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa−1 (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model. PMID:24344166

  4. Aberrant post-translational modifications compromise human myosin motor function in old age.

    PubMed

    Li, Meishan; Ogilvie, Hannah; Ochala, Julien; Artemenko, Konstantin; Iwamoto, Hiroyuki; Yagi, Naoto; Bergquist, Jonas; Larsson, Lars

    2015-04-01

    Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

  5. Cross-reactivity of termite myosin; a potential allergen

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Catch-slip bonds can be dispensable for motor force regulation during skeletal muscle contraction

    NASA Astrophysics Data System (ADS)

    Dong, Chenling; Chen, Bin

    2015-07-01

    It is intriguing how multiple molecular motors can perform coordinated and synchronous functions, which is essential in various cellular processes. Recent studies on skeletal muscle might have shed light on this issue, where rather precise motor force regulation was partly attributed to the specific stochastic features of a single attached myosin motor. Though attached motors can randomly detach from actin filaments either through an adenosine triphosphate (ATP) hydrolysis cycle or through "catch-slip bond" breaking, their respective contribution in motor force regulation has not been clarified. Here, through simulating a mechanical model of sarcomere with a coupled Monte Carlo method and finite element method, we find that the stochastic features of an ATP hydrolysis cycle can be sufficient while those of catch-slip bonds can be dispensable for motor force regulation.

  7. Myosin II Activity Softens Cells in Suspension.

    PubMed

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

    2015-04-21

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  8. Myosin II Activity Softens Cells in Suspension

    PubMed Central

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

    2015-01-01

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  9. Myosin II Activity Softens Cells in Suspension.

    PubMed

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

    2015-04-21

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

  10. Disrupted myosin cross-bridge cycling kinetics triggers muscle weakness in nebulin-related myopathy.

    PubMed

    Ochala, Julien; Lehtokari, Vilma-Lotta; Iwamoto, Hiroyuki; Li, Meishan; Feng, Han-Zhong; Jin, Jian-Ping; Yagi, Naoto; Wallgren-Pettersson, Carina; Pénisson-Besnier, Isabelle; Larsson, Lars

    2011-06-01

    Nebulin is a giant protein expressed at high levels in skeletal muscle. Mutations in the nebulin gene (NEB) lead to muscle weakness and various congenital myopathies. Despite increasing clinical and scientific interest, the pathogenesis of weakness remains unknown. The present study, therefore, aims at unraveling the underlying molecular mechanisms. Hence, we recorded and analyzed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibers expressing nebulin mutations. Results demonstrated that, during contraction, the cycling rate of myosin heads attaching to actin is dramatically perturbed, causing a reduction in the fraction of myosin-actin interactions in the strong binding state. This phenomenon prevents complete thin-filament activation, more especially proper and full tropomyosin movement, further limiting additional binding of myosin cross-bridges. At the cell level, this reduces the force-generating capacity and, overall, provokes muscle weakness. To reverse such a negative cascade of events, future potential therapeutic interventions should, therefore, focus on the triggering component, the altered myosin cross-bridge cycling kinetics. PMID:21350120

  11. A mechanochemical model for myosin VI

    NASA Astrophysics Data System (ADS)

    Tehver, Riina; Jack, Amanda; Lowe, Ian

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

  12. Unique sequences and predicted functions of myosins in Tetrahymena thermophila.

    PubMed

    Sugita, Maki; Iwataki, Yoshinori; Nakano, Kentaro; Numata, Osamu

    2011-07-01

    Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.

  13. Platelet contractile proteins: separation and characterization of the actin and myosin-like components.

    PubMed

    Cove, D H; Crawford, N

    1975-01-01

    Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution

  14. Characterization of Amoeba proteus myosin VI immunoanalog.

    PubMed

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

    2005-07-01

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

  15. Dynamic exchange of myosin molecules between thick filaments.

    PubMed

    Saad, A D; Pardee, J D; Fischman, D A

    1986-12-01

    To examine thick filament assembly and myosin exchange, a fluorescence energy transfer assay has been established. Assembly-competent myosin molecules labeled with the sulfhydryl-specific fluorochromes 5-(2-[(iodoacetyl)-amino]ethyl)aminonaphthalene-1-sulfonic acids (IAEDANS) or 5-iodoacetamidofluorescein (IAF) were prepared. Using IAEDANS-labeled myosin as fluorescence donor and IAF-labeled myosin as acceptor, thick filament formation was followed by the decrease in donor fluorescence at 0.1 M KCl/10 mM potassium phosphate, pH 6.9. The critical concentration of myosin--i.e., that concentration that remained unassembled at equilibrium with fully formed filaments--was 40 nM. In FET and 125I-labeled myosin incorporation assays, extensive exchange of myosin between thick filaments was observed. The presence of a critical concentration and the measurements of extensive exchange suggest a dynamic equilibrium between fully polymerized myosin and a small pool of soluble myosin.

  16. Major nonhistone proteins of rat liver chromatin: preliminary identification of myosin, actin, tubulin, and tropomyosin.

    PubMed Central

    Douvas, A S; Harrington, C A; Bonner, J

    1975-01-01

    Two major nonhistone polypeptides from rat liver chromatin have been identified as myosin and actin. Preliminary observations indicate that three other chromatin polypeptides of molecular weights 50,000, 34,000, and 32,000 are tubulin and heavy and light tropomyosin, respectively. A sixth component of molecular weight 65,000 which has been purified and electrophoreses as a single band on sodium dodecyl sulfate-polyacrylamide gels may be composed in part of protease-digested myosin. These six polypeptides together account for as much as 38% of the nonhistone protein mass of chromatin in this tissue. Images PMID:1060072

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

    PubMed Central

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

    2016-01-01

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

  18. Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

    PubMed Central

    Schoffstall, Brenda; Brunet, Nicolas M; Williams, Shanedah; Miller, Victor F; Barnes, Alyson T; Wang, Fang; Compton, Lisa A; McFadden, Lori A; Taylor, Dianne W; Seavy, Margaret; Dhanarajan, Rani; Chase, P Bryant

    2006-01-01

    Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca2+ activation. To determine whether Ca2+ sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and α-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca2+ sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca2+ sensitivity. PMID:17008370

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

    PubMed

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

    2016-03-29

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

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

    PubMed Central

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

    2016-01-01

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

  1. Modulating myosin restores muscle function in a mouse model of nemaline myopathy

    PubMed Central

    Lindqvist, Johan; Levy, Yotam; Pati‐Alam, Alisha; Hardeman, Edna C.; Gregorevic, Paul

    2016-01-01

    Objective Nemaline myopathy, one of the most common congenital myopathies, is associated with mutations in various genes including ACTA1. This disease is also characterized by various forms/degrees of muscle weakness, with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force‐generating capacity of muscle fibers. ACTA1 mutations are also associated with variations in myofiber size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (1) would be directly due to the inefficient actomyosin complex and (2) could be restored by manipulating myosin expression. Methods We used a knockin mouse model expressing the ACTA1 His40Tyr actin mutation found in human patients. We then performed in vivo intramuscular injections of recombinant adeno‐associated viral vectors harboring a myosin transgene known to facilitate muscle contraction. Results We observed that in the presence of the transgene, the intrinsic force‐generating capacity was restored and myofiber size was normal. Interpretation This demonstrates a direct link between disrupted attachment of myosin molecules to actin monomers and muscle fiber atrophy. These data also suggest that further therapeutic interventions should primarily target myosin dysfunction to alleviate the pathology of ACTA1‐related nemaline myopathy. Ann Neurol 2016;79:717–725 PMID:26891371

  2. Chlamydia trachomatis inclusion membrane protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms

    PubMed Central

    Lutter, Erika I.; Barger, Alexandra C.; Nair, Vinod; Hackstadt, Ted

    2013-01-01

    Summary Chlamydia trachomatis replicates within a membrane bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion while the phosphorylated inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), and myosin IIA and IIB also colocalized with inactive MYPT1. The role of these proteins was examined in the context of host-cell exit mechanisms; cell lysis or extrusion of intact inclusions. Inhibition of myosin II or siRNA depletion of myosin IIA and IIB, MLC2, or MLCK reduced chlamydial extrusion thus favoring lytic events as the primary means of release. These studies provide insights into regulation of egress mechanisms by C. trachomatis. PMID:23727243

  3. Phototherapy in skeletal muscle performance and recovery after exercise: effect of combination of super-pulsed laser and light-emitting diodes.

    PubMed

    Antonialli, Fernanda Colella; De Marchi, Thiago; Tomazoni, Shaiane Silva; Vanin, Adriane Aver; dos Santos Grandinetti, Vanessa; de Paiva, Paulo Roberto Vicente; Pinto, Henrique Dantas; Miranda, Eduardo Foschini; de Tarso Camillo de Carvalho, Paulo; Leal-Junior, Ernesto Cesar Pinto

    2014-11-01

    Recent studies with phototherapy have shown positive results in enhancement of performance and improvement of recovery when applied before exercise. However, several factors still remain unknown such as therapeutic windows, optimal treatment parameters, and effects of combination of different light sources (laser and LEDs). The aim of this study was to evaluate the effects of phototherapy with the combination of different light sources on skeletal muscle performance and post-exercise recovery, and to establish the optimal energy dose. A randomized, double-blinded, placebo-controlled trial with participation of 40 male healthy untrained volunteers was performed. A single phototherapy intervention was performed immediately after pre-exercise (baseline) maximum voluntary contraction (MVC) with a cluster of 12 diodes (4 of 905 nm lasers-0.3125 mW each, 4 of 875 nm LEDs-17.5 mW each, and 4 of 670 nm LEDs-15 mW each- manufactured by Multi Radiance Medical™) and dose of 10, 30, and 50 J or placebo in six sites of quadriceps. MVC, delayed onset muscle soreness (DOMS), and creatine kinase (CK) activity were analyzed. Assessments were performed before, 1 min, 1, 24, 48, 72, and 96 h after eccentric exercise protocol employed to induce fatigue. Phototherapy increased (p < 0.05) MVC was compared to placebo from immediately after to 96 h after exercise with 10 or 30 J doses (better results with 30 J dose). DOMS was significantly decreased compared to placebo (p < 0.05) with 30 J dose from 24 to 96 h after exercise, and with 50 J dose from immediately after to 96 h after exercise. CK activity was significantly decreased (p < 0.05) compared to placebo with all phototherapy doses from 1 to 96 h after exercise (except for 50 J dose at 96 h). Pre-exercise phototherapy with combination of low-level laser and LEDs, mainly with 30 J dose, significantly increases performance, decreases DOMS, and improves biochemical marker related to skeletal muscle

  4. Change in the actin-myosin subfragment 1 interaction during actin polymerization.

    PubMed

    Chaussepied, P; Kasprzak, A A

    1989-12-01

    To better characterize the conformational differences of G- and F-actin, we have compared the interaction between G- and F-actin with myosin subfragment 1 (S1) which had part of its F-actin binding site (residues 633-642) blocked by a complementary peptide or "antipeptide" (Chaussepied, P., and Morales, M. F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475). Light scattering, sedimentation, and electron microscopy measurements showed that, with the antipeptide covalently attached to the S1 heavy chain, S1 was not capable of inducing G-actin polymerization in the absence of salt. Moreover, the antipeptide-carrying S1 did not change the fluorescence polarization of 5-[2-(iodoacetyl)-aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled G-actin or of 1,5-IAEDANS-labeled actin dimer, compared to the control S1. This result, interpreted as a lack of interaction between G-actin and antipeptide-carrying S1, was confirmed further by the following experiments: in the presence of G-actin, antipeptide.S1 heavy chain was not protected against trypsin and papain proteolysis, and G-actin could not be cross-linked to antipeptide.S1 by 1-ethyl-3[-3-(dimethylamino)propyl]carbodiimide. In contrast, similar experiments showed that antipeptide.S1 was able to interact with nascent F-actin and with F-actin. Thus, blocking the stretch 633-642 of S1 heavy chain by the antipeptide strongly inhibits G-actin-S1 interaction but only slightly alters F-actin-S1 contact. We, therefore postulate that this stretch of skeletal S1 heavy chain is essential for G-actin-S1 interaction and that the G-F transformation generates new S1 binding site(s) on the actin molecule.

  5. Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

    PubMed

    Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

    2013-03-01

    In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

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

    PubMed Central

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

    2015-01-01

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

  7. Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck?

    PubMed

    Ratti, Joyce; Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2011-04-01

    Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

  8. Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

    PubMed

    Mizutani, Takeomi; Li, Rui; Haga, Hisashi; Kawabata, Kazushige

    2015-09-18

    The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

  9. Human myoblasts from skeletal muscle biopsies: in vitro culture preparations for morphological and cytochemical analyses at light and electron microscopy.

    PubMed

    Malatesta, Manuela; Giagnacovo, Marzia; Cardani, Rosanna; Meola, Giovanni; Pellicciari, Carlo

    2013-01-01

    We describe protocols for the isolation of satellite cells from human muscle biopsies, for the in vitro culture of proliferating and differentiating myoblasts, and for the preparation of cell samples suitable for morphological and cytochemical analyses at light and electron microscopy. The procedures described are especially appropriate for processing small muscle biopsies, and allow obtaining myoblast/myotube monolayers on glass coverslips, thus preserving good cell morphology and immunoreactivity for protein markers of myoblast proliferation, differentiation, and senescence.These cell preparations are suitable for cytochemical, immunocytochemical, and FISH procedures at light microscopy, and can be observed not only in bright field, phase contrast, and differential interference contrast but also in fluorescence (which can hardly be used for cells grown on conventional plastic surfaces, which generally exhibit intense autofluorescence). In their ultrastructural cytochemical application, the protocols are intended for post-embedding techniques, by which ultrathin sections from a single sample may be used for detecting a wide variety of molecular markers. PMID:23400435

  10. Skeletal Dysplasias

    PubMed Central

    Krakow, Deborah

    2015-01-01

    Synoposis The skeletal dysplasias are a group of more than 450 heritable disorders of bone. They frequently present in the newborn period with disproportion, radiographic abnormalities, and occasionally other organ system abnormalities. For improved clinical care it is important to determine a precise diagnosis to aid in management, familial recurrence and identify those disorders highly associated with mortality. Long-term management of these disorders is predicated on an understanding of the associated skeletal system abnormalities and these children are best served by a team approach to health care surveillance. PMID:26042906

  11. Cell and molecular biology of the fastest myosins.

    PubMed

    Higashi-Fujime, Sugie; Nakamura, Akio

    2009-01-01

    Chara myosin is a class XI plant myosin in green algae Chara corallina and responsible for fast cytoplasmic streaming. The Chara myosin exhibits the fastest sliding movement of F-actin at 60 mum/s as observed so far, 10-fold of the shortening speed of muscle. It has some distinct properties differing from those of muscle myosin. Although knowledge about Chara myosin is very limited at present, we have tried to elucidate functional bases of its characteristics by comparing with those of other myosins. In particular, we have built the putative atomic model of Chara myosin by using the homology-based modeling system and databases. Based on the putative structure of Chara myosin obtained, we have analyzed the relationship between structure and function of Chara myosin to understand its distinct properties from various aspects by referring to the accumulated knowledge on mechanochemical and structural properties of other classes of myosin, particularly animal and fungal myosin V. We will also discuss the functional significance of Chara myosin in a living cell.

  12. The fraction of strongly bound cross-bridges is increased in mice that carry the myopathy-linked myosin heavy chain mutation MYH4L342Q

    PubMed Central

    Lindqvist, Johan; Iwamoto, Hiroyuki; Blanco, Gonzalo; Ochala, Julien

    2013-01-01

    SUMMARY Myosinopathies have emerged as a new group of diseases and are caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. One major hallmark of these diseases is skeletal muscle weakness or paralysis, but the underlying molecular mechanisms remain unclear. Here, we have undertaken a detailed functional study of muscle fibers from Myh4arl mice, which carry a mutation that provokes an L342Q change within the catalytic domain of the type IIb skeletal muscle myosin protein MYH4. Because homozygous animals develop rapid muscle-structure disruption and lower-limb paralysis, they must be killed by postnatal day 13, so all experiments were performed using skeletal muscles from adult heterozygous animals (Myh4arl/+). Myh4arl/+ mice contain MYH4L342Q expressed at 7% of the levels of the wild-type (WT) protein, and are overtly and histologically normal. However, mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized fibers revealed, upon maximal Ca2+ activation, higher stiffness as well as altered meridional and equatorial reflections in Myh4arl/+ mice when compared with age-matched WT animals. Under rigor conditions, by contrast, no difference was observed between Myh4arl/+ and WT mice. Altogether, these findings prove that, in adult MYH4L342Q heterozygous mice, the transition from weak to strong myosin cross-bridge binding is facilitated, increasing the number of strongly attached myosin heads, thus enhancing force production. These changes are predictably exacerbated in the type IIb fibers of homozygous mice, in which the embryonic myosin isoform is fully replaced by MYH4L342Q, leading to a hypercontraction, muscle-structure disruption and lower-limb paralysis. Overall, these findings provide important insights into the molecular pathogenesis of skeletal myosinopathies. PMID:23335206

  13. Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction.

    PubMed

    Burghardt, Thomas P; Josephson, Matthew P; Ajtai, Katalin

    2011-09-13

    Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.

  14. [Role of myosins in depression of sensitivity of Helix neurons to acetylcholine in a cellular analog of habituation].

    PubMed

    Pivovarov, A S; Murzina, G B; Makhnovskiĭ, D A; Vasil'eva, N A; Tret'iakova, M S

    2013-01-01

    We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

  15. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    SciTech Connect

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-05-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with (..gamma..-/sup 32/P)ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo.

  16. Skeletal muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are approximately 650-850 muscles in the human body these include skeletal (striated), smooth and cardiac muscle. The approximation is based on what some anatomists consider separate muscle or muscle systems. Muscles are classified based on their anatomy (striated vs. smooth) and if they are v...

  17. Visualizing Key Hinges and a Potential Major Source of Compliance in the Lever Arm of Myosin

    SciTech Connect

    J Brown; V Senthil Kumar; E ONeall-Hennessey; L Reshetnikova; H Robinson; M Nguyen-McCarty; A Szent-Gyorgyi; C Cohen

    2011-12-31

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  18. Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

    SciTech Connect

    Brown, J.H.; Robinson, H.; Senthil Kumar, V. S.; O'Neall-Hennessey, E.; Reshetnikova, L.; Nguyen-McCarty, M.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-01-04

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  19. Diffraction rings obtained from a suspension of skeletal myofibrils by laser light illumination. Study of internal structure of sarcomeres.

    PubMed Central

    Ishiwata, S; Okamura, N

    1989-01-01

    Diffraction rings corresponding to the first, second, and third order were obtained by laser light illumination from a suspension of rabbit glycerinated psoas myofibrils (diameter, 1-2 microns; average length of the straight region, 44 microns; average sarcomere length, 2.2-2.6 microns) of which the optical thickness was appropriately chosen. Dispersed myofibrils were nearly randomly oriented in two dimensions, so that the effects of muscle volume were minimized; these effects usually interfere significantly with a quantitative analysis of laser optical diffraction in the fiber system. The diameters of diffraction rings represented the average sarcomere length. By using this system, we confirmed the ability of the unit cell (sarcomere) structure model to explain the intensity change of diffraction lines accompanying the dissociation from both ends of thick filaments in a high salt solution. The length of an A-band estimated from the relative intensity of diffraction rings and that directly measured on phase-contrast micrographs coincided well with each other. Also, we found that myofibrils with a long sarcomere length shorten to a slack length accompanying the decrease in overlap between thick and thin filaments produced by the dissociation of thick filaments. Images FIGURE 4 FIGURE 1 FIGURE 8 PMID:2692720

  20. Separation of cardiac myosin heavy chains by gradient SDS-PAGE.

    PubMed

    Esser, K A; Boluyt, M O; White, T P

    1988-09-01

    Separation of alpha- and beta-myosin heavy chains (MHCs) in cardiac ventricles of rats by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was accomplished and compared with the separation of myosin isozymes obtained with pyrophosphate gels. Whole muscle homogenates were electrophoresed on a 4-9% linear gradient SDS polyacrylamide gel for 3-4 h. MHC bands were identified by the migration distance relative to a MHC standard and immunoblot results with a monoclonal antibody to MHC. The MHC bands were further identified as alpha and beta based on the electrophoretic mobility of ventricular homogenates from hypothyroid and hyperthyroid rats and ventricular and slow soleus skeletal muscle homogenates from control rats. The beta-MHC migrated faster than alpha-MHC, and laser densitometry revealed separate peaks when both MHCs were present. With homogenates containing MHC ranging from 0 to 100% alpha, the separation of MHCs with gradient SDS-PAGE correlated highly (r = 0.97) with separation of myosin isozymes by pyrophosphate gel electrophoresis. The SDS-PAGE technique reported herein is a quick, valid, and direct method for the identification and quantification of ventricular MHCs.

  1. Preparation and Characterization of Myosin Proteins.

    ERIC Educational Resources Information Center

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  2. Laing early onset distal myopathy: slow myosin defect with variable abnormalities on muscle biopsy

    PubMed Central

    Lamont, P J; Udd, B; Mastaglia, F L; de Visser, M; Hedera, P; Voit, T; Bridges, L R; Fabian, V; Rozemuller, A; Laing, N G

    2006-01-01

    Background Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7. It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one form of dilated cardiomyopathy. However, the clinical picture of MPD1 is distinct from these three conditions. Objective To collate and discuss the histological features reported in the muscle biopsies of MPD1 patients and to outline the clinical features. Results The phenotype of MPD1 was consistent, with initial weakness of great toe/ankle dorsiflexion, and later development of weakness of finger extension and neck flexion. Age of onset was the only variable, being from birth up to the 20s, but progression was always very slow. The pathological features were variable. In this retrospective series, there were no pathognomonic diagnostic features, although atrophic type I fibres were found in half the families. Rimmed vacuoles are consistently seen in all other distal myopathies with the exception of Myoshi distal myopathy. However, they were found in a minority of patients with MPD1, and were not prominent when present. Immunohistochemical staining for slow and fast myosin showed co‐expression of slow and fast myosin in some type I fibres, possibly indicating a switch to type II status. This may be a useful aid to diagnosis. Conclusions The pathological findings in MPD1 are variable and appear to be affected by factors such as the specific muscle biopsied, the age of the patient at biopsy, and the duration of disease manifestations. PMID:16103042

  3. The contributions of filaments and cross-bridges to sarcomere compliance in skeletal muscle

    PubMed Central

    Brunello, Elisabetta; Caremani, Marco; Melli, Luca; Linari, Marco; Fernandez-Martinez, Manuel; Narayanan, Theyencheri; Irving, Malcolm; Piazzesi, Gabriella; Lombardi, Vincenzo; Reconditi, Massimo

    2014-01-01

    Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (Ccb) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa−1, close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa−1). Ccb calculated using these estimates is 0.37 ± 0.12 nm pN−1, a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN−1, obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to Ccb from the myosin tail that joins the head to the thick filament is excluded. The low Ccb value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle. PMID:25015916

  4. Mechanics of the power stroke in myosin II

    NASA Astrophysics Data System (ADS)

    Marcucci, L.; Truskinovsky, L.

    2010-05-01

    Power stroke in skeletal muscles is a result of a conformational change in the globular portion of the molecular motor myosin II. In this paper we show that the fast tension recovery data reflecting the inner working of the power stroke mechanism can be quantitatively reproduced by a Langevin dynamics of a simple mechanical system with only two structural states. The proposed model is a generalization of the two state model of Huxley and Simmons. The main idea is to replace the rigid bistable device of Huxley and Simmons with an elastic bistable snap spring. In this setting the attached configuration of a cross bridge is represented not only by the discrete energy minima but also by a continuum of intermediate states where the fluctuation induced dynamics of the system takes place. We show that such soft-spin approach explains the load dependence of the power stroke amplitude and removes the well-known contradiction inside the conventional two state model regarding the time scale of the power stroke.

  5. Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle

    SciTech Connect

    Giometti, C.S.; Barany, M.; Danon, M.J.; Anderson, N.G.

    1980-07-01

    High-resolution two-dimensional electrophoresis was used to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

  6. Skeletal muscle plasticity induced by seasonal acclimatization involves IGF1 signaling: implications in ribosomal biogenesis and protein synthesis.

    PubMed

    Fuentes, Eduardo N; Zuloaga, Rodrigo; Valdes, Juan Antonio; Molina, Alfredo; Alvarez, Marco

    2014-10-01

    One of the most fundamental biological processes in living organisms that are affected by environmental fluctuations is growth. In fish, skeletal muscle accounts for the largest proportion of body mass, and the growth of this tissue is mainly controlled by the insulin-like growth factor (IGF) system. By using the carp (Cyprinus carpio), a fish that inhabits extreme conditions during winter and summer, we assessed the skeletal muscle plasticity induced by seasonal acclimatization and the relation of IGF signaling with protein synthesis and ribosomal biogenesis. The expression of igf1 in muscle decreased during winter in comparison with summer, whereas the expression for both paralogues of igf2 did not change significantly between seasons. The expression of igf1 receptor a (igf1ra), but not of igf1rb, was down-regulated in muscle during the winter as compared to the summer. A decrease in protein contents and protein phosphorylation for IGF signaling molecules in muscle was observed in winter-acclimatized carp. This was related with a decreased expression in muscle for markers of myogenesis (myoblast determination factor (myod), myogenic factor 5 (myf5), and myogenin (myog)); protein synthesis (myosin heavy chain (mhc) and myosin light chain (mlc3 and mlc1b)); and ribosomal biogenesis (pre-rRNA and ribosomal proteins). IGF signaling, and key markers of ribosomal biogenesis, protein synthesis, and myogenesis were affected by seasonal acclimatization, with differential regulation in gene expression and signaling pathway activation observed in muscle between both seasons. This suggests that these molecules are responsible for the muscle plasticity induced by seasonal acclimatization in carp.

  7. Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

    PubMed

    Billington, Neil; Beach, Jordan R; Heissler, Sarah M; Remmert, Kirsten; Guzik-Lendrum, Stephanie; Nagy, Attila; Takagi, Yasuharu; Shao, Lin; Li, Dong; Yang, Yi; Zhang, Yingfan; Barzik, Melanie; Betzig, Eric; Hammer, John A; Sellers, James R

    2015-03-30

    Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

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

    PubMed Central

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

    2015-01-01

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

  9. Homozygous MYH7 R1820W mutation results in recessive myosin storage myopathy: scapuloperoneal and respiratory weakness with dilated cardiomyopathy.

    PubMed

    Yüceyar, Nur; Ayhan, Özgecan; Karasoy, Hatice; Tolun, Aslıhan

    2015-04-01

    Myosin storage myopathy (MSM) is a protein aggregate myopathy caused by the accumulation of myosin in muscle fibres and results from MYH7 mutation. Although MYH7 mutation is also an established cause of variable cardiomyopathy with or without skeletal myopathy, cardiomyopathy with MSM is a rare combination. Here, we update the clinical findings in the two brothers that we previously reported as having recessively inherited MSM characterized by scapuloperoneal distribution of weakness and typical hyaline-like bodies in type 1 muscle fibres. One of the patients, weak from childhood but not severely symptomatic until 28 years of age, had an unusual combination of MSM, severe dilated cardiomyopathy, and respiratory impairment at the age of 44 years. We identified homozygous missense mutation c.5458C>T (p.R1820W) in exon 37 in these patients as the second recessive MYH7 mutation reported to date.

  10. Altered cross-bridge properties in skeletal muscle dystrophies

    PubMed Central

    Guellich, Aziz; Negroni, Elisa; Decostre, Valérie; Demoule, Alexandre; Coirault, Catherine

    2014-01-01

    Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies (MDs) have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal MDs and discuss their ultimate impacts on striated muscle function. PMID:25352808

  11. Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

    PubMed Central

    Schnittler, H J; Wilke, A; Gress, T; Suttorp, N; Drenckhahn, D

    1990-01-01

    1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability. 2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16.2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC). 3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro- and macrovascular endothelial cells in situ and in vitro. 4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulin-binding drugs trifluperazine (TFP) and W7. 5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM). 6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 PMID:2100310

  12. [Caldesmon and myosin subfragment-1 act differently on the structural state of 1,5-IAEDANS-modified tropomyosin in ghost muscle fibers].

    PubMed

    Borovikov, Iu S; Novak, E; Khoroshev, M I; Dabrowska, R

    1990-07-01

    The effect of caldesmon (CD) and subfragment 1 of myosin (S1) on the structural state of tropomyosin (TM) modified with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylene-diamine (1.5-IAEDANS) in single myosin-free skeletal muscle fibers was studied using polarized microfluorimetry. S1 was performed from skeletal muscles of rabbits, whereas CD and TM were prepared from the smooth muscle of chicken gizzards. An analysis of experimental data revealed that CD initiates and increases the motility of 1.5-IAEDANS-TM, while S1 decreases it. In the presence of CD S1 binding to actin is accompanied by significant changes in the fluorescent label motility. It is supposed that CD and S1 induce in TM conformational changes which interfere with the protein interaction with F-actin.

  13. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  14. Chara myosin and the energy of cytoplasmic streaming.

    PubMed

    Yamamoto, Keiichi; Shimada, Kiyo; Ito, Khoji; Hamada, Saeko; Ishijima, Akio; Tsuchiya, Takayoshi; Tazawa, Masashi

    2006-10-01

    Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.

  15. Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor.

    PubMed Central

    Koubassova, Natalia A; Tsaturyan, A K

    2002-01-01

    Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction. PMID:12124288

  16. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-08-02

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.

  17. Nuclear myosin I regulates cell membrane tension

    PubMed Central

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  18. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  19. The On-off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

    SciTech Connect

    Himmel, D.; Mui, S; O' Neall-Hennessey, E; Szent-Györgyi, A; Cohen, C

    2009-01-01

    In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long {alpha}-helical 'heavy chain' stabilized by a 'regulatory' light chain (RLC) and an 'essential' light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca{sup 2+} to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca{sup 2+}. Our results indicate that loss of Ca{sup 2+} abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.

  20. The on-off switch in regulated myosins: different triggers but related mechanisms.

    PubMed

    Himmel, Daniel M; Mui, Suet; O'Neall-Hennessey, Elizabeth; Szent-Györgyi, Andrew G; Cohen, Carolyn

    2009-12-01

    In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long alpha-helical "heavy chain" stabilized by a "regulatory" light chain (RLC) and an "essential" light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca(2+) to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca(2+). Our results indicate that loss of Ca(2+) abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins. PMID:19769984

  1. In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

    PubMed

    Bhuiyan, Md Shenuarin; McLendon, Patrick; James, Jeanne; Osinska, Hanna; Gulick, James; Bhandary, Bidur; Lorenz, John N; Robbins, Jeffrey

    2016-10-01

    Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

  2. Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

    PubMed Central

    Armel, Thomas Z.; Leinwand, Leslie A.

    2009-01-01

    Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, β-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the α-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule. PMID:19336582

  3. In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

    PubMed

    Bhuiyan, Md Shenuarin; McLendon, Patrick; James, Jeanne; Osinska, Hanna; Gulick, James; Bhandary, Bidur; Lorenz, John N; Robbins, Jeffrey

    2016-10-01

    Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart. PMID:27568194

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

    SciTech Connect

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    2014-08-01

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

  5. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

    SciTech Connect

    Mahmood, R.

    1985-01-01

    The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz/sub 2/ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz/sub 2/ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF/sub 1/) with a binding constant of 3.2 x 10/sup 5/ M/sup -1/. Bz/sub 2/ATP is also a substrate for the ATPase activity of SF/sub 1/ in the presence of different cations. The irradiation of SF/sub 1/ with (/sup 3/H)Bz/sub 2/ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF/sub 1/ after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling (/sup 3/H)Bz/sub 2/ATP is trapped on SF/sub 1/ by cross-linking the two reactive thiols, SH/sub 1/ and SH/sub 2/, by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified (/sup 14/C)Bz/sub 2/ATP-SF/sub 1/, after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography.

  6. Ca2+-dependent regulations and signaling in skeletal muscle: from electro-mechanical coupling to adaptation.

    PubMed

    Gehlert, Sebastian; Bloch, Wilhelm; Suhr, Frank

    2015-01-05

    Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca(2+) is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca(2+) regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca(2+)-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca(2+) ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca(2+) ions in adult muscle but also highlight recent findings of critical Ca(2+)-dependent mechanisms essential for skeletal muscle-regulation and maintenance.

  7. Ca2+-Dependent Regulations and Signaling in Skeletal Muscle: From Electro-Mechanical Coupling to Adaptation

    PubMed Central

    Gehlert, Sebastian; Bloch, Wilhelm; Suhr, Frank

    2015-01-01

    Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance. PMID:25569087

  8. Ca2+-dependent regulations and signaling in skeletal muscle: from electro-mechanical coupling to adaptation.

    PubMed

    Gehlert, Sebastian; Bloch, Wilhelm; Suhr, Frank

    2015-01-01

    Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca(2+) is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca(2+) regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca(2+)-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca(2+) ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca(2+) ions in adult muscle but also highlight recent findings of critical Ca(2+)-dependent mechanisms essential for skeletal muscle-regulation and maintenance. PMID:25569087

  9. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

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

    2002-01-01

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

  10. Genetics Home Reference: myosin storage myopathy

    MedlinePlus

    ... proteins accumulate in type I skeletal muscle fibers, forming the protein clumps characteristic of the disorder. It ... Epub 2007 Mar 2. Citation on PubMed Tajsharghi H, Oldfors A. Myosinopathies: pathology and mechanisms. Acta Neuropathol. ...

  11. SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.

    PubMed

    Kojima, S; Fujiwara, K; Onishi, H

    1999-09-01

    To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.

  12. Gene Regions Responding to Skeletal Muscle Atrophy

    NASA Technical Reports Server (NTRS)

    Booth, Frank W.

    1997-01-01

    Our stated specific aims for this project were: 1) Identify the region(s) of the mouse IIb myosin heavy chain (MHC) promoter necessary for in vivo expression in mouse fast-twitch muscle, and 2) Identify the region(s) of the mouse IIb MHC promoter responsive to immobilization in mouse slow-twitch muscle in vivo. We sought to address these specific aims by introducing various MHC IIb promoter/reporter gene constructs directly into the tibialis anterior and gastrocnemius muscles of living mice. Although the method of somatic gene transfer into skeletal muscle by direct injection has been successfully used in our laboratory to study the regulation of the skeletal alpha actin gene in chicken skeletal muscle, we had many difficulties utilizing this procedure in the mouse. Because of the small size of the mouse soleus and the difficulty in obtaining consistent results, we elected not to study this muscle as first proposed. Rather, our MHC IIb promoter deletion experiments were performed in the gastrocnemius. Further, we decided to use hindlimb unloading via tail suspension to induce an upregulation of the MHC IIb gene, rather than immobilization of the hindlimbs via plaster casts. This change was made because tail suspension more closely mimics spaceflight, and this procedure in our lab results in a smaller loss of overall body mass than the mouse hindlimb immobilization procedure. This suggests that the stress level during tail suspension is less than during immobilization. This research has provided an important beginning point towards understanding the molecular regulation of the MHC lIb gene in response to unweighting of skeletal muscle Future work will focus on the regulation of MHC IIb mRNA stability in response to altered loading of skeletal muscle

  13. Lighting.

    SciTech Connect

    United States. Bonneville Power Administration.

    1992-09-01

    Since lighting accounts for about one-third of the energy used in commercial buildings, there is opportunity to conserve. There are two ways to reduce lighting energy use: modify lighting systems so that they used less electricity and/or reduce the number of hours the lights are used. This booklet presents a number of ways to do both. Topics covered include: reassessing lighting levels, reducing lighting levels, increasing bulb & fixture efficiency, using controls to regulate lighting, and taking advantage of daylight.

  14. Biochemical and histochemical adaptations of skeletal muscle to rat suspension

    NASA Technical Reports Server (NTRS)

    Templeton, G. H.

    1984-01-01

    The influence of rat suspension on soleus disuse and atrophy was investigated to measure changes in fiber area and number and to determine if suspension elicited changes in lysosomal protease activity and rate of calcium uptake by the sarcoplasmic reticulum. The infuence of rat suspension on myosin light chain phosphorylation and succinate dehydrogenase activity are determined.

  15. Identification of morphological markers of sarcopenia at early stage of aging in skeletal muscle of mice.

    PubMed

    Sayed, Ramy K A; de Leonardis, Erika Chacin; Guerrero-Martínez, José A; Rahim, Ibtissem; Mokhtar, Doaa M; Saleh, Abdelmohaimen M; Abdalla, Kamal E H; Pozo, María J; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-10-01

    The gastrocnemius muscle (GM) of young (3months) and aged (12months) female wild-type C57/BL6 mice was examined by light and electron microscopy, looking for the presence of structural changes at early stage of the aging process. Morphometrical parameters including body and gastrocnemius weights, number and type of muscle fibers, cross section area (CSA), perimeter, and Feret's diameter of single muscle fiber, were measured. Moreover, lengths of the sarcomere, A-band, I-band, H-zone, and number and CSA of intermyofibrillar mitochondria (IFM), were also determined. The results provide evidence that 12month-old mice had significant changes on skeletal muscle structure, beginning with the reduction of gastrocnemius weight to body weight ratio, compatible with an early loss of skeletal muscle function and strength. Moreover, light microscopy revealed increased muscle fibers size, with a significant increase on their CSA, perimeter, and diameter of both type I and type II muscle fibers, and a reduction in the percentage of muscle area occupied by type II fibers. Enhanced connective tissue infiltrations, and the presence of centrally nucleated muscle fibers, were also found in aged mice. These changes may underlie an attempt to compensate the loss of muscle mass and muscle fibers number. Furthermore, electron microscopy discovered a significant age-dependent increase in the length of sarcomeres, I and H bands, and reduction on the overlapped actin/myosin length, supporting contractile force loss with age. Electron microscopy also showed an increased number and CSA of IFM with age, which may reveal more endurance at 12months of age. Together, mice at early stage of aging already show significant changes in gastrocnemius muscle morphology and ultrastructure that are suggestive of the onset of sarcopenia.

  16. Identification of morphological markers of sarcopenia at early stage of aging in skeletal muscle of mice.

    PubMed

    Sayed, Ramy K A; de Leonardis, Erika Chacin; Guerrero-Martínez, José A; Rahim, Ibtissem; Mokhtar, Doaa M; Saleh, Abdelmohaimen M; Abdalla, Kamal E H; Pozo, María J; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-10-01

    The gastrocnemius muscle (GM) of young (3months) and aged (12months) female wild-type C57/BL6 mice was examined by light and electron microscopy, looking for the presence of structural changes at early stage of the aging process. Morphometrical parameters including body and gastrocnemius weights, number and type of muscle fibers, cross section area (CSA), perimeter, and Feret's diameter of single muscle fiber, were measured. Moreover, lengths of the sarcomere, A-band, I-band, H-zone, and number and CSA of intermyofibrillar mitochondria (IFM), were also determined. The results provide evidence that 12month-old mice had significant changes on skeletal muscle structure, beginning with the reduction of gastrocnemius weight to body weight ratio, compatible with an early loss of skeletal muscle function and strength. Moreover, light microscopy revealed increased muscle fibers size, with a significant increase on their CSA, perimeter, and diameter of both type I and type II muscle fibers, and a reduction in the percentage of muscle area occupied by type II fibers. Enhanced connective tissue infiltrations, and the presence of centrally nucleated muscle fibers, were also found in aged mice. These changes may underlie an attempt to compensate the loss of muscle mass and muscle fibers number. Furthermore, electron microscopy discovered a significant age-dependent increase in the length of sarcomeres, I and H bands, and reduction on the overlapped actin/myosin length, supporting contractile force loss with age. Electron microscopy also showed an increased number and CSA of IFM with age, which may reveal more endurance at 12months of age. Together, mice at early stage of aging already show significant changes in gastrocnemius muscle morphology and ultrastructure that are suggestive of the onset of sarcopenia. PMID:27435496

  17. Effect of ATP analogues on the actin-myosin interface.

    PubMed

    Van Dijk, J; Fernandez, C; Chaussepied, P

    1998-06-01

    The interaction between skeletal myosin subfragment 1 (S1) and filamentous actin was examined at various intermediate states of the actomyosin ATPase cycle by chemical cross-linking experiments. Reaction of the actin-S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626-647 and 567-578 were cross-linked independently to the N-terminal segment of residues 1-12 of one actin monomer, and of 265 kDa, in which the two loops were bound to the N termini of two adjacent monomers. In strong-binding complexes, i.e., without nucleotide or with ADP, S1 was sequentially cross-linked to one and then to two actin monomers. In the weak-binding complexes, two types of cross-linking pattern were observed. First, during steady-state hydrolysis of ATP or ATPgammaS at 20 degreesC, the cross-linking reaction gave rise to a small amount of unknown 200 kDa product. Second, in the presence of AMPPNP, ADP.BeFx, ADP.AlF4-, or ADP.VO43- or with S1 internally cross-linked by N,N'-p-phenylenedimaleimide, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak- and strong-binding states, while it dissociated only weak-binding complexes. These results indicate that, in the weak-binding state populated with the ADP.Pi analogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while these ionic contacts are much less significant in stabilizing the rigor actin-S1 complexes. They also suggest that the electrostatic actin-S1 interface is not influenced by the type of ADP.Pi analogue bound to the active site.

  18. On the kinetics that moves Myosin V

    NASA Astrophysics Data System (ADS)

    Maes, Christian; O'Kelly de Galway, Winny

    2015-10-01

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

  19. Neuromuscular Development and Regulation of Myosin Expression

    NASA Technical Reports Server (NTRS)

    Bodine, Sue

    1997-01-01

    The proposed experiments were designed to determine whether the absence of gravity during embryogenesis influences the postnatal development of the neuromuscular system. Further, we examined the effects of reduced gravity on hindlimb muscles of the pregnant rats. Microgravity may have short and long-term effects on the development of muscle fiber type differentiation and force producing capabilities. Microgravity will reduce muscle fiber size and cause a shift in myosin heavy chain expression from slow to fast in hindlimb muscles of the adult pregnant rats.

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

    NASA Technical Reports Server (NTRS)

    Baldwin, Kenneth M.; Haddad, Fadia

    2002-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  2. Structural insight into the UNC-45-myosin complex.

    PubMed

    Fratev, Filip; Osk Jónsdóttir, Svava; Pajeva, Ilza

    2013-07-01

    The UNC-45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC-45-myosin complex using various in silico methods. Initially, the human UNC-45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B-MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosin-actin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15-16 hypertrophic cardiomyopathy mutations and provide directions for drug targeting. PMID:23408646

  3. Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

    PubMed

    Forer, Arthur; Fabian, Lacramioara

    2005-04-01

    BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro. We discuss how the two sets of apparently contradictory results might be resolved, and we suggest possible experiments that might clarify the contradictory interpretations. PMID:15868207

  4. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells.

    PubMed

    Pedersen, S F; Hoffmann, E K

    2002-07-01

    Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization. In contrast, the previously reported shrinkage-induced actin polymerization [Pedersen et al. (1999) Exp. Cell Res. 252, 63-74] was independent of Rho kinase, p38, myosin light chain kinase (MLCK), and protein kinase C (PKC), which thus do not exert their effects on the shrinkage-activated transporters via effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both by osmotic shrinkage and by the serine/threonine phosphatase inhibitor Calyculin A (CL-A). Both stimuli caused Rho kinase-dependent myosin II relocation to the cortical cytoplasm, but in contrast to the shrinkage-induced F-actin polymerization, CL-A treatment elicited a slight F-actin depolymerization. Moreover, Rho kinase inhibition did not significantly affect NHE1 activation, neither by shrinkage nor by CL-A. Implications for the possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation are discussed. PMID:12061817

  5. Marsupial cardiac myosins are similar to those of eutherians in subunit composition and in the correlation of their expression with body size.

    PubMed

    Hoh, Joseph F Y; Kim, Yoonah; Lim, Jacqueline H Y; Sieber, Louise G; Lucas, Christine A; Zhong, Wendy W H

    2007-02-01

    Cardiac myosins and their subunit compositions were studied in ten species of marsupial mammals. Using native gel electrophoresis, ventricular myosin in macropodoids showed three isoforms, V(1), V(2) and V(3), and western blots using specific anti-alpha- and anti-beta-cardiac myosin heavy chain (MyHC) antibodies showed their MyHC compositions to be alphaalpha, alphabeta and betabeta, respectively. Atrial myosin showed alphaalpha MyHC composition but differed from V(1) in light chain composition. Small marsupials (Sminthopsis crassicaudata, Antechinus stuartii, Antechinus flavipes) showed virtually pure V(1), while the larger (1-3 kg) Pseudocheirus peregrinus and Trichosurus vulpecula showed virtually pure V(3). The five macropodoids (Bettongia penicillata, Macropus eugenii, Wallabia bicolour, M. rufus and M. giganteus), ranging in body mass from 2 to 66 kg, expressed considerably more alpha-MyHC (22.8%) than expected for their body size. These results show that cardiac myosins in marsupial mammals are substantially the same as their eutherian counterparts in subunit composition and in the correlation of their expression with body size, the latter feature underlies the scaling of resting heart rate and cardiac cross-bridge kinetics with specific metabolic rate. The data from macropodoids further suggest that expression of cardiac myosins in mammals may also be influenced by their metabolic scope.

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

    PubMed

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

    2012-06-01

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

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

    PubMed

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

    2012-06-01

    Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships. PMID:25363580

  8. Developmental Changes is Expression of Beta-Adrenergic Receptors in Cultures of C2C12 Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, K. Y.; Vaughn, J. R.

    2000-01-01

    beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

  9. Cardiac assistance from skeletal muscle: a reappraisal.

    PubMed

    Salmons, Stanley

    2009-02-01

    Cardiac assistance from skeletal muscle offers an attractive surgical solution to the problem of end-stage heart failure, yet it is widely regarded as a failed approach. I argue here that this is an outdated assessment. Systematic progress has been made over the last 25 years in understanding the relevant basic science. In the light of these advances we should be reconsidering the place of skeletal muscle assist in the surgical armamentarium. PMID:18954996

  10. Reversible movement of switch 1 loop of myosin determines actin interaction

    PubMed Central

    Kintses, Bálint; Gyimesi, Máté; Pearson, David S; Geeves, Michael A; Zeng, Wei; Bagshaw, Clive R; Málnási-Csizmadia, András

    2007-01-01

    The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin–myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general. PMID:17213877

  11. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

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

    PubMed Central

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

    2016-01-01

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

  13. Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres.

    PubMed

    Avrova, Stanislava V; Rysev, Nikita A; Matusovsky, Oleg S; Shelud'ko, Nikolay S; Borovikov, Yurii S

    2012-05-01

    The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.

  14. Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer.

    PubMed

    Yengo, C M; Chrin, L R; Berger, C L

    2000-09-01

    Fluorescence resonance energy transfer (FRET) experiments were carried out in the absence of nucleotide (rigor) or in the presence of MgADP between fluorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-naphthalene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules (FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester) at Lys-553 of skeletal muscle myosin subfragment 1. The critical Förster distance (R(0)) was determined to be 44 and 38 A for the IAEDANS-FHS and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy transfer between the acceptor molecules at Lys-553 of myosin and donor probes at Cys-374 or Gln-41 of actin was calculated to be 0.78 +/- 0.01 or 0.94 +/- 0.01, respectively, corresponding to distances of 35.6 +/- 0.4 A and 24.0 +/- 1.6 A, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrangements in the acto-myosin interface are likely to occur elsewhere than in the lower 50-kDa subdomain of myosin as its affinity for actin is weakened by MgADP binding.

  15. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  16. Single-molecule studies of unconventional motor protein myosin VI

    NASA Astrophysics Data System (ADS)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  17. Myosin-V stepping kinetics: a molecular model for processivity.

    PubMed

    Rief, M; Rock, R S; Mehta, A D; Mooseker, M S; Cheney, R E; Spudich, J A

    2000-08-15

    Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

  18. The effects of rearing light level and duration differences on the optic nerve, brain, and associated structures in developing zebrafish larvae: a light and transmission electron microscope study.

    PubMed

    Chapman, George B; Tarboush, Rania; Connaughton, Victoria P

    2012-03-01

    The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature.

  19. Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle.

    PubMed

    Malinchik, S; Xu, S; Yu, L C

    1997-11-01

    By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered

  20. O-GlcNAcylation, contractile protein modifications and calcium affinity in skeletal muscle

    PubMed Central

    Cieniewski-Bernard, Caroline; Lambert, Matthias; Dupont, Erwan; Montel, Valérie; Stevens, Laurence; Bastide, Bruno

    2014-01-01

    O-GlcNAcylation, a generally undermined atypical protein glycosylation process, is involved in a dynamic and highly regulated interplay with phosphorylation. Akin to phosphorylation, O-GlcNAcylation is also involved in the physiopathology of several acquired diseases, such as muscle insulin resistance or muscle atrophy. Recent data underline that the interplay between phosphorylation and O-GlcNAcylation acts as a modulator of skeletal muscle contractile activity. In particular, the O-GlcNAcylation level of the phosphoprotein myosin light chain 2 seems to be crucial in the modulation of the calcium activation properties, and should be responsible for changes in calcium properties observed in functional atrophy. Moreover, since several key structural proteins are O-GlcNAc-modified, and because of the localization of the enzymes involved in the O-GlcNAcylation/de-O-GlcNAcylation process to the nodal Z disk, a role of O-GlcNAcylation in the modulation of the sarcomeric structure should be considered. PMID:25400587

  1. Skeletal myoblasts for cardiac repair

    PubMed Central

    Durrani, Shazia; Konoplyannikov, Mikhail; Ashraf, Muhammad; Haider, Khawaja Husnain

    2011-01-01

    Stem cells provide an alternative curative intervention for the infarcted heart by compensating for the cardiomyocyte loss subsequent to myocardial injury. The presence of resident stem and progenitor cell populations in the heart, and nuclear reprogramming of somatic cells with genetic induction of pluripotency markers are the emerging new developments in stem cell-based regenerative medicine. However, until safety and feasibility of these cells are established by extensive experimentation in in vitro and in vivo experimental models, skeletal muscle-derived myoblasts, and bone marrow cells remain the most well-studied donor cell types for myocardial regeneration and repair. This article provides a critical review of skeletal myoblasts as donor cells for transplantation in the light of published experimental and clinical data, and indepth discussion of the advantages and disadvantages of skeletal myoblast-based therapeutic intervention for augmentation of myocardial function in the infarcted heart. Furthermore, strategies to overcome the problems of arrhythmogenicity and failure of the transplanted skeletal myoblasts to integrate with the host cardiomyocytes are discussed. PMID:21082891

  2. Skeletal trauma in child abuse.

    PubMed

    Swoboda, Sara L; Feldman, Kenneth W

    2013-11-01

    Fractures and other skeletal injuries are common in childhood. Most are the result of falls, motor vehicle accidents, and other forms of accidental trauma. However, skeletal trauma is present in a significant number of abused children. Age and developmental abilities are key components in raising clinical suspicion for child abuse. Children who are unable to provide their own history because of age or developmental delay require increased attention. Younger children are more likely to have abusive fractures, whereas accidental fractures increase with age and developmental abilities. The consequences of missing abuse are high because children returned to their homes without intervention are likely to face further abuse and have an increased mortality risk. Because of the potentially high cost of undiagnosed child abuse, diagnosis of a skeletal injury is incomplete without diagnosing its etiology. All health providers for children should be able to recognize patterns of skeletal injury secondary to abusive trauma and understand the process for initiating Child Protective Services (CPS) investigations when necessary. Although they can occur accidentally, fractures in nonmobile children should always increase the clinician's concern for abusive trauma. In light of the significant consequences for children when abuse is missed by a primary care provider, abuse should be on the differential diagnosis for all presenting childhood injuries.

  3. X-ray recordings reveal how a human disease-linked skeletal muscle α-actin mutation leads to contractile dysfunction.

    PubMed

    Ochala, Julien; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J; Iwamoto, Hiroyuki

    2015-12-01

    In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction. PMID:26407659

  4. The myosin filament XIV backbone structure.

    PubMed Central

    Ashton, F T; Weisel, J; Pepe, F A

    1992-01-01

    The substructure of the thick filaments of chemically skinned chicken pectoralis muscle was investigated by electron microscopy. Images of transverse sections of the myosin filaments were determined to have threefold symmetry by cross-correlation analysis, which gives an unbiased determination of the rotational symmetry of the images. Resolution, using the phase residual test (Frank et al. 1981. Science [Wash. DC]. 214:1353-1355), was found to be between 3.2 and 3.6 nm. Three arrangements of nine subfilaments in the backbone were found in all regions of the filament at ionic strengths of 20 and 200 mM. In the average images of two of these, there were three dense central subfilaments and three pairs of subfilaments on the surface of the thick filament. In the average image of the third arrangement, all of the protein mass of the nine subfilaments was on the surface of the filament with three of them showing less variation in position than the others. A fourth arrangement appearing to be transitional between two of these was seen often at 200 mM ionic strength and only rarely at 20 mM. On average, the myosin subfilaments were parallel to the long axis of the filament. The different arrangements of subfilaments appear to be randomly distributed among the filaments in a transverse section of the A-band. Relative rotational orientations with respect to the hexagonal filament lattice, using the three densest subfilaments as reference showed a major clustering (32%) of filaments within one 10 degrees spread, a lesser clustering (15%) at 90 degrees to the first, and the remainder scattered thinly over the rest of the 120 degrees range. There was no obvious pattern of distribution of the two predominant orientations that could define a superlattice in the filament lattice. Images FIGURE 2 FIGURE 6 FIGURE 8 PMID:1617136

  5. Making Skeletal Muscle from Progenitor and Stem Cells: Development versus Regeneration

    PubMed Central

    Li, Lydia; Rozo, Michelle E.; Lepper, Christoph

    2012-01-01

    For locomotion, vertebrate animals use the force generated by contractile skeletal muscles. These muscles form an actin/myosin-based bio-machinery that is attached to skeletal elements to effect body movement and maintain posture. The mechanics, physiology, and homeostasis of skeletal muscles in normal and disease states are of significant clinical interest. How muscles originate from progenitors during embryogenesis has attracted considerable attention from developmental biologists. How skeletal muscles regenerate and repair themselves after injury by the use of stem cells is an important process to maintain muscle homeostasis throughout lifetime. In recent years, much progress has been made towards uncovering the origins of myogenic progenitors and stem cells as well as the regulation of these cells during development and regeneration. PMID:22737183

  6. Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact.

    PubMed

    Rehfeldt, C; Te Pas, M F W; Wimmers, K; Brameld, J M; Nissen, P M; Berri, C; Valente, L M P; Power, D M; Picard, B; Stickland, N C; Oksbjerg, N

    2011-04-01

    Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only

  7. Kinetic Adaptations of Myosins for Their Diverse Cellular Functions.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2016-08-01

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

  8. Emergent Systems Energy Laws for Predicting Myosin Ensemble Processivity

    PubMed Central

    Egan, Paul; Moore, Jeffrey; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

    2015-01-01

    In complex systems with stochastic components, systems laws often emerge that describe higher level behavior regardless of lower level component configurations. In this paper, emergent laws for describing mechanochemical systems are investigated for processive myosin-actin motility systems. On the basis of prior experimental evidence that longer processive lifetimes are enabled by larger myosin ensembles, it is hypothesized that emergent scaling laws could coincide with myosin-actin contact probability or system energy consumption. Because processivity is difficult to predict analytically and measure experimentally, agent-based computational techniques are developed to simulate processive myosin ensembles and produce novel processive lifetime measurements. It is demonstrated that only systems energy relationships hold regardless of isoform configurations or ensemble size, and a unified expression for predicting processive lifetime is revealed. The finding of such laws provides insight for how patterns emerge in stochastic mechanochemical systems, while also informing understanding and engineering of complex biological systems. PMID:25885169

  9. Functional roles for myosin 1c in cellular signaling pathways

    PubMed Central

    Bond, Lisa M.; Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Cellular signaling pathways underlie the transfer of information throughout the cell and to adjoining cells and so govern most critical cellular functions. Increasing evidence points to the molecular motor myosin 1c as a prominent player in many signaling cascades, from the integrin-dependent signaling involved in cell migration to the signaling events underlying insulin resistance. Myosin 1c functions on these pathways both via an important role in regulating lipid raft recycling and also via direct involvement in signaling cascades. This review provides an overview of the functional involvement of myosin 1c in cellular signaling and discusses the possible potential for myosin 1c as a target for drug-based treatments for human diseases. PMID:23022959

  10. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  11. Axial disposition of myosin heads in isometrically contracting muscles.

    PubMed Central

    Juanhuix, J; Bordas, J; Campmany, J; Svensson, A; Bassford, M L; Narayanan, T

    2001-01-01

    Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time. PMID:11222303

  12. Actin filaments on myosin beds: The velocity distribution

    NASA Astrophysics Data System (ADS)

    Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

    1995-12-01

    In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

  13. Neural-Thyroid Interaction on Skeletal Isomyosin in Zero Gravity

    NASA Technical Reports Server (NTRS)

    Baldwin, Kenneth M.

    2000-01-01

    The primary goal of the project was to develop a ground based model to first study the role of the nerve and of thyroid hormone (T3) in the regulation of body growth and skeletal muscle growth and differentiation in rodents. A primary objective was to test the hypothesis that normal weight bearing activity is essential for the development of antigravity, slow twitch skeletal muscle and the corresponding slow myosin heavy chain (MHC) gene; whereas, T3 was obligatory for general body and muscle growth and the establishment of fast MHC phenotype in typically fast locomoter muscles. These ground based experiments would provide both the efficacy and background for a spaceflight experiment (referred to as the Neurolab Mission) jointly sponsored by the NIH and NASA.

  14. Non-muscle myosin II takes centre stage in cell adhesion and migration

    PubMed Central

    Vicente-Manzanares, Miguel; Ma, Xuefei; Adelstein, Robert S.; Horwitz, Alan Rick

    2010-01-01

    Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases. PMID:19851336

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

    PubMed

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

    2016-06-21

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

  16. A monomeric myosin VI with a large working stroke.

    PubMed

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

    2004-04-21

    Myosin VI is involved in a wide variety of intracellular processes such as endocytosis, secretion and cell migration. Unlike almost all other myosins so far studied, it moves towards the minus end of actin filaments and is therefore likely to have unique cellular properties. However, its mechanism of force production and movement is not understood. Under our experimental conditions, both expressed full-length and native myosin VI are monomeric. Electron microscopy using negative staining revealed that the addition of ATP induces a large conformational change in the neck/tail region of the expressed molecule. Using an optical tweezers-based force transducer we found that expressed myosin VI is nonprocessive and produces a large working stroke of 18 nm. Since the neck region of myosin VI is short (it contains only a single IQ motif), it is difficult to reconcile the 18 nm working stroke with the classical 'lever arm mechanism', unless other structures in the molecule contribute to the effective lever. A possible model to explain the large working stroke of myosin VI is presented. PMID:15044955

  17. An invertebrate smooth muscle with striated muscle myosin filaments

    PubMed Central

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-01-01

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components. PMID:26443857

  18. Myosin heavy chain-2b transcripts and isoform are expressed in human laryngeal muscles.

    PubMed

    Smerdu, Vika; Cvetko, Erika

    2013-01-01

    Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.

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

    PubMed Central

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

    2000-01-01

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

  20. Wave biomechanics of the skeletal muscle

    NASA Astrophysics Data System (ADS)

    Rudenko, O. V.; Sarvazyan, A. P.

    2006-12-01

    Results of acoustic measurements in skeletal muscle are generalized. It is shown that assessment of the pathologies and functional condition of the muscular system is possible with the use of shear waves. The velocity of these waves in muscles is much smaller than the velocity of sound; therefore, a higher symmetry type is formed for them. In the presence of a preferential direction (along muscle fibers), it is characterized by only two rather than five (as in usual media with the same anisotropy) moduli of elasticity. A covariant form of the corresponding wave equation is presented. It is shown that dissipation properties of skeletal muscles can be controlled by contracting them isometrically. Pulsed loads (shocks) and vibrations are damped differently, depending on their frequency spectrum. Characteristic frequencies on the order of tens and hundreds of hertz are attenuated due to actin-myosin bridges association/dissociation dynamics in the contracted muscle. At higher (kilohertz) frequencies, when the muscle is tensed, viscosity of the tissue increases by a factor of several tens because of the increase in friction experienced by fibrillar structures as they move relative to the surrounding liquid; the tension of the fibers changes the hydrodynamic conditions of the flow around them. Finally, at higher frequencies, the attenuation is associated with the rheological properties of biological molecules, in particular, with their conformational dynamics in the wave field. Models that describe the controlled shock dissipation mechanisms are proposed. Corresponding solutions are found, including those that allow for nonlinear effects.

  1. Effect of trypsin treatments on the structure and binding capacity of volatile compounds of myosin.

    PubMed

    Lv, Tong; Wang, Ying; Pan, Daodong; Cao, Jinxuan; Zhang, Xin; Sun, Yangying; Chen, Yinji; Liu, Yuan

    2017-01-01

    In order to investigate the mechanism between flavor binding and proteins degradation during meat processing, the influence of different trypsin contents on the structure of myosin and the adsorption capacity on aldehydes and ketones was determined. The 1% treatment produced subfragment 2 (S2), light meromyosin (LMM) and decreased 18 and 16kDa light chains; 5% and 10% treatments produced 100 and 65kDa new bands and more S2, LMM and cleaned light chains. With the rising trypsin contents, β-sheet, β-turn, random coil, hydrophobicity and total sulfydryl content increased; solubility, α-helix and free percentages of aldehydes and ketones decreased. The increase of absorbing capacity could be attributed to the increased hydrophobicity and total sulphydryl and the unfolding of secondary structures by exposing reactive amino and thiol groups and hydrophobic sites; the decreased solubility was related to the increased hydrophobicity. The trypsin-dose dependent proteolysis of myosin increased the retention of volatile compounds. PMID:27507529

  2. Three distinct actin-attached structural states of myosin in muscle fibers.

    PubMed

    Mello, Ryan N; Thomas, David D

    2012-03-01

    We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M'.D.P, we used pPDM to cross-link SH1 (Cys(707)) to SH2 (Cys(697)) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M'.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M'D.P analog on the force generation pathway. PMID:22404931

  3. Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer.

    PubMed

    Palm, T; Sale, K; Brown, L; Li, H; Hambly, B; Fajer, P G

    1999-10-01

    The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65]. To test this model we have measured, using frequency-modulated FRET, three distances between the catalytic domain and regulatory domains and within the regulatory domain of myosin. The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; and ELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled light chains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in the putative prepower stroke states (in the presence of MgATP or ADP/AlF(4-)) and the postpower stroke states (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairs were reversed to minimize uncertainty in the distance measured, arising from probe orientational factors. The distances obtained from FRET were in close agreement with the distances in the crystal structure. Importantly, none of the measured distances varied by more than 2 A, putting a strong constraint on the extent of conformational changes within S1. The maximum axial movement of the distal part of myosin head was modeled using FRET distance changes within the myosin head reported here and previously. These models revealed an upper bound of 85 A for a swing of the regulatory domain with respect to the catalytic domain during the power stroke. Additionally, an upper bound of 22 A could be contributed to the power stroke by a reorientation of RLC with respect to the ELC during the power stroke.

  4. Regenerating tail muscles in lizard contain Fast but not Slow Myosin indicating that most myofibers belong to the fast twitch type for rapid contraction.

    PubMed

    Alibardi, L

    2015-10-01

    During tail regeneration in lizards a large mass of muscle tissue is formed in form of segmental myomeres of similar size located under the dermis of the new tail. These muscles accumulate glycogen and a fast form of myosin typical for twitch myofibers as it is shown by light and ultrastructural immunocytochemistry using an antibody directed against a Fast Myosin Heavy Chain. High resolution immunogold labeling shows that an intense labeling for fast myosin is localized over the thick filaments of the numerous myofibrils in about 70% of the regenerated myofibers while the labeling becomes less intense in the remaining muscle fibers. The present observations indicate that at least two subtypes of Fast Myosin containing muscle fibers are regenerated, the prevalent type was of the fast twitch containing few mitochondria, sparse glycogen, numerous smooth endoplasmic reticulum vesicles. The second, and less frequent type was a Fast-Oxidative-Glycolitic twitch fiber containing more mitochondria, a denser cytoplasm and myofibrils. Since their initial differentiation, myoblasts, myotubes and especially the regenerated myofibers do not accumulate any immuno-detectable Slow Myosin Heavy Chain. The study indicates that most of the segmental muscles of the regenerated tail serve for the limited bending of the tail during locomotion and trashing after amputation of the regenerated tail, a phenomenon that facilitates predator escape.

  5. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

    PubMed

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  6. Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines

    PubMed Central

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca2+ influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement. PMID:24465547

  7. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

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

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

    PubMed

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

    2014-11-01

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

  9. Skeletal muscle hypertrophy and structure and function of skeletal muscle fibres in male body builders

    PubMed Central

    D'Antona, Giuseppe; Lanfranconi, Francesca; Pellegrino, Maria Antonietta; Brocca, Lorenza; Adami, Raffaella; Rossi, Rosetta; Moro, Giorgio; Miotti, Danilo; Canepari, Monica; Bottinelli, Roberto

    2006-01-01

    Needle biopsy samples were taken from vastus lateralis muscle (VL) of five male body builders (BB, age 27.4 ± 0.93 years; mean ±s.e.m.), who had being performing hypertrophic heavy resistance exercise (HHRE) for at least 2 years, and from five male active, but untrained control subjects (CTRL, age 29.9 ± 2.01 years). The following determinations were performed: anatomical cross-sectional area and volume of the quadriceps and VL muscles in vivo by magnetic resonance imaging (MRI); myosin heavy chain isoform (MHC) distribution of the whole biopsy samples by SDS-PAGE; cross-sectional area (CSA), force (Po), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n= 524) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE; actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays. In BB a preferential hypertrophy of fast and especially type 2X fibres was observed. The very large hypertrophy of VL in vivo could not be fully accounted for by single muscle fibre hypertrophy. CSA of VL in vivo was, in fact, 54% larger in BB than in CTRL, whereas mean fibre area was only 14% larger in BB than in CTRL. MHC isoform distribution was shifted towards 2X fibres in BB. Po/CSA was significantly lower in type 1 fibres from BB than in type 1 fibres from CTRL whereas both type 2A and type 2X fibres were significantly stronger in BB than in CTRL. Vo of type 1 fibres and Vf of myosin 1 were significantly lower in BB than in CTRL, whereas no difference was observed among fast fibres and myosin 2A. The findings indicate that skeletal muscle of BB was markedly adapted to HHRE through extreme hypertrophy, a shift towards the stronger and more powerful fibre types and an increase in specific force of muscle fibres. Such adaptations could not be fully accounted for by well known mechanisms of muscle plasticity, i.e. by the hypertrophy of single muscle fibre (quantitative mechanism) and by a

  10. Skeletal stem cells

    PubMed Central

    Bianco, Paolo; Robey, Pamela G.

    2015-01-01

    Skeletal stem cells (SSCs) reside in the postnatal bone marrow and give rise to cartilage, bone, hematopoiesis-supportive stroma and marrow adipocytes in defined in vivo assays. These lineages emerge in a specific sequence during embryonic development and post natal growth, and together comprise a continuous anatomical system, the bone-bone marrow organ. SSCs conjo