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1

Substitution Mutations in the Myosin Essential Light Chain Lead to Reduced Actin-activated ATPase Activity Despite Stoichiometric  

E-print Network

Substitution Mutations in the Myosin Essential Light Chain Lead to Reduced Actin-activated ATPase Biology, Northwestern University Medical School, Chicago, Illinois 60611 Myosin essential light chain (ELC) wraps around an -helix that extends from the myosin head, where it is believed to play a structural

Chisholm, Rex L.

2

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain  

E-print Network

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain), and kinetics simula- tions to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin

Thomas, David D.

3

Biochemical Kinetic Characterization of the Acanthamoeba Myosin-I ATPase  

E-print Network

constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We-IA and myosin- IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway also deter- mined equilibrium constants for myosin-I binding to ac- tin filaments, ADP binding

4

Force generation, but not myosin ATPase activity, declines with age in rat muscle fibers  

E-print Network

Force generation, but not myosin ATPase activity, declines with age in rat muscle fibers DAWN A Lowe, Dawn A., David D. Thomas, and LaDora V. Thompson. Force generation, but not myosin ATPase activ-associated decline in muscle function is related to a change in myosin ATPase activity. Single, glycerinated semi

Thomas, David D.

5

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility.  

PubMed

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ?1M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, Vmax, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover. PMID:23954499

Wazawa, Tetsuichi; Yasui, Shin-ichiro; Morimoto, Nobuyuki; Suzuki, Makoto

2013-12-01

6

Blebbistatin, a Myosin II Inhibitor, Is Photoinactivated by Blue Light Takeshi Sakamoto, John Limouze, Christian A. Combs, Aaron F. Straight,| and James R. Sellers*,  

E-print Network

Blebbistatin, a Myosin II Inhibitor, Is Photoinactivated by Blue Light Takeshi Sakamoto, John is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in Vitro motility of class II myosins. In cells, it has

Straight, Aaron

7

Myofibrillar ATPase activity of feline muscle fibers expressing slow and fast myosin heavy chains.  

PubMed

The interrelationships among myofibrillar ATPase activity (Quant-mATPase), qualitative myofibrillar ATPase staining after acid (Acid-mATPase) and alkaline (Alk-mATPase) preincubations, and myosin heavy chain (MHC) composition were determined in frozen sections of soleus (Sol) and medial gastrocnemius (MG) muscle fibers from adult control cats and cats 6 months after complete spinal cord transection (Sp). Fibers were categorized as either fast, slow, or fast and slow (Fast-Slow) based on monoclonal antibody labeling. Slow fibers had low Quant-mATPase activity and stained lightly with Alk-mATPase and darkly with Acid-mATPase. Fast fibers had high Quant-mATPase activity (approximately twice that of slow fibers) and stained darkly with Alk-mATPase and lightly with Acid-mATPase. Fast-Slow fibers had intermediate Quant-mATPase activity and stained intermediately for Acid-mATPase and darkly for Alk-mATPase. There was a positive linear relationship between Alk-mATPase and Quant-mATPase for all fibers of Sol and MG from control and Sp cats. There was a negative linear relationship between Acid-mATPase and Quant-mATPase for all fibers of Sol and MG. However, within the fast fiber population of the MG there was a positive relationship between these two measures of mATPase. In summary, quantitative and qualitative measures of mATPase are highly correlated with the types of MHC expressed by single fibers from control and Sp cat muscles. PMID:7542680

Talmadge, R J; Roy, R R; Jiang, B; Edgerton, V R

1995-08-01

8

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin  

SciTech Connect

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

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

1987-07-28

9

Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase  

SciTech Connect

It was established that the dialdehyde derivative of ATP (oxo-ATP) is a good substrate of the Ca-ATPase of heavy meromyosin: (1.2-1.4) x 10/sup -4/ M; V = V/sub ATP/. At the same time, it is capable of inducing irreversible inhibition of the enzyme. Since oxo-ATP is rapidly digested by myosin, forming oxo-ADP, this inhibition is a consequence of the interaction of the enzyme with oxo-ADP. It was shown that the inhibition of heavy meromyosin (HMM), by oxo-ADP occurs according to the kinetics characteristic of affinity modification; moreover, ATP entirely protects HMM from the loss of activity. Similar data on the irreversible inhibition of ATPase activity under the action of oxo-ADP were obtained in the case of myosin, heavy meromyosin, subfragment-1, and natural actomyosin, as well as in the absence of divalent cations, which is evidence of modification of the active site of myosin ATPase.

Grishin, M.N.; Kodentsova, V.M.; Abdraimova, U.A.; Nikolaeva, O.P.; Petushkova, E.V.

1986-03-20

10

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

SciTech Connect

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

Koppole, Sampath [University of Heidelberg; Smith, Jeremy C [ORNL; Fischer, S. [University of Heidelberg

2007-07-01

11

Calcium and cargoes as regulators of myosin 5a activity  

SciTech Connect

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.

Sellers, James R. [Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-1762 (United States)], E-mail: sellersj@nhlbi.nih.gov; Thirumurugan, Kavitha [Institute of Molecular and Cellular Biology, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Sakamoto, Takeshi [Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-1762 (United States); Hammer, John A. [Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, MD 20892-1762 (United States); Knight, Peter J. [Institute of Molecular and Cellular Biology, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

2008-04-25

12

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

PubMed Central

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

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

2014-01-01

13

Erythrocyte Protein 4.1 Binds and Regulates Myosin  

NASA Astrophysics Data System (ADS)

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

Pasternack, Gary R.; Racusen, Richard H.

1989-12-01

14

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label*  

PubMed Central

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation. PMID:23250750

Moen, Rebecca J.; Thomas, David D.; Klein, Jennifer C.

2013-01-01

15

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function  

PubMed Central

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK. PMID:3032987

1987-01-01

16

Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells.  

PubMed

Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle. PMID:24578340

Natarajan, Paramasivam; Crothers, James M; Rosen, Jared E; Nakada, Stephanie L; Rakholia, Milap; Okamoto, Curtis T; Forte, John G; Machen, Terry E

2014-04-15

17

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle.  

PubMed

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac ?-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in ?-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these ?-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type ?-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction. PMID:22155441

Rysev, Nikita A; Karpicheva, Olga E; Redwood, Charles S; Borovikov, Yurii S

2012-02-01

18

Metabolic types of muscle in the sheep: I. Myosin ATPase, gloycolytic, and mitochondrial enzyme activities  

Microsoft Academic Search

Summary  The metabolic characteristics of 12 skeletal muscles of the sheep were studied.Glycolytic activities (hexokinase, glycogen synthetase I and D, phosphorylase a and b, phosphofructokinase) were measured. Myofibrillar ATPase activity was evaluated. Oxygen consumption, respiratory control and carnitine palmity1 transferase, isocitrate dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities were measured in isolated mitochondria.Three metabolic types could be distinguished;(1) \\u000aessentially oxidative slow

M. Briand; A Talmant; Y. Briand; G. Monin; R. Durand

1981-01-01

19

Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert  

PubMed Central

Smooth muscle myosin gene products include two isoforms, SMA and SMB, differing by a 7-residue peptide in loop 1 (i7) at the myosin active site where ATP is hydrolyzed. Using chicken isoforms, previous work indicated that the i7 deletion in SMA prolongs strong actin binding by inhibiting active site ingress and egress of nucleotide when compared to i7 inserted SMB. Additionally, i7 deletion inhibits Pi release associated with the switch 2 closed ? open transition in actin-activated ATPase. Switch 2 is far from loop 1 indicating i7 deletion has an allosteric effect on Pi release. Chicken SMA and SMB have unknown and robust nucleotide-sensitive tryptophan (NST) fluorescence increments, respectively. Human SMA and SMB both lack NST increments while Pi release in Ca2+ ATPase is not impacted by i7 deletion. The NST reports relay helix movement following conformation change in switch 2 but in the open ? closed transition. The NST is common to all known myosin isoforms except human smooth muscle. Other independent works on human SMA and SMB motility indicate no functional effect of i7 deletion. Smooth muscle myosin is a stunning example of species-specific myosin structure/function divergence underscoring the danger in extrapolating disease-linked mutant effects on myosin across species. PMID:24587982

Ajtai, Katalin; Mayanglambam, Azad; Wang, Yihua; Burghardt, Thomas P.

2014-01-01

20

Filamentous smooth muscle myosin is regulated by phosphorylation  

SciTech Connect

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to fix dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.

Trybus, K.M. (Brandeis Univ., Waltham, MA (USA))

1989-12-01

21

Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines  

PubMed Central

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

Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

2014-01-01

22

The effect of the dilated cardiomyopathy-causing mutation Glu54Lys of alpha-tropomyosin on actin-myosin interactions during the ATPase cycle.  

PubMed

In order to understand how the Glu54Lys mutation of alpha-tropomyosin affects actomyosin interactions, we labeled SH1 helix of myosin subfragment-1 (S1) and the actin subdomain-1 with fluorescent probes. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. The addition of wild-type alpha-tropomyosin to actin filaments increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of work of each cross-bridge. The Glu54Lys mutation inhibits this effect. The Glu54Lys mutation also results in the coupling of the weak-binding sub-state of S1 to the strong-binding sub-state of actin thus altering the concerted conformational changes during the ATPase cycle. We suggest that these alterations will result in reduced force production, which is likely to underlie at least in part the contractile deficit observed in human dilated cardiomyopathy. PMID:19646950

Borovikov, Yurii S; Karpicheva, Olga E; Avrova, Stanislava V; Robinson, Paul; Redwood, Charles S

2009-09-01

23

Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.  

PubMed

The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-?PDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-?PDZ proteins bound to actin with K(d) values of 2.61 and 1.04 ?M, respectively, but only about 50-75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein. PMID:21498886

Guzik-Lendrum, Stephanie; Nagy, Attila; Takagi, Yasuharu; Houdusse, Anne; Sellers, James R

2011-06-17

24

pH lability of myosin ATPase activity permits discrimination of different muscle fibre types in crustaceans.  

PubMed

Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria. PMID:6236181

Maier, L; Rathmayer, W; Pette, D

1984-01-01

25

Myosin types in human skeletal muscle fibers  

Microsoft Academic Search

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and

R. Billeter; H. Weber; H. Lutz; H. Howald; H. M. Eppenberger; E. Jenny

1980-01-01

26

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss.  

PubMed

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene. PMID:25080041

Kwon, Tae-Jun; Oh, Se-Kyung; Park, Hong-Joon; Sato, Osamu; Venselaar, Hanka; Choi, Soo Young; Kim, SungHee; Lee, Kyu-Yup; Bok, Jinwoong; Lee, Sang-Heun; Vriend, Gert; Ikebe, Mitsuo; Kim, Un-Kyung; Choi, Jae Young

2014-07-01

27

The mechanism of pentabromopseudilin inhibition of myosin motor activity  

E-print Network

#12;The mechanism of pentabromopseudilin inhibition of myosin motor activity Roman Fedorov1, Markus pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin

Manstein, Dietmar J.

28

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

SciTech Connect

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

Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)] [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)] [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan) [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

2013-11-29

29

The essential light chain is required for full force production by skeletal muscle myosin.  

PubMed Central

Myosin, a molecular motor that is responsible for muscle contraction, is composed of two heavy chains each with two light chains. The crystal structure of subfragment 1 indicates that both the regulatory light chains (RLCs) and the essential light chains (ELCs) stabilize an extended alpha-helical segment of the heavy chain. It has recently been shown in a motility assay that removal of either light chain markedly reduces actin filament sliding velocity without a significant loss in actin-activated ATPase activity. Here we demonstrate by single actin filament force measurements that RLC removal has little effect on isometric force, whereas ELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose leyer arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of ELC-deficient myosins. Images Fig. 2 PMID:7809049

VanBuren, P; Waller, G S; Harris, D E; Trybus, K M; Warshaw, D M; Lowey, S

1994-01-01

30

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle  

Microsoft Academic Search

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac ?-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin

Yurii S. Borovikov; Stanislava V. Avrova; Olga E. Karpicheva; Paul Robinson; Charles S. Redwood

2011-01-01

31

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle.  

PubMed

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac ?-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and ?-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these ?-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM(??)·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy. PMID:21741356

Borovikov, Yurii S; Avrova, Stanislava V; Karpicheva, Olga E; Robinson, Paul; Redwood, Charles S

2011-08-01

32

Myosin chaperones?  

PubMed Central

The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure–function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review. PMID:24440450

Hellerschmied, Doris; Clausen, Tim

2014-01-01

33

Myosin light chain genes in the turkey (Meleagris gallopavo)  

Microsoft Academic Search

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

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

2003-01-01

34

Dictyostelium Myosin 25-50K Loop Substitutions Specifically Affect ADP Release Rates  

E-print Network

Dictyostelium Myosin 25-50K Loop Substitutions Specifically Affect ADP Release Rates Coleen TVed March 10, 1998 ABSTRACT: While most of the sequence of myosin's motor domain is highly conserved among of ATPase activity. Given the function of myosin as a molecular motor, it was proposed that the 25-50K loop

Murphy, Coleen

35

Structural Dynamics of Actin during Active Interaction with Myosin: Different Effects of Weakly and Strongly Bound Myosin Heads  

E-print Network

Structural Dynamics of Actin during Active Interaction with Myosin: Different Effects of Weakly energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374

Thomas, David D.

36

Myosin isoenzyme redistribution in chronic heart overload  

Microsoft Academic Search

Since the first observation by Spann et al.1, it has become clear that in cardiac hypertrophy induced by a mechanical overloading, the velocity of shortening of the cardiac muscle (Vmax) is reduced (see ref. 2 for review). Most authors agree that this mechanical alteration is accompanied by a decrease in the Ca2+-dependent ATPase activity of myosin (see ref. 3 for

Anne-Marie Lompre; Ketty Schwartz; Anne D'Albis; Gabrielle Lacombe; Nguyen van Thiem; Bernard Swynghedauw

1979-01-01

37

Preparation and Characterization of Myosin Proteins.  

ERIC Educational Resources Information Center

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)

Caldwell, Elizabeth; Eftink, Maurice R.

1985-01-01

38

William M. Shih and James A. Spudich Actomyosin ATPase Cycle  

E-print Network

is cited· to choose from all of JBC's e-mail alertsClick here http://www.jbc.org/content/276/22/19491.full, structural infor- mation is lacking on whether this contact is maintained throughout the actin on actin binding, ATP-in- duced actin release, and actin-activated ATPase. These re- sults demonstrate

Spudich, James A.

39

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site.  

PubMed

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes. PMID:24550279

Amrute-Nayak, Mamta; Lambeck, Katharina-Antonia; Radocaj, Ante; Huhnt, Helen Elisabeth; Scholz, Tim; Hahn, Nils; Tsiavaliaris, Georgios; Walter, Wilhelm J; Brenner, Bernhard

2014-02-18

40

Thiol groups of gizzard myosin heavy chains  

SciTech Connect

Proteolysis of phosphorylated and /sup 3/H-labeled dinitrophenylated chicken gizzard myosin with trypsin released major fragments of M/sub r/ 25,000, 50,000 and 66,000 in a 1:1 ratio. They contained 57% of the dinitrophenyl (N/sub 2/ph) group bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The fragments of M/sub r/ 25,000 and M/sub r/ 66,000 were dinitrophenylated predominantly when the K/sup +/-ATPase activity was inhibited. Thiolysis of phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol removed 60% and 25% of the N/sub 2/ph group from the N-terminal and M/sub r/ 66,000 fragments of the heavy chain, respectively, when 48% of the K/sup +/-ATPase activity was restored. Papain proteolysis of the tryptic digest of modified myosin released a C-terminal segment from the fragment of M/sub r/ 66,000 and it contained most of the remaining label. Proteolysis of /sup 3/H-labeled dinitrophenylated myosin alone resulted in the same digestion pattern but less of the label was bound to the heavy chain fragments. In this case, restoration of enzymic activity occurred in thiolyzed dinitrophenylated myosin when the N/sub 2/ph group was removed from the light chains, predominantly. Conformational changes in gizzard myosin, mediated by phosphorylation, altered the reactivity of the thiols in specific fragments of the heavy chain. Thiol groups of the N- and C-terminal heavy chain regions are involved in maintaining the ATPase activity of myosin.

Bailin, G.

1986-05-01

41

Flexibility of Acanthamoeba Myosin Rod Minifilaments M. Jolanta Redowicz,, John A. Hammer, III, Blair Bowers, Michal Zolkiewski, Ann Ginsburg,  

E-print Network

Flexibility of Acanthamoeba Myosin Rod Minifilaments M. Jolanta Redowicz,, John A. Hammer, III-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly

Rau, Don C.

42

Myosins: a diverse superfamily  

Microsoft Academic Search

Myosins constitute a large superfamily of actin-dependent molecular motors. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosins which form filaments in muscle and non-muscle cells form class II. There has been extensive characterization of these myosins and much is known about their function. With the exception of class I and class V myosins, little is known about

James R Sellers

2000-01-01

43

Germinal Center Specific Protein HGAL Directly Interacts with Both Myosin and Actin and Increases the Binding of Myosin to Actin  

PubMed Central

HGAL is a germinal center (GC)-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classicHodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on the actomyosin ATPase activity and on the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of GC-specific HGAL protein on lymphocyte and lymphoma cell motility. PMID:21447067

Lu, Xiaoqing; Kazmierczak, Katarzyna; Jiang, Xiaoyu; Jones, Michelle; Watt, James; Helfman, David M.; Moore, Jeffrey R.; Szczesna-Cordary, Danuta; Lossos, Izidore S.

2011-01-01

44

Magnesium Regulates ADP Dissociation From Myosin V  

E-print Network

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release, and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V:MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin in order to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.

Steven S. Rosenfeld; Anne Houdusse; H. Lee Sweeney

45

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain  

Microsoft Academic Search

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the

Katusya Hirano; Dmitry N. Derkach; Mayumi Hirano; Junji Nishimura; Hideo Kanaide

2003-01-01

46

Evaluation of myosin light chain phosphorylation in isolated pancreatic acini  

SciTech Connect

The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K{sup +}-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 {mu}mol{center dot}min{sup {minus}1}{center dot}mg protein {sup {minus}1}. Three major myosin subunits of apparent M{sub r} of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of M{sub r} 21,000 was also present. Purification of myosin from {sup 32}P-labeled acini revealed that M{sub r} 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from {sup 32}P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.

Burnham, D.B.; Soeling, H.D.; Williams, J.A. (Univ. of California, San Francisco (USA) Universitaet Goettingen (West Germany))

1988-01-01

47

Temperature induced denaturation of myosin: evidence of structural alterations of myosin subfragment-1.  

PubMed

Denaturation of myofibrillar proteins in porcine longissimus thoracis et lumborum muscle was investigated after pre-rigor temperature incubation at 20, 30 and 40°C. At 24h myofibrils were isolated and myosin was further cleaved by chymotrypsin. High temperature pre-rigor induced release of myosin S1 (subfragment-1), less (P < 0.05) Ca(2+)-ATPase activity and structural alterations of the region of the myosin molecule that harbors S1. Surface hydrophobicity of myofibrils from the 40°C group increased (P<0.001), suggesting a temperature-induced structural rearrangement exposing hydrophobic groups on the surface of myofibrils which in turn may explain the reduced water-holding of PSE meat. PMID:24927048

Liu, Jiao; Puolanne, Eero; Ertbjerg, Per

2014-10-01

48

Theoretical studies of the ATP hydrolysis mechanism of myosin.  

PubMed Central

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis. PMID:11606291

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

2001-01-01

49

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin.  

PubMed

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility. PMID:21447067

Lu, Xiaoqing; Kazmierczak, Katarzyna; Jiang, Xiaoyu; Jones, Michelle; Watt, James; Helfman, David M; Moore, Jeffrey R; Szczesna-Cordary, Danuta; Lossos, Izidore S

2011-06-01

50

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin  

PubMed Central

The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network. PMID:17704147

Halstead, Miriam F.; Ajtai, Katalin; Penheiter, Alan R.; Spencer, Joshua D.; Zheng, Ye; Morrison, Emma A.; Burghardt, Thomas P.

2007-01-01

51

Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity  

NASA Technical Reports Server (NTRS)

The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

Mcdonald, K. S.; Fitts, R. H.

1993-01-01

52

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites.  

PubMed

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition. PMID:16733094

Cruz-Rivera, M; Reyes-Torres, A; Reynoso-Ducoing, O; Flisser, A; Ambrosio, J R

2006-07-01

53

Rare, Non-Synonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11, R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells  

PubMed Central

Rationale Mutations in MYH11 cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, non-synonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. Objective To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). Methods and Results The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11R247C/R247C mice were de-differentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11R247C/R247C SMCs to a Rho activator rescued the de-differentiated phenotype of the SMCs. Conclusions These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury. PMID:22511748

Kuang, Shao-Qing; Kwartler, Callie S.; Byanova, Katerina L.; Pham, John; Gong, Limin; Prakash, Siddharth K.; Huang, Jian; Kamm, Kristine E.; Stull, James T.; Sweeney, H. Lee; Milewicz, Dianna M.

2013-01-01

54

Myosins and deafness  

Microsoft Academic Search

The discovery in the past few years of a huge diversity within the myosin superfamily has been coupled with an understanding of the role of these motor proteins in various cellular functions. Extensive studies have revealed that myosin isoforms are not only involved in muscle contraction but also in crucial functions of many specialized mammalian cells such as melanocytes, kidney

MARIA J. R?DOWICZ

1999-01-01

55

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

SciTech Connect

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.

Maruta, H.; Korn, E.D.

1981-01-10

56

Instructions for use Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms  

E-print Network

in the head region of skeletal muscle myosin, and the proteolytic cleavage of the myosin heavy chain (MHC to the 25/50-kDa and 50/20-kDa junctions were not resolved in the crystal structure of chicken skeletal, and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence

Tsunogai, Urumu

57

Two ATPases  

PubMed Central

In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. PMID:22822068

Senior, Alan E.

2012-01-01

58

Effect of an increase in occlusal vertical dimension on the rate of cyclic actin-myosin interaction in guinea-pig masseter muscle  

Microsoft Academic Search

To study the effects of increased occlusal vertical dimension on these kinetics, the actin-filament sliding velocity on masseter myosins in an in vitro motility assay and the ATPase activity of masseter myosins from normal (control [and bite-opened (5.6 mm increase in the vertical dimension for 1 week (guinea.-pigs were measured. In control myosin preparations, the average value (mean ± SD,

K. Kawasaki; Y. Saeki; Y. Ohnuki

1997-01-01

59

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

PubMed Central

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. PMID:22178692

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

2011-01-01

60

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

PubMed Central

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

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

2008-01-01

61

Rotary ATPases  

PubMed Central

Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

2013-01-01

62

Global Fit Analysis of Myosin-5b Motility Reveals Thermodynamics of Mg2+-Sensitive Acto-Myosin-ADP States  

PubMed Central

Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg2+-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg2+-ions. Free Mg2+-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg2+-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (?HMg and ?SMg) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (?H1‡ and ?H2‡). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions. PMID:23738001

Chizhov, Igor; Hartmann, Falk K.; Hundt, Nikolas; Tsiavaliaris, Georgios

2013-01-01

63

Functional analysis of the mutations in the human cardiac beta-myosin that are responsible for familial hypertrophic cardiomyopathy. Implication for the clinical outcome.  

PubMed Central

More than 30 missense mutations in the beta-cardiac myosin heavy chain gene have been shown to be responsible for familial hypertrophic cardiomyopathy. To clarify the effects of these point mutations on myosin motor function, we expressed wild-type and mutant human beta-cardiac myosin heavy chains in insect cells with human cardiac light chains. The wild-type myosin was well purified with similar enzymatic and motor activities to those of the naturally isolated V3 cardiac myosin. Arg249-->Gln and Arg453-->Cys mutations resulted in decreased actin translocating activity (61 and 23% of the wild-type, respectively) with decreased intrinsic ATPase activity. Arg403-->Gln mutation greatly decreased actin translocating activity (27% of wild type) with a 3.3-fold increased dissociation constant for actin, while intrinsic ATPase activity was unchanged. Val606-->Met mutation only mildly affected the actin translocating activity as well as ATPase activity of myosin. The degree of deterioration by each mutation was closely correlated with the prognosis of the affected kindreds, indicating that myosin dysfunction caused by the point mutations is responsible for the pathogenesis of the disease. Structure/function relationship of myosin is discussed. PMID:8981935

Sata, M; Ikebe, M

1996-01-01

64

Ultraslow Myosin Molecular Motors of Placental Contractile Stem Villi in Humans  

PubMed Central

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n?=?20) and electrical tetanic stimulation (n?=?20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue. PMID:25268142

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

2014-01-01

65

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

PubMed

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n?=?20) and electrical tetanic stimulation (n?=?20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue. PMID:25268142

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

2014-01-01

66

Myosin phosphatase: Structure, regulation and function  

Microsoft Academic Search

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PP1c; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a

Masaaki Ito; Takeshi Nakano; Ferenc Erd?di; David J. Hartshorne

2004-01-01

67

Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin.  

PubMed Central

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407-416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361-4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126-->Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative. PMID:12765546

Colegrave, Melanie; Patel, Hitesh; Offer, Gerald; Chantler, Peter D

2003-01-01

68

Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin.  

PubMed

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407-416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361-4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126-->Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative. PMID:12765546

Colegrave, Melanie; Patel, Hitesh; Offer, Gerald; Chantler, Peter D

2003-08-15

69

Mammalian myosin-18A, a highly divergent myosin.  

PubMed

The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, ? and ?, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18A? isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18A? and -18? species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18A?-S1 and -18A?-S1 molecules bound actin weakly with Kd values of 4.9 and 54 ?m, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells. PMID:23382379

Guzik-Lendrum, Stephanie; Heissler, Sarah M; Billington, Neil; Takagi, Yasuharu; Yang, Yi; Knight, Peter J; Homsher, Earl; Sellers, James R

2013-03-29

70

Original article Myosin expression in semitendinosus muscle  

E-print Network

Original article Myosin expression in semitendinosus muscle during fetal development of cattle types of myosin and the development of different muscle cell populations were studied to different myosin heavy chains were used. Two cell generations were identified during myogenesis

Paris-Sud XI, Université de

71

Genetics Home Reference: Myosin storage myopathy  

MedlinePLUS

... Genetic testing OMIM Genetic disorder catalog Conditions > Myosin storage myopathy On this page: Description Genetic changes Inheritance ... Glossary definitions Reviewed February 2013 What is myosin storage myopathy? Myosin storage myopathy is a condition that ...

72

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

PubMed

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

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

2014-10-01

73

Myosin-VIIb, a Novel Unconventional Myosin, Is a Constituent of Microvilli in Transporting Epithelia  

E-print Network

Myosin-VIIb, a Novel Unconventional Myosin, Is a Constituent of Microvilli in Transporting Haven, Connecticut 06520 Received September 5, 2000; accepted November 23, 2000 Mouse myosin-VIIb, a novel unconventional myosin, was cloned from the inner ear and kidney. The human myosin-VIIb (HGMW

Corey, David P.

74

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

PubMed Central

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

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

2004-01-01

75

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

PubMed

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

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

2011-06-01

76

Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments.  

PubMed

Regulation of muscle contraction via the myosin filaments occurs in vertebrate smooth and many invertebrate striated muscles. Studies of unphosphorylated vertebrate smooth muscle myosin suggest that activity is switched off through an intramolecular interaction between the actin-binding region of one head and the converter and essential light chains of the other, inhibiting ATPase activity and actin interaction. The same interaction (and additional interaction with the tail) is seen in three-dimensional reconstructions of relaxed, native myosin filaments from tarantula striated muscle, suggesting that such interactions are likely to underlie the off-state of myosin across a wide spectrum of the animal kingdom. We have tested this hypothesis by carrying out cryo-electron microscopy and three-dimensional image reconstruction of myosin filaments from horseshoe crab (Limulus) muscle. The same head-head and head-tail interactions seen in tarantula are also seen in Limulus, supporting the hypothesis. Other data suggest that this motif may underlie the relaxed state of myosin II in all species (including myosin II in nonmuscle cells), with the possible exception of insect flight muscle. The molecular organization of the myosin tails in the backbone of muscle thick filaments is unknown and may differ between species. X-ray diffraction data support a general model for crustaceans in which tails associate together to form 4-nm-diameter subfilaments, with these subfilaments assembling together to form the backbone. This model is supported by direct observation of 4-nm-diameter elongated strands in the tarantula reconstruction, suggesting that it might be a general structure across the arthropods. We observe a similar backbone organization in the Limulus reconstruction, supporting the general existence of such subfilaments. PMID:18976661

Zhao, Fa-Qing; Craig, Roger; Woodhead, John L

2009-01-16

77

Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications  

PubMed Central

Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca2+ binding to the essential light chains, contributing to on–off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca2+ binding. Cryo-electron microscopy and single-particle image reconstruction of Ca2+-regulated (scallop) filaments reveals a helical array of myosin head-pair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation—involving phosphorylation or Ca2+ binding—share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca2+-binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca2+-regulated myosin. PMID:23650385

Woodhead, John L.; Zhao, Fa-Qing; Craig, Roger

2013-01-01

78

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

NASA Technical Reports Server (NTRS)

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.

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

1981-01-01

79

The hypertrophic cardiomyopathy myosin mutation R453C alters ATP binding and hydrolysis of human cardiac ?-myosin.  

PubMed

The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human ?-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607-12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis. PMID:24344137

Bloemink, Marieke; Deacon, John; Langer, Stephen; Vera, Carlos; Combs, Ariana; Leinwand, Leslie; Geeves, Michael A

2014-02-21

80

The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac ?-Myosin*  

PubMed Central

The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human ?-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607–12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis. PMID:24344137

Bloemink, Marieke; Deacon, John; Langer, Stephen; Vera, Carlos; Combs, Ariana; Leinwand, Leslie; Geeves, Michael A.

2014-01-01

81

Effect of Hindlimb Unweighting on Single Soleus Fiber Maximal Shortening Velocity and ATPase Activity  

NASA Technical Reports Server (NTRS)

This study characterizes the time course of change in single soleus muscle fiber size and function elicited by hindlimb un weighting (HU) and analyzes the extent to which varying durations of HU altered maximal velocity of shortening (V(sub o)), myofibrillar adenosinetriphosphatase (ATPase), and relative content of slow and fast myosin in individual soleus fibers. After 1, 2, or 3 weeks of HU, soleus muscle bundles were prepared and stored in skinning solution at -20 C. Single fibers were isolated and mounted between a motor arm and a transducer, and fiber force, V(sub o), and ATPase activity were measured. Fiber myosin content was determined by one-dimensional sodium dodecyl sulfate- (SDS) polyacrylamide gel electrophoresis. After 1, 2, and 3 weeks of HU, soleus fibers exhibited a progressive reduction in fiber diameter (16, 22, and 42%, respectively) and peak force (42, 48, and 7%, respectively). Peak specific tension was significantly reduced after 1 week of HU (18%) and showed no further change in 2-3 weeks of HU. During 1 and 3 wk of HU, fiber V(sub o) and ATPase showed a significant increase. By 3 week, V(sub o) had increased from 1.32 +/- 0.04 to 2.94 +/- 0.17 fiber lengths/s and fiber ATPase from 291 +/- 16 to 1064 +/- 128 micro-M min(sub -1) mm(sub -3). The percent fibers expressing fast myosin heavy chain increased from 4% to 29% by 3 week of HU, and V(sub o) and ATPase activity within a fiber were highly correlated. However, a large population of fibers after 1, 2, and 3 weeks of HU showed increases in V(sub o) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers. The mechanism eliciting increased fiber V(sub o) and ATPase activity was not obvious but may have been due to increases in fast myosin that went undetected on SDS gels and/or other factors unrelated to the myosin filament.

McDonald, K. S.; Fitts, R. H.

1993-01-01

82

550 Research Article Introduction  

E-print Network

of the p21-activated kinase (PAK) family occurs at a serine or threonine residue that is located 16 prevalent and most widely expressed myosins are the monomeric, non-filamentous class I myosins phosphorylation is required for efficient actin activation of steady-state ATPase activity and actin

Manstein, Dietmar J.

83

Modulation of the effects of tropomyosin on actin and myosin conformational changes by troponin and Ca2+.  

PubMed

The molecular mechanisms by which troponin (TN)-tropomyosin (TM) regulates the myosin ATPase cycle were investigated using fluorescent probes specifically bound to Cys36 of TM, Cys707 of myosin subfragment-1, and Cys374 of actin incorporated into ghost muscle fibers. Intermediate states of actomyosin were simulated by using nucleotides and non-hydrolysable ATP analogs. Multistep changes in mobility and spatial arrangement of SH1 helix of myosin motor domain and actin subdomain-1 during the ATPase cycle were observed. Each intermediate state of actomyosin induced a definite conformational state and specific position of TM strands on the surface of thin filament. TM increased the amplitude of myosin SH1 helix and actin subdomain-1 movements at transition from weak- to strong-binding states shifting to the center of thin filament at strong-binding and to the periphery of thin filament at weak-binding states. TN modulated those movements in a capital ES, Cyrillicsmall a, Cyrillic(2+)-dependent manner. At high-Ca(2+), TN enhanced the effect of TM on SH1 helix and subdomain-1 movements by transferring TM further to the center of thin filament at strong-binding states. In contrast, at low-Ca(2+), TN inhibited the effect of TM movements, "freezing" actin structure in "OFF" state and TM in the position typical for weak-binding states, resulting in disturbing the interplay of actin and myosin. PMID:19100866

Borovikov, Yurii S; Karpicheva, Olga E; Avrova, Stanislava V; Redwood, Charles S

2009-07-01

84

A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate  

PubMed Central

Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and Pi) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent. PMID:22947934

Zhang, Chong; Yusuf Ali, M.; Warshaw, David M.; Kad, Neil M.

2012-01-01

85

Visualization of the mechanochemical coupling in myosin V using deac-aminoATP  

NASA Astrophysics Data System (ADS)

MyosinV is a twoheaded motor, which moves processively along an actin with ADP-release as the rate limiting step. The kinetic cycles of the two heads are gated by the internal strain each places on the other Mechanical studies suggest that there is tight coupling (i.e., one ATP is hydrolyzed per power stroke). We investigated the coordination between the ATPase mechanism of the two heads-myosin Va and directly visualized the binding and dissociation of nucleotide molecules, while simultaneously observing the stepping motion of the myosin V as it moved along an actin filament. To do this, we used an fluorescent labeled ATP analog, deac-aminoATP, which shows a 20fold increase in fluorescent intensity when bound to the active site of myosinV I directly demonstrate tight coupling between myosin V movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision. Supported by K99/R00 NIH grant.

Sakaoto, Takeshi; Webb, Martin; Forgacs, Eva; White, Howard; Sellers, Jim

2010-03-01

86

Motor Proteins: Myosin Mechanosensors Mechanosensation is emerging as a general principle of myosin motors. As  

E-print Network

Dispatches Motor Proteins: Myosin Mechanosensors Mechanosensation is emerging as a general principle of myosin motors. As demonstrated in a recent study, the single-headed myosin I molecule remodeling and cell-shape control. At the molecular level, ion channels, myosin and kinesin motors

Robinson, Douglas N.

87

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase  

SciTech Connect

Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca/sup 2 +/-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca/sup 2 +/-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10/sup -6/ M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10/sup -4/ M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 ..mu..M ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using (..gamma..-/sup 32/P)ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase.

Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

1988-05-05

88

Myosin: A Link between Streptococci and Heart  

NASA Astrophysics Data System (ADS)

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.

Krisher, Karen; Cunningham, Madeleine W.

1985-01-01

89

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance  

Microsoft Academic Search

SUMMARY Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins

Eva van Rooij; Daniel Quiat; Brett A. Johnson; Lillian B. Sutherland; Xiaoxia Qi; James A. Richardson; Robert J. Kelm Jr.; Eric N. Olson

2009-01-01

90

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

SciTech Connect

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.

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

1987-10-01

91

Directional Mechanosensing in Myosin VI  

NASA Astrophysics Data System (ADS)

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.

Yang, Yubo; Tehver, Riina

2013-03-01

92

Five models for myosin V  

E-print Network

Myosin V was the first discovered processive motor from the myosin family. It has therefore been subject of a number of mechanical, kinetic, optical and structural studies and now belongs to the best characterised motor proteins. This effort has been accompanied by a number of different theoretical models. In this article we give an overview of them and discuss what they have in common and where the open questions are. The latter include the existence of sub-steps, the process that limits the run length, the nature of backward steps, the flexibility of the lever arm and the state of the lead head.

Vilfan, Andrej

2008-01-01

93

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules  

PubMed Central

Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (?12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation. PMID:24268149

Debold, Edward P.; Walcott, Sam; Woodward, Mike; Turner, Matthew A.

2013-01-01

94

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load.  

PubMed

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ?50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ?310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (?13 s(-1)) is similar to the maximum ATPase activity (?12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (?0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ?17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ?35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor. PMID:24753602

Takagi, Yasuharu; Farrow, Rachel E; Billington, Neil; Nagy, Attila; Batters, Christopher; Yang, Yi; Sellers, James R; Molloy, Justin E

2014-05-01

95

The molecular cloning and characterization of Drosophila melanogaster myosin-IA and myosin-IB.  

PubMed

In this paper we describe the isolation and characterization of myosin-IA and myosin-IB, two distinct class I myosins from Drosophila melanogaster. A polymerase chain reaction based strategy using degenerate primers directed against two highly-conserved regions in the head domain of most myosins resulted in the isolation of these two novel myosins-I in addition to a number of previously identified myosins from three Drosophila cDNA libraries. A approximately 3.9 kilobase cDNA clone encoding the putative full-length myosin-IA gene product was isolated from an early embryonic library. Its deduced amino acid sequence predicts a protein of 1011 residues (117,094 Da) with a typical although highly basic myosin head, a neck composed of two IQ motifs, and a unique tail. A approximately 3.4 kilobase cDNA clone encoding the putative full-length myosin-IB gene product was isolated from an adult head library. Its deduced amino acid sequence predicts a protein of 1026 residues (117,741 Da) with a canonical head, three IQ motifs constituting the neck, and a distinct tail. Although both are myosins-I from fly, myosin-IA at cytological locus 31D-F and myosin-IB at cytological locus 61F appear to be more similar to their vertebrate homologs than they are to each other. Primary sequence analyses of both the head and tail domains of the known class I myosins illustrate a division of the metazoan myosin-I family into four distinct subclasses with myosin-IA and myosin-IB as members of two of these groups. Just as the sequence comparisons demonstrate a disparity between myosin-IA and myosin-IB, Northern blot analysis of these two unconventional myosins indicates distinct patterns of temporal expression. PMID:8201616

Morgan, N S; Skovronsky, D M; Artavanis-Tsakonas, S; Mooseker, M S

1994-06-10

96

Small-molecule inhibitors of myosin proteins  

PubMed Central

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

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

2014-01-01

97

Small molecule-mediated refolding and activation of myosin motor function  

PubMed Central

The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated ?-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001 PMID:24520162

Radke, Michael B; Taft, Manuel H; Stapel, Britta; Hilfiker-Kleiner, Denise; Preller, Matthias; Manstein, Dietmar J

2014-01-01

98

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

SciTech Connect

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A. (UPENN); (Duke); (MRCLMB); (FSU); (Jikei-Med)

2010-10-22

99

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

SciTech Connect

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by {approx}60 degrees. This recovery stroke is coupled to the activation of myosin's ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a see-saw motion of the relay helix, followed by a piston/seesaw motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery stroke by using Principal Component Analysis. This reveals that the only principal motions of these two helices that make a large amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions.

Mesentean, Sidonia [University of Heidelberg; Koppole, Sampath [University of Heidelberg; Smith, Jeremy C [ORNL; Fischer, S. [University of Heidelberg

2006-12-01

100

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human ?-cardiac myosin motor function  

PubMed Central

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including ?-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human ?-cardiac myosin. We found that this mutation causes a ?30% decrease in the maximum ATPase of the human ?-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human ?-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle. PMID:23798412

Sommese, Ruth F.; Sung, Jongmin; Nag, Suman; Sutton, Shirley; Deacon, John C.; Choe, Elizabeth; Leinwand, Leslie A.; Ruppel, Kathleen; Spudich, James A.

2013-01-01

101

Myosin lever arm directs collective motion on cellular actin network.  

PubMed

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

Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

2014-03-18

102

Myosin lever arm directs collective motion on cellular actin network  

PubMed Central

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

Hariadi, Rizal F.; Cale, Mario; Sivaramakrishnan, Sivaraj

2014-01-01

103

Phosphorylation of Myosin-binding Subunit (MBS) of Myosin Phosphatase by Rho-Kinase In Vivo  

Microsoft Academic Search

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphory- lation state of the substrates including myosin light chain (MLC), ERM (ezrin\\/radixin\\/moesin) family pro- teins and adducin by their direct phosphorylation and by the inactivation of myosin

Yoji Kawano; Yuko Fukata; Noriko Oshiro; Mutsuki Amano; Toshikazu Nakamura; Masaaki Ito; Fumio Matsumura; Masaki Inagaki; Kozo Kaibuchi

1999-01-01

104

Myosin is involved in postmitotic cell spreading  

Microsoft Academic Search

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video micros- copy, and immunofluorescence. We have also deter- mined the spatial organization and polarity of actin fila- ments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and

L. P. Cramer; T. J. Mitchison

1995-01-01

105

Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target  

PubMed Central

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

Lorenzo-Morales, Jacob; Lopez-Arencibia, Atteneri; Reyes-Batlle, Maria; Pinero, Jose E.; Valladares, Basilio; Maciver, Sutherland K.

2014-01-01

106

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities*  

PubMed Central

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila. PMID:21680742

Bloemink, Marieke J.; Melkani, Girish C.; Dambacher, Corey M.; Bernstein, Sanford I.; Geeves, Michael A.

2011-01-01

107

[The causes of the nonuniform thermostability of Mg-ATPase and of the contractility of muscle models].  

PubMed

With longer periods of preliminary heat-treatment of actomyosin suspension the decrease in the rate of superprecipitation (SPP) is followed by that in the extent of SPP, and, finally, in the Mg-ATPase activity. A similar uncoupling of mechanical and enzymatic activities is observed when the ratio between the native and the inactivated myosin in reconstructed actomyosin varied. This uncoupling is supposed to result from the formation during heat-treatment of myosin bridges incapable of dissociating in the presence of Mg-ATP. The bridges affect largely the mechanical properties of actomyosin, and in a lesser degree, its enzymatic properties. PMID:2525292

Shelud'ko, N S; Stadnikov, V L

1989-02-01

108

Myosin II Dynamics during Embryo Morphogenesis  

NASA Astrophysics Data System (ADS)

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.

Kasza, Karen

2013-03-01

109

New insights into myosin evolution and classification.  

PubMed

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. PMID:16505385

Foth, Bernardo J; Goedecke, Marc C; Soldati, Dominique

2006-03-01

110

New insights into myosin evolution and classification  

PubMed Central

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. PMID:16505385

Foth, Bernardo J.; Goedecke, Marc C.; Soldati, Dominique

2006-01-01

111

Proteomics Analysis of the Non-Muscle Myosin Heavy Chain IIa-Enriched Actin-Myosin Complex Reveals Multiple Functions within the Podocyte  

PubMed Central

MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA‘s function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA’s functions goes beyond the podocyte, providing important insights into its general molecular role. PMID:24949636

Hays, Thomas; Ma'ayan, Avi; Clark, Neil R.; Tan, Christopher M.; Teixeira, Avelino; Teixeira, Angela; Choi, Jae W.; Burdis, Nora; Jung, Sung Yun; Bajaj, Amol O.; O'Malley, Bert W.; He, John C.; Hyink, Deborah P.; Klotman, Paul E.

2014-01-01

112

Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores.  

PubMed

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion. PMID:2138933

Wu, B Y; Yu, F X; Lynch, T J; Taylor, J D; Tchen, T T

1990-01-01

113

Living markers for actin block myosin-dependent motility of plant organelles and auxin.  

PubMed

Expression-based techniques using recombinant actin-binding proteins (ABPs) have been developed as advantageous means of visualising actin filaments. As actin function is linked to the movement of cellular cargoes, and overexpression of ABPs may compete with endogenous cytoskeletal proteins, such as myosins, secondary effects on cellular motility might be observed during actin visualisation. Cytoplasmic streaming and auxin transport were chosen as examples of cargo movement and investigated in two Arabidopsis thaliana lines stably transformed with fluorescently labelled talin (GFP-mTn) or fimbrin (GFP-FABD2). In both lines, the maximal streaming velocity of organelles was reduced to 80% in hypocotyl epidermal cells, where actin was broadly equally labelled by both ABPs. In contrast, observations of streaming and actin organisation during treatments with cytochalasin D (CD) suggested GFP-mTn-labelled actin to remain more stable. Furthermore, basipetal auxin transport was undisturbed in the GFP-FABD2 line but reduced by GFP-mTn. Remarkably, treatments with CD and 2,3-butanedione monoxime, which immobilizes myosin by impairing its ATPase, produced not only failures in organelle movement but also in basipetal auxin transport in the wild-type. These observations suggest that myosin is involved in processes of auxin translocation. In parallel, reduced motility in transgenic plants may be explained by a disturbed acto-myosin interplay, if overexpressed ABPs block the processive movement of myosin along actin filaments. This report shows that the use of live markers for actin visualisation may affect motility of cellular compounds and underlines the general need for critical investigation of actin-related processes in wild-type as well as transgenic plants prior to further interpretation. PMID:17009330

Holweg, Carola L

2007-02-01

114

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

PubMed

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(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(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. PMID:22120626

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

2011-12-16

115

Minireview 501 Kinesin and myosin: molecular motors with similar engines  

E-print Network

Minireview 501 Kinesin and myosin: molecular motors with similar engines Ivan Rayment Structure of molecular motor exhibits the same architecture as myosin and suggests that these microtubule- and actin or superfamilies: myosin, dyenin and kinesin. Myosin moves along actin filaments and kinesin and dyenin operate

Rayment, Ivan

116

A kinetic transport model for actin-myosin interaction  

E-print Network

A kinetic transport model for actin-myosin interaction A. Manhart1 and C. Schmeiser2 Abstract: Based on the behavior of individual myosin monomers imbedded in myosin bundles and acting on actin both for the attachment/detachment kinetics of myosin heads and for the mechan- ics leading

Schmeiser, Christian

117

Defocused orientation and position imaging (DOPI) of myosin V  

E-print Network

Defocused orientation and position imaging (DOPI) of myosin V Erdal Toprak*, Joerg Enderlein-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD and is consistent with a 37-nm forward step size of myosin V. When changes, the probe rotates 27° azimuthally around

Enderlein, Jörg

118

INTRODUCTION The intracellular localization of myosin-II in vertebrate  

E-print Network

INTRODUCTION The intracellular localization of myosin-II in vertebrate nonmuscle cells is well established by fluorescent antibody staining and by microinjection of fluorescent myosin (Fujiwara and Pollard-Stewart, 1974). Myosin- II is a double-headed myosin with an alpha-helical coiled-coil tail that associates

119

Growth Cones Contain Myosin II Bipolar Filament Arrays  

E-print Network

Growth Cones Contain Myosin II Bipolar Filament Arrays Paul C. Bridgman* Dept. of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B

Bridgman, Paul C.

120

Mechanism, Regulation, and Functional Properties of Dictyostelium Myosin-1B*  

E-print Network

Mechanism, Regulation, and Functional Properties of Dictyostelium Myosin-1B* Received, United Kingdom Myosin-1B is one of three long tailed class-1 myosins contain- ing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes

Manstein, Dietmar J.

121

Rotational dynamics of actin-bound myosin heads in active myofibrils.  

PubMed

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo. PMID:8385491

Berger, C L; Thomas, D D

1993-04-13

122

Cold-sensitive mutations of Dictyostelium myosin heavy chain highlight functional domains of the myosin motor  

SciTech Connect

Dictyostelium provides a powerful environment for characterization of myosin II function. It provides well-established biochemical methods for in vitro analysis of myosin`s properties as well as an array of molecular genetic tools. The absence of myosin function results in an array of phenotypes that can be used to genetically manipulate myosin function. We have previously reported methods for the isolation and identification of rapid-effect cold-sensitive myosin II mutations in Dictyostelium. Here, we report the development and utilization of a rapid method for localizing these point mutations. We have also sequenced 19 mutants. The mutations show distinct clustering with respect to three-dimensional location and biochemically characterized functional domains of the protein. We conclude that these mutants represent powerful tools for understanding the mechanisms driving this protein motor. 36 refs., 5 figs., 2 tabs.

Patterson, B.; Spudich, J.A. [Stanford Univ., CA (United States)

1996-06-01

123

Coupling of Two Non-processive Myosin 5c Dimers Enables Processive Stepping along Actin Filaments  

PubMed Central

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30–36?nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells. PMID:24809456

Gunther, Laura K.; Furuta, Ken'ya; Bao, Jianjun; Urbanowski, Monica K.; Kojima, Hiroaki; White, Howard D.; Sakamoto, Takeshi

2014-01-01

124

BIOPHYSICS: Myosin Motors Walk the Walk  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Myosin molecular motors move along actin filaments to drive, for example, muscle contraction or the intracellular trafficking of vesicles. However, it has not been clear whether myosin V moves along actin filaments in a hand-over-hand or inchworm fashion. In their Perspective, Molloy and Veigel explain new work (Yildiz et al.) that provides evidence in support of the hand-over-hand model for how myosin V walks along actin.

Justin E. Molloy (National Institute for Medical Research;Division of Physical Biochemistry); Claudia Veigel (National Institute for Medical Research;Division of Physical Biochemistry)

2003-06-27

125

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains  

PubMed Central

We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain. PMID:21151434

Markov, Denis I.; Zubov, Eugene O.; Nikolaeva, Olga P.; Kurganov, Boris I.; Levitsky, Dmitrii I.

2010-01-01

126

Expression of myosin VIIA during mouse embryogenesis  

Microsoft Academic Search

The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In\\u000a humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular\\u000a dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s)

Iman Sahly; Aziz El-Amraoui; Marc Abitbol; Christine Petit; Jean-Louis Dufier

1997-01-01

127

Myosin filament structure in vertebrate smooth muscle  

PubMed Central

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts. PMID:8698822

1996-01-01

128

Progress in Cardiology myosin binding protein C (cMBPC),6 ventricular myosin  

E-print Network

Progress in Cardiology myosin binding protein C (cMBPC),6 ventricular myosin essential light chain actin, -tropomyosin,4 cardiac troponin T (cTnT),4 cardiac troponin I,5 cardiac From the aCardiology, Kurume, and the dDepartment of Cardiology, Research Institute of Environmen- tal Medicine, Nagoya

Lee, Won-Ha

129

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

SciTech Connect

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60{sup o}. This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motion of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.

Mesentean, Sidonia [University of Heidelberg; Koppole, Sampath [University of Heidelberg; Smith, Jeremy C [ORNL; Fischer, S. [University of Heidelberg

2007-03-01

130

Statistical mechanics of myosin molecular motors in skeletal muscles.  

PubMed

Statistical mechanics provides the link between microscopic properties of matter and its bulk properties. The grand canonical ensemble formalism was applied to contracting rat skeletal muscles, the soleus (SOL, n = 30) and the extensor digitalis longus (EDL, n = 30). Huxley's equations were used to calculate force (pi) per single crossbridge (CB), probabilities of six steps of the CB cycle, and peak muscle efficiency (Eff(max)). SOL and EDL were shown to be in near-equilibrium (CB cycle affinity 2.5 kJ/mol) and stationary state (linearity between CB cycle affinity and myosin ATPase rate). The molecular partition function (z) was higher in EDL (1.126+/-0.005) than in SOL (1.050+/-0.003). Both pi and Eff(max) were lower in EDL (8.3+/-0.1 pN and 38.1+/-0.2%, respectively) than in SOL (9.2+/-0.1 pN and 42.3+/-0.2%, respectively). The most populated step of the CB cycle was the last detached state (D3) (probability P(D3): 0.890+/-0.004 in EDL and 0.953+/-0.002 in SOL). In each muscle group, both pi and Eff(max) linearly decreased with z and statistical entropy and increased with P(D3). We concluded that statistical mechanics and Huxley's formalism provided a powerful combination for establishing an analytical link between chemomechanical properties of CBs, molecular partition function and statistical entropy. PMID:15882700

Lecarpentier, Y; Blanc, F-X; Quillard, J; Hébert, J-L; Krokidis, X; Coirault, C

2005-08-01

131

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

PubMed

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 regulation of acto-myosin contractility may define a control point for conditional uncoupling of differentiation and the cell cycle. PMID:15252113

Dhawan, Jyotsna; Helfman, David M

2004-08-01

132

The 3-(bromoacetamido)-propylamine hydrochloride: A novel sulfhydryl reagent and its future potential in the configurational study of S1-myosin  

NASA Technical Reports Server (NTRS)

Configurational study of S1-Myosin is an important step towards understanding force generation in muscle contraction. Previously reported NMR studies were corroborated. A new compound was synthesized, 3-(Bromoacetamido)-propylamine hydrochloride. Its potential as a sulfhydryl reagent provides an indirect but elegant approach towards future structural elucidation of S1-Myosin. The preliminary investigation has shown that this compound, BAAP, reacted with S1 in the absence of MgADP. The modified enzyme had a 2-fold increase in CaATPase activity and no detectable K-EDTA ATPase activity. Reaction of BAAP with S1 in the presence of MgADP resulted in a modified enzyme which retained a Ca-ATPase activity that was about 60 percent of the unmodified S1 and had essentially zero K-EDTA ATPase activity. Sulfhydryl titration indicated that about 1.5 and 3.5 SH groups per S1 molecule were blocked by BAAP in the absence and presence of MgADP, respectively. When coupled to a carboxyl group of EDTA, the resulting reagent could become a useful SH reagent in which chelated paramagnetic or luminescent lanthanide ions can be exploited to probe S1 conformation.

Sharma, Prasanta; Cheung, Herbert C.

1989-01-01

133

Light-Triggered Myosin Activation for Probing Dynamic Cellular Processes  

E-print Network

Shining light on myosin: The incorporation of a caging group onto the essential phosphoserine residue of myosin by protein semisynthesis enables light-triggered activation of the protein (see picture). Caging eliminates ...

Goguen, Brenda N.

134

Mechanical Coupling in Myosin V: A Simulation Study  

E-print Network

Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The interdomain coupling in myosin V is studied with restrained targeted molecular ...

Ovchinnikov, Victor

135

Structure of the Rigor Actin-Tropomyosin-Myosin Complex  

PubMed Central

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

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

2014-01-01

136

Folding and regulation in myosins II and V  

Microsoft Academic Search

The enzymatic activity of many myosins is regulated by various means including calcium binding, phosphorylation or binding\\u000a of receptor molecules. In this review we compare and contrast the regulation of smooth muscle myosin II and myosin Va with\\u000a particular emphasis on the structural basis for the regulation. Both myosins adopt folded compact conformations in their off\\u000a states, but the details

James R. Sellers; Peter J. Knight

2007-01-01

137

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

SciTech Connect

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.

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.; (RPI); (IIT); (SDSU); (Vermont)

2009-07-01

138

Atomic model of a myosin filament in the relaxed state  

Microsoft Academic Search

Contraction of muscle involves the cyclic interaction of myosin heads on the thick filaments with actin subunits in the thin filaments(1). Muscles relax when this interaction is blocked by molecular switches on either or both filaments(2). Insight into the relaxed ( switched OFF) structure of myosin has come from electron microscopic studies of smooth muscle myosin molecules, which are regulated

John L. Woodhead; Fa-Qing Zhao; Roger Craig; Edward H. Egelman; Lorenzo Alamo; R. Padron

2005-01-01

139

Myosin Motors: The Chemical Restraints Imposed by ATP  

E-print Network

2 Myosin Motors: The Chemical Restraints Imposed by ATP I. Rayment and J. Allingham University organization of linear molecular motors seen today with a focus on myosin. This chapter is written to ask@biochem.wisc.edu I. Rayment and J. Allingham: Myosin Motors: The Chemical Restraints Imposed by ATP, Lect. Notes Phys

Rayment, Ivan

140

Sarcomere Lattice Geometry Influences Cooperative Myosin Binding in Muscle  

E-print Network

Sarcomere Lattice Geometry Influences Cooperative Myosin Binding in Muscle Bertrand C. W. Tanner1, United States of America In muscle, force emerges from myosin binding with actin (forming a cross there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along

Daniel, Tom

141

ORIGINAL PAPER Allosteric communication in Dictyostelium myosin II  

E-print Network

ORIGINAL PAPER Allosteric communication in Dictyostelium myosin II Piyali Guhathakurta · Ewa May 2012 � Springer Science+Business Media B.V. 2012 Abstract Myosin's affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect

Thomas, David D.

142

COMMUNICATION Engineered Myosin VI Motors Reveal Minimal Structural  

E-print Network

COMMUNICATION Engineered Myosin VI Motors Reveal Minimal Structural Determinants of Directionality; accepted 16 July 2009 Available online 22 July 2009 Myosins have diverse mechanical properties reflecting functions from a common motor core. Myosin VI (M6) is specialized for processive motion toward the (-) end

Delp, Scott

143

Original article Myosin-heavy-chain DNA polymorphisms  

E-print Network

Original article Myosin-heavy-chain DNA polymorphisms of subterranean mole rats of the Spalax-fragment-length polymorphisms (RFLPs) of the sarcomeric- myosin heavy chain multigene family were studied in 13 populations of 4, probably even 8, different loci of the sarcomeric myosin heavy chain. The level of polymorphism was31

Paris-Sud XI, Université de

144

Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge  

E-print Network

Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge and Specific Domains of the Myosin Rod Daniel Hostetter1[¤1 , Sarah Rice1[¤2 , Sara Dean1 , David Altman1 , Peggy M. McMahon2 of America Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating

Spudich, James A.

145

Myosin individualized: single nucleotide polymorphisms in energy transduction  

Microsoft Academic Search

BACKGROUND: Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power

Thomas P Burghardt; Kevin L Neff; Eric D Wieben; Katalin Ajtai

2010-01-01

146

Precise Positioning of Myosin VI on Endocytic Vesicles In Vivo  

E-print Network

with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function myosins are found in a variety of organisms from Caenorhabditis elegans to human, and in a variety of cell clathrin-mediated endocytosis, myosin VI is implicated in trafficking vesicles that have recently shed

Spudich, James A.

147

Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice.  

PubMed

We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype. PMID:24992035

Huang, Wenrui; Liang, Jingsheng; Kazmierczak, Katarzyna; Muthu, Priya; Duggal, Divya; Farman, Gerrie P; Sorensen, Lars; Pozios, Iraklis; Abraham, Theodore P; Moore, Jeffrey R; Borejdo, Julian; Szczesna-Cordary, Danuta

2014-09-01

148

Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea  

E-print Network

1 Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea Neil J. Rzechorzek, John K. Blackwood, Sian M. Bray, Joseph D. Maman, Luca Pellegrini* and Nicholas P. Robinson... -sensing, and inter-subunit interactions in AAA and AAA+ ATPases. J Struct Biol 146, 106 (Apr-May, 2004). 11. J. E. Walker, M. Saraste, M. J. Runswick, N. J. Gay, Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases...

Rzechorzek, Neil J.; Blackwood, John K.; Bray, Sian M.; Maman, Joseph D.; Pellegrini, Luca; Robinson, Nicholas P.

2014-01-01

149

Acetylation at the N-terminus of actin strengthens weak interaction between actin and myosin.  

PubMed

The N-terminus of all actins so far studied is acetylated. Although the pathways of acetylation have been well studied, its functional importance has been unclear. A negative charge cluster in the actin N-terminal region is shown to be important for the function of actomyosin. Acetylation at the N-terminus removes a positive charge and increases the amount of net negative charges in the N-terminal region. This may augment the role of the negative charge cluster. To examine this possibility, actin with a nonacetylated N-terminus (nonacetylated actin) was produced. The nonacetylated actin polymerized and depolymerized normally. In actin-activated heavy meromyosin ATPase assays, the nonacetylated actin showed higher K(app) without significantly changing V(max), compared with those of wild-type actin. This is in contrast to the effect of the N-terminal negative charge cluster, which increases V(max) without changing K(app). These results indicate that the acetylation at the N-terminus of actin strengthens weak actomyosin interaction. PMID:10652204

Abe, A; Saeki, K; Yasunaga, T; Wakabayashi, T

2000-02-01

150

Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change  

E-print Network

Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change Ste¡en Nock, Wenchuan Liang, Hans M. Warrick, James A. Spudich/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization

Spudich, James A.

151

Characterization of Monoclonal Antibodies to Acanthamoeba Myosin-I that Cross-react with Both Myosin-II and Low  

E-print Network

Characterization of Monoclonal Antibodies to Acanthamoeba Myosin-I that Cross-react with Both Myosin-II and Low Molecular Mass Nuclear Proteins Susan J. Hagen, Daniel P. Kiehart, Donald A. Kaiser antibod- ies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind

152

Site-Specific Mutations in the Myosin Binding Sites of Actin Affect Structural Transitions That Control Myosin Binding  

E-print Network

Site-Specific Mutations in the Myosin Binding Sites of Actin Affect Structural Transitions That Control Myosin Binding Ewa Prochniewicz* and David D. Thomas Department of Biochemistry, Molecular Biology ReceiVed July 23, 2001 ABSTRACT: We have examined the effects of actin mutations on myosin binding

Thomas, David D.

153

Characterization of myosin-IA and myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton.  

PubMed

The expression patterns of myosin-IA (MIA) and myosin-IB (MIB), two novel unconventional myosins from Drosophila melanogaster, have been characterized through immunoblot analysis and immunocytochemistry of embryos, larvae, and adults. The appearance and distribution of both proteins during embryogenesis is correlated with the formation of a brush border within the alimentary canal as documented at the ultrastructural level. MIA and MIB, both found predominantly at the basolateral domain of immature enterocytes, exhibit increased expression at the apical domain of differentiated enterocytes co-incident with microvillus assembly. Colocalization of MIA and MIB to larval and adult gut by confocal microscopy demonstrates distinct but overlapping subcellular distributions of these two proteins. In the larval brush border, MIA is enriched in the subapical terminal web domain whereas MIB is found predominantly in the apical microvillar domain. In the adult gut, MIA and MIB both exhibit a microvillar component as MIA attains a more apical position in addition to its previous terminal web locale. MIB is also found in egg chambers at both the basolateral and apical surfaces of the somatic follicle cells during oogenesis. MIA and MIB both demonstrate ATP-dependent extraction from the larval brush border cytoskeleton and exogenous F-actin, biochemical properties characteristic of functional myosins-I. PMID:7589814

Morgan, N S; Heintzelman, M B; Mooseker, M S

1995-11-01

154

Myosin VI Is Required for Targeted Membrane Transport during Cytokinesis  

PubMed Central

Myosin VI plays important roles in endocytic and exocytic membrane-trafficking pathways in cells. Because recent work has highlighted the importance of targeted membrane transport during cytokinesis, we investigated whether myosin VI plays a role in this process during cell division. In dividing cells, myosin VI undergoes dramatic changes in localization: in prophase, myosin VI is recruited to the spindle poles; and in cytokinesis, myosin VI is targeted to the walls of the ingressing cleavage furrow, with a dramatic concentration in the midbody region. Furthermore, myosin VI is present on vesicles moving into and out of the cytoplasmic bridge connecting the two daughter cells. Inhibition of myosin VI activity by small interfering RNA (siRNA)-mediated knockdown or by overexpression of dominant-negative myosin VI tail leads to a delay in metaphase progression and a defect in cytokinesis. GAIP-interacting protein COOH terminus (GIPC), a myosin VI binding partner, is associated with the function(s) of myosin VI in dividing cells. Loss of GIPC in siRNA knockdown cells results in a more than fourfold increase in the number of multinucleated cells. Our results suggest that myosin VI has novel functions in mitosis and that it plays an essential role in targeted membrane transport during cytokinesis. PMID:17881731

Arden, Susan D.; Puri, Claudia; Au, Josephine Sui-Yan; Kendrick-Jones, John

2007-01-01

155

Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome  

Microsoft Academic Search

Summary Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis

Jose S. Ramalho; Vanda S. Lopes; Abul K. Tarafder; Miguel C. Seabra; Alistair N. Hume

156

Human single masseter muscle fibers contain unique combinations of myosin and myosin binding protein C isoforms  

Microsoft Academic Search

Striated craniofacial and limb muscles differ in their embryological origin, regulatory program during myogenesis, and innervation.\\u000a In an attempt to explore the effects of these differences on the striated muscle phenotype in humans, the expression of myosin\\u000a and myosin-associated thick filament proteins were studied at the single fiber level both in the human jaw-closing masseter\\u000a muscle and in two limb

F. Yu; P. Stål; L.-E. Thornell; L. Larsson

2002-01-01

157

Myosin V and the endoplasmic reticulum  

PubMed Central

In this issue, Estrada et al. (2003) provide new and important insights into how the endoplasmic reticulum (ER) of budding yeast cells is inherited. Together with other studies in plant and animal cells, the results of Estrada et al. (2003) support the idea that myosin V acts as a universal motor for the transport of ER membranes. PMID:14691131

Wagner, Wolfgang; Hammer, John A.

2003-01-01

158

Phosphorylation of human skeletal muscle myosin  

SciTech Connect

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.

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

1986-03-01

159

Duplex RNA activated ATPases (DRAs)  

PubMed Central

Double-stranded RNAs are an important class of functional macromolecules in living systems. They are usually found as part of highly specialized intracellular machines that control diverse cellular events, ranging from virus replication, antiviral defense, RNA interference, to regulation of gene activities and genomic integrity. Within different intracellular machines, the RNA duplex is often found in association with specific RNA-dependent ATPases, including Dicer, RIG-I and DRH-3 proteins. These duplex RNA-activated ATPases represent an emerging group of motor proteins within the large and diverse super family 2 nucleic acid-dependent ATPases (which are historically defined as SF2 helicases). The duplex RNA-activated ATPases share characteristic molecular features for duplex RNA recognition, including motifs (e.g., motifs IIa and Vc) and an insertion domain (HEL2i), and they require double-strand RNA binding for their enzymatic activities. Proteins in this family undergo large conformational changes concomitant with RNA binding, ATP binding and ATP hydrolysis in order to achieve their functions, which include the release of signaling domains and the recruitment of partner proteins. The duplex RNA-activated ATPases represent a distinct and fascinating group of nanomechanical molecular motors that are essential for duplex RNA sensing and processing in diverse cellular pathways. PMID:23228901

Luo, Dahai; Kohlway, Andrew; Pyle, Anna Marie

2013-01-01

160

Cardiac beta myosin heavy chain diversity in normal and chronically hypertensive baboons.  

PubMed Central

We have identified two distinct beta-myosin heavy chains (MHCs) present in baboon myocardium by electrophoresis in gradient pore gels and by Western blots with anti-MHC MAb. The two beta-MHCs have molecular masses of 210 and 200 kD and share several antigenic determinants including an epitope recognized by a beta-MHC-specific MAb. A fivefold increase in the level of the 200-kD beta-MHC was observed in the hypertrophied left ventricles of baboons with chronic (5.3 +/- 0.7 yr) renal hypertension. A 60% increase (P less than 0.01) in BP and a 100% increase (P less than 0.001) in left ventricular mass to body weight ratio occurred in hypertensive baboons compared with normotensive animals. The Ca2+-activated myosin ATPase activity in hypertrophied left ventricles was decreased by 35% (P less than 0.05) compared with controls. Normal levels of the 200-kD MHC were detected in the right ventricles and intraventricular septa of the hypertensive animals. These data suggest that cardiac MHCs of primates may exist in alternative molecular forms that are indistinguishable by nondenaturing gel electrophoresis and that increased concentration of a second beta-MHC is associated with ventricular hypertrophy (r = 0.55). The functional significance and mechanisms that control the concentration of beta-MHC subspecies remain to be determined. Images PMID:2523412

Henkel, R D; VandeBerg, J L; Shade, R E; Leger, J J; Walsh, R A

1989-01-01

161

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity.  

PubMed

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+). PMID:12011043

Tobacman, Larry S; Nihli, Mahta; Butters, Carol; Heller, Mark; Hatch, Victoria; Craig, Roger; Lehman, William; Homsher, Earl

2002-08-01

162

The effect of myosin antibody on the division of starfish blastomeres  

Microsoft Academic Search

Antiserum against starfish egg myosin was produced in rabbits. Antibody specific- ity to myosin was demonstrated by Ouchterlony's immunodiffusion test and by immunoelectrophoresis in the presence of sodium dodecylsulfate (SDS). The latter technique showed that the antibody binds to both heavy and light chains of egg myosin. Furthermore, the antibody reacted with starfish sperm myosin and starfish adult muscle myosin

ISSEI MABUCHI; MAKOTO OKUNO

1977-01-01

163

Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization  

PubMed Central

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells. PMID:16571671

Lenartowska, Marta; Miller, Kathryn G.

2006-01-01

164

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers  

Microsoft Academic Search

Combined histochemical and biochemical analyses were performed on single fibers of rabbit soleus muscle. Histochemically, four fiber types (I, IC, IIC, IIA) were defined. Of these, types I and IIA were separate, histochemically homogeneous groups. A heterogeneous C fiber population exhibited a continuum of staining intensities between types I and IIA. Microelectrophoretic analyses of specific, histochemically defined fibers revealed that

R. S. Staron; D. Pette

1986-01-01

165

Comparison of orientation and rotational motion of skeletal muscle cross-bridges containing phosphorylated and dephosphorylated myosin regulatory light chain.  

PubMed

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca(2+) binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33-45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430-435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969-1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca(2+) saturation. A simple theory was developed to account for this fact. PMID:23319584

Midde, Krishna; Rich, Ryan; Marandos, Peter; Fudala, Rafal; Li, Amy; Gryczynski, Ignacy; Borejdo, Julian

2013-03-01

166

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain*  

PubMed Central

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca2+ saturation. A simple theory was developed to account for this fact. PMID:23319584

Midde, Krishna; Rich, Ryan; Marandos, Peter; Fudala, Rafal; Li, Amy; Gryczynski, Ignacy; Borejdo, Julian

2013-01-01

167

Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics  

NASA Astrophysics Data System (ADS)

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.

Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

2013-03-01

168

Stochastic dynamics and mechanosensitivity of myosin II minifilaments  

E-print Network

Tissue cells are in a state of permanent mechanical tension that is maintained mainly by myosin II minifilaments, which are bipolar assemblies of tens of myosin II molecular motors contracting actin networks and bundles. Here we introduce a stochastic dynamics model for myosin II minifilaments as two small myosin II motor ensembles engaging in a stochastic tug-of-war. Each of the two ensembles is described by the parallel cluster model that allows us to use exact stochastic simulations and at the same time to keep important molecular details of the myosin II crossbridge cycle. Our simulation and analytical results reveal a strong dependance of myosin II minifilament dynamics on environmental stiffness that is reminiscent of the cellular response to substrate stiffness. For small stiffness, minifilaments form transient crosslinks exerting short spikes of force with negligible mean. For large stiffness, minifilaments form near permanent crosslinks exerting a mean force which hardly depends on environmental elas...

Albert, Philipp J; Schwarz, Ulrich S

2014-01-01

169

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.  

PubMed Central

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions. Images PMID:2523077

Nagai, R; Zarain-Herzberg, A; Brandl, C J; Fujii, J; Tada, M; MacLennan, D H; Alpert, N R; Periasamy, M

1989-01-01

170

Oxidation-induced unfolding facilitates Myosin cross-linking in myofibrillar protein by microbial transglutaminase.  

PubMed

Myofibrillar protein from pork Longissimus muscle was oxidatively stressed for 2 and 24 h at 4 °C with mixed 10 ?M FeCl(3)/100 ?M ascorbic acid/1, 5, or 10 mM H(2)O(2) (which produces hydroxyl radicals) and then treated with microbial transglutaminase (MTG) (E:S = 1:20) for 2 h at 4 °C. Oxidation induced significant protein structural changes (P < 0.05) as evidenced by suppressed K-ATPase activity, elevated Ca-ATPase activity, increased carbonyl and disulfide contents, and reduced conformational stability, all in a H(2)O(2) dose-dependent manner. The structural alterations, notably with mild oxidation, led to stronger MTG catalysis. More substantial amine reductions (19.8-27.6%) at 1 mM H(2)O(2) occurred as compared to 11.6% in nonoxidized samples (P < 0.05) after MTG treatment. This coincided with more pronounced losses of myosin in oxidized samples (up to 33.2%) as compared to 21.1% in nonoxidized (P < 0.05), which was attributed to glutamine-lysine cross-linking as suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID:22809283

Li, Chunqiang; Xiong, Youling L; Chen, Jie

2012-08-15

171

Atrial and ventricular myosins from human hearts II. Isoenzyme distribution after myocardial infarction  

Microsoft Academic Search

Summary Human atrial and ventricular myosins were prepared from autopsy specimens from subjects with coronary heart disease. Cardiac myosin light chain isotypes were resolved using twodimensional gel electrophoresis, whereas myosin isozymes were detected by pyrophosphate gel electrophoresis.

U. Hoffmann; C. Axmann; N. Palm

1987-01-01

172

Slow myosin in developing rat skeletal muscle  

Microsoft Academic Search

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. Immuno- blots of MHC peptide

M. Narusawa; R. B. Fitzsimons; S. Izumo; B. Nadal-Ginard; N. A. Rubinstein; A. M. Kelly

1987-01-01

173

Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes  

PubMed Central

Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times within bilaterian evolution. PMID:24498429

Kollmar, Martin; Hatje, Klas

2014-01-01

174

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana  

PubMed Central

Background The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response. Results Wild type and phot2 mutant plants were irradiated with strong (36 µEm?2s?1) and/or weak (0.8 µEm?2s?1) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant. Conclusions Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements. PMID:19516997

Krzeszowiec, Weronika

2007-01-01

175

Dynamic Exchange of Myosin VI on Endocytic Structures*  

PubMed Central

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI. PMID:22992744

Bond, Lisa M.; Arden, Susan D.; Kendrick-Jones, John; Buss, Folma; Sellers, James R.

2012-01-01

176

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle.  

PubMed

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca(2+)/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca(2+) binding to calmodulin forming a (Ca(2+))(4)•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca(2+) results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca(2+)/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca(2+)-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

Stull, James T; Kamm, Kristine E; Vandenboom, Rene

2011-06-15

177

Phosphate release coupled to rotary motion of F1-ATPase  

PubMed Central

F1-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three ??-dimers creates torque on its central ?-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation. In response to torque-driven rotation of the ?-subunit in the hydrolysis direction, the nucleotide-free ??E interface forming the “empty” E site loosens and singly charged Pi readily escapes to the P loop. By contrast, the interface stays closed with doubly charged Pi. The ?-rotation tightens the ATP-bound ??TP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the ??E interface 120° after ATP hydrolysis closely matches the ?1-ms functional timescale. Conversely, Pi release from the ADP-bound ??DP interface postulated in earlier models would occur through a kinetically infeasible inward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of Pi exit, its pathway and kinetics, associated changes in Pi protonation, and changes of the F1-ATPase structure in the 40° substep. Important elements of the molecular mechanism of Pi release emerging from our simulations appear to be conserved in myosin despite the different functional motions. PMID:24062450

Okazaki, Kei-ichi; Hummer, Gerhard

2013-01-01

178

Cargo recognition and cargo-mediated regulation of unconventional myosins.  

PubMed

Conspectus Organized motions are hallmarks of living organisms. Such motions range from collective cell movements during development and muscle contractions at the macroscopic scale all the way down to cellular cargo (e.g., various biomolecules and organelles) transportation and mechanoforce sensing at more microscopic scales. Energy required for these biological motions is almost invariably provided by cellular chemical fuels in the form of nucleotide triphosphate. Biological systems have designed a group of nanoscale engines, known as molecular motors, to convert cellular chemical fuels into mechanical energy. Molecular motors come in various forms including cytoskeleton motors (myosin, kinesin, and dynein), nucleic-acid-based motors, cellular membrane-based rotary motors, and so on. The main focus of this Account is one subfamily of actin filament-based motors called unconventional myosins (other than muscle myosin II, the remaining myosins are collectively referred to as unconventional myosins). In general, myosins can use ATP to fuel two types of mechanomotions: dynamic tethering actin filaments with various cellular compartments or structures and actin filament-based intracellular transport. In contrast to rich knowledge accumulated over many decades on ATP hydrolyzing motor heads and their interactions with actin filaments, how various myosins recognize their specific cargoes and whether and how cargoes can in return regulate functions of motors are less understood. Nonetheless, a series of biochemical and structural investigations in the past few years, including works from our own laboratory, begin to shed lights on these latter questions. Some myosins (e.g., myosin-VI) can function both as cellular transporters and as mechanical tethers. To function as a processive transporter, myosins need to form dimers or multimers. To be a mechanical tether, a monomeric myosin is sufficient. It has been shown for myosin-VI that its cellular cargo proteins can play critical roles in determining the motor properties. Dab2, an adaptor protein linking endocytic vesicles with actin-filament-bound myosin-VI, can induce the motor to form a transport competent dimer. Such a cargo-mediated dimerization mechanism has also been observed in other myosins including myosin-V and myosin-VIIa. The tail domains of myosins are very diverse both in their lengths and protein domain compositions and thus enable motors to engage a broad range of different cellular cargoes. Remarkably, the cargo binding tail of one myosin alone often can bind to multiple distinct target proteins. A series of atomic structures of myosin-V/cargo complexes solved recently reveals that the globular cargo binding tail of the motor contains a number of nonoverlapping target recognition sites for binding to its cargoes including melanophilin, vesicle adaptors RILPL2, and vesicle-bound GTPase Rab11. The structures of the MyTH4-FERM tandems from myosin-VIIa and myosin-X in complex with their respective targets reveal that MyTH4 and FERM domains extensively interact with each other forming structural and functional supramodules in both motors and demonstrate that the structurally similar MyTH4-FERM tandems of the two motors display totally different target binding modes. These structural studies have also shed light on why numerous mutations found in these myosins can cause devastating human diseases such as deafness and blindness, intellectual disabilities, immune disorders, and diabetes. PMID:25230296

Lu, Qing; Li, Jianchao; Zhang, Mingjie

2014-10-21

179

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases  

NASA Technical Reports Server (NTRS)

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

180

Relationship of the membrane ATPase from Halobacterium saccharovorum to vacuolar ATPases  

NASA Technical Reports Server (NTRS)

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-chloro-7-nitrobenzofurazan inhibited the halobacterial ATPase also in a nucleotide-protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggest that this halobacterial ATPase may have conserved structural features from both the vacuolar and the F-type ATPases.

Stan-Lotter, Helga; Hochstein, Lawrence I.; Bowman, Emma J.

1991-01-01

181

Myoglobin levels and mATPase activity in pectoral muscles of spruce and ruffed grouse (Aves: Tetraoninae).  

PubMed

Myoglobin concentration and myosin ATPase activity were measured in the pectoral muscle of wild spruce grouse (Dendragapus canadensis) and ruffed grouse (Bonasa umbellus), together with the weight of the Mm. pectoralis, supracoracoideus and heart. mATPase activities were similar in both species, but spruce grouse contained 15 times more myoglobin in the pectoralis muscle and the heart was three times heavier than that of the ruffed grouse. The relative mass of the flight muscles and wing loading were similar between species. Characteristics of the pectoral muscle of both grouse species reflect adaptations to predation and advertising displays. The glycolytic nature of the ruffed grouse pectoral muscle and small heart size is an adaptation to a sedentary existence within a small home range. The more oxidative pectoral muscle of spruce grouse together with its larger heart are adaptations to seasonal dispersals requiring more sustained flight. PMID:2859952

Thomas, V G

1985-01-01

182

Avian Synaptopodin 2 (Fesselin) Stabilizes Myosin Filaments and Actomyosin in the Presence of ATP  

PubMed Central

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins have been identified that stabilize myosin filaments and that stabilize actin-myosin interactions. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not HMM, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin. PMID:24083890

Kingsbury, Nathanial L.; Renegar, Randall H.; Chalovich, Joseph M.

2013-01-01

183

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.  

PubMed

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin. PMID:24083890

Kingsbury, Nathanial L; Renegar, Randall H; Chalovich, Joseph M

2013-10-29

184

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

NASA Technical Reports Server (NTRS)

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.

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

2001-01-01

185

Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras.  

PubMed

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility. PMID:15962288

Helfman, David M; Pawlak, Geraldine

2005-08-01

186

Light Chains from Fast and Slow Muscle Myosins  

Microsoft Academic Search

Myosin from fast skeletal muscles consists of two large subunits (each about 200,000 daltons) and approximately four smaller subunits (in the range of 20,000 daltons). The light chains are heterogeneous in both size and charge, irrespective of the muscle type of origin. Moreover, the myosins from fast muscles of several species show a characteristic light chain pattern which is distinct

Susan Lowey; Dennis Risby

1971-01-01

187

Functional Characterization of Myosin I Tail Regions in Candida albicans  

Microsoft Academic Search

The molecular motor myosin I is required for hyphal growth in the pathogenic yeast Candida albicans. Specific myosin I functions were investigated by a deletion analysis of five neck and tail regions. Hyphal formation requires both the TH1 region and the IQ motifs. The TH2 region is important for optimal hyphal growth. All of the regions, except for the SH3

Ursula Oberholzer; Tatiana L. Iouk; David Y. Thomas; Malcolm Whiteway

2004-01-01

188

Immunolocalization of myosin I? in the hair cell's hair bundle  

Microsoft Academic Search

The hair bundle, the hair cell's sensory organelle, transduces acoustical or vestibular stimulation into a change in membrane potential. The actin-based stereociliary processes of the hair bundle contain a number of myosin isoforms that may be important to the bundle's function. One of these isoforms, myosin I b, has been proposed to constitute an adaptation motor controling sensitivity of the

Anne B. Metcalf

1998-01-01

189

A new model of cooperative myosin-thin filament binding.  

PubMed

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction. PMID:10864931

Tobacman, L S; Butters, C A

2000-09-01

190

Myofibrillar ATPase activity and mechanical performance of skinned fibres from rabbit psoas muscle.  

PubMed Central

1. The relationship between energy turnover and mechanical performance was investigated in chemically skinned single fibres from rabbit psoas muscle at 15 degrees C, pH = 7.1, with MgATP, 5 mM; free Mg2+, 1 mM; ionic strength, 200 mM and sarcomere length, 2.4 microns by measuring force production and myofibrillar ATP turnover during isometric contractions as well as during repetitive changes in length. ATP hydrolysis was stoichiometrically coupled to the breakdown of NADH, which was measured photometrically via the absorption of near UV light at 340 nm. 2. Force and ATPase activity were measured during square-wave length changes of different amplitudes (1-10% of the fibre length, Lo) and different frequencies (2.5-167 Hz). The average force during the length changes was less than the isometric value and decreased with increasing amplitude and frequency. At full activation (pCa 4.5), the isometric ATP turnover rate (+/- S.E.M.) was 2.30 +/- 0.05 s-1 per myosin head. ATP turnover increased monotonically with increasing amplitude as well as with increasing frequency until saturation was reached. The greatest increase observed was 2.4 times the isometric value. 3. Force and ATPase activity were also determined for ramp shortenings followed by fast restretches. The average force decreased with increasing shortening velocity in a hyperbolic fashion. The ATP turnover increased with ramp velocity up to 0.5 L0 s-1 and stayed almost constant (at 2.2 times the isometric value) for larger velocities. 4. Isometric force and ATPase activity both decreased as the calcium concentration was decreased. They did not vary in proportion at low Ca2+ concentrations, but this could largely be accounted for by the presence of a residual, Ca(2+)-dependent, membrane-bound ATPase. At high calcium concentrations ATPase activity during square-wave length changes was higher than the isometric value, but at low calcium concentrations (pCa > 6.1), the ATPase activity during the length changes decreased below the isometric value and reached a minimum of 40% of the isometric level. 5. ATPase activity and average force obtained during changes in length show a high, movement protocol-independent correlation. During the length changes the rate of ATP turnover divided by the average force level (tension cost) was larger than the isometric tension cost. The largest value found, for 10% length changes at 23 Hz, was 17 times the tension cost under isometric conditions.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8006817

Potma, E J; Stienen, G J; Barends, J P; Elzinga, G

1994-01-01

191

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

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

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

2013-01-01

192

Atomic model of the human cardiac muscle myosin filament  

PubMed Central

Of all the myosin filaments in muscle, the most important in terms of human health, and so far the least studied, are those in the human heart. Here we report a 3D single-particle analysis of electron micrograph images of negatively stained myosin filaments isolated from human cardiac muscle in the normal (undiseased) relaxed state. The resulting 28-Å resolution 3D reconstruction shows axial and azimuthal (no radial) myosin head perturbations within the 429-Å axial repeat, with rotations between successive 132 Å-, 148 Å-, and 149 Å-spaced crowns of heads close to 60°, 35°, and 25° (all would be 40° in an unperturbed three-stranded helix). We have defined the myosin head atomic arrangements within the three crown levels and have modeled the organization of myosin subfragment 2 and the possible locations of the 39 Å-spaced domains of titin and the cardiac isoform of myosin-binding protein-C on the surface of the myosin filament backbone. Best fits were obtained with head conformations on all crowns close to the structure of the two-headed myosin molecule of vertebrate chicken smooth muscle in the dephosphorylated relaxed state. Individual crowns show differences in head-pair tilts and subfragment 2 orientations, which, together with the observed perturbations, result in different intercrown head interactions, including one not reported before. Analysis of the interactions between the myosin heads, the cardiac isoform of myosin-binding protein-C, and titin will aid in understanding of the structural effects of mutations in these proteins known to be associated with human cardiomyopathies. PMID:23251030

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

2013-01-01

193

A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase  

PubMed Central

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3? to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3? overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3? inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3? overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity. PMID:18094049

Koga, Yasuhiko

2008-01-01

194

Organelle Targeting of Myosin XI Is Mediated by Two Globular Tail Subdomains with Separate Cargo  

E-print Network

Organelle Targeting of Myosin XI Is Mediated by Two Globular Tail Subdomains with Separate Cargo 37996 Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding

Nebenführ, Andreas

195

A new model for myosin dimeric motors incorporating Brownian ratchet and powerstroke mechanisms  

E-print Network

A new model for myosin dimeric motors incorporating Brownian ratchet and powerstroke mechanisms in Myosins V and VI can be explained by varying the physical parameters describing the coupling between-headed processive motors. Keywords: Molecular motors, Brownian motors, power stroke, myosin V, myosin VI 1

Kawai, Ryoichi

196

Bidirectional cooperative motion of myosin-II motors on actin tracks with randomly alternating polarities  

E-print Network

Bidirectional cooperative motion of myosin-II motors on actin tracks with randomly alternating- myosin II systems, fast directional motion was observed, reflecting the tendency of myosin II motors to the stretching of the actin track by the myosin II motors. The new model yields a very good quantitative

Farago, Oded

197

An elastically tethered viscous load imposes a regular gait on the motion of myosin-V.  

E-print Network

An elastically tethered viscous load imposes a regular gait on the motion of myosin-V. Simulation NW7 1AA, UK Myosin-V is a processive molecular motor that moves membrane vesicles along actin tracks of the elastic connection are similar to those of the myosin-II coiled-coil domain, the myosin-V motor, tether

198

Organization and Ligand Binding Properties of the Tail of Acanthamoeba Myosin-IA  

E-print Network

Organization and Ligand Binding Properties of the Tail of Acanthamoeba Myosin-IA IDENTIFICATION The Acanthamoeba myosin-IA heavy chain gene en- codes a 134-kDa protein with a catalytic domain, three potential/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC

199

Band-like Stress Fiber Propagation in a Continuum and Implications for Myosin Contractile Stresses  

E-print Network

Band-like Stress Fiber Propagation in a Continuum and Implications for Myosin Contractile Stresses-like features that form with sarcomere-like actin and myosin arrangement between cell regions, resisting myosin contractility. We consider three aspects of stress fiber formation: (1) they form by cytoskeletal actin­myosin

Mofrad, Mohammad R. K.

200

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium.  

PubMed

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin-dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE. PMID:19137585

Lin-Jones, Jennifer; Sohlberg, Lorraine; Dosé, Andréa; Breckler, Jennifer; Hillman, David W; Burnside, Beth

2009-03-10

201

Enhancement of Force Generated by Individual Myosin Heads in Skinned Rabbit Psoas Muscle Fibers at Low Ionic Strength  

PubMed Central

Although evidence has been presented that, at low ionic strength, myosin heads in relaxed skeletal muscle fibers form linkages with actin filaments, the effect of low ionic strength on contraction characteristics of Ca2+-activated muscle fibers has not yet been studied in detail. To give information about the mechanism of muscle contraction, we have examined the effect of low ionic strength on the mechanical properties and the contraction characteristics of skinned rabbit psoas muscle fibers in both relaxed and maximally Ca2+-activated states. By progressively decreasing KCl concentration from 125 mM to 0 mM (corresponding to a decrease in ionic strength ? from 170 mM to 50 mM), relaxed fibers showed changes in mechanical response to sinusoidal length changes and ramp stretches, which are consistent with the idea of actin-myosin linkage formation at low ionic strength. In maximally Ca2+-activated fibers, on the other hand, the maximum isometric force increased about twofold by reducing KCl concentration from 125 to 0 mM. Unexpectedly, determination of the force-velocity curves indicated that, the maximum unloaded shortening velocity Vmax, remained unchanged at low ionic strength. This finding indicates that the actin-myosin linkages, which has been detected in relaxed fibers at low ionic strength, are broken quickly on Ca2+ activation, so that the linkages in relaxed fibers no longer provide any internal resistance against fiber shortening. The force-velocity curves, obtained at various levels of steady Ca2+-activated isometric force, were found to be identical if they are normalized with respect to the maximum isometric force. The MgATPase activity of muscle fibers during isometric force generation was found not to change appreciably at low ionic strength despite the two-fold increase in Ca2+-activated isometric force. These results can be explained in terms of enhancement of force generated by individual myosin heads, but not by any changes in kinetic properties of cyclic actin-myosin interaction. PMID:23691080

Sugi, Haruo; Abe, Takahiro; Kobayashi, Takakazu; Chaen, Shigeru; Ohnuki, Yoshiki; Saeki, Yasutake; Sugiura, Seiryo

2013-01-01

202

Evidence for expression of IIb myosin heavy chain isoform in some skeletal muscles of Blonde d'Aquitaine bulls.  

PubMed

In cattle, expression of the IIb myosin heavy chain (MyHC) isoform has been demonstrated only in extraocular muscles. In this study, we demonstrated for the first time its expression in the Semitendinosus and Longissimus thoracis muscles of a French beef breed, Blonde d'Aquitaine. Several techniques were used: RT-PCR, electrophoresis, western blotting, histochemistry with ATPase staining and immunohistochemistry using a combination of anti MyHC antibodies on serial sections. We found that MyHC IIb was expressed at the mRNA level in the two muscles of the cattle studied. However, the protein was observed at the tissue and cellular levels in only five of the 22 young bulls analysed, suggesting complex regulation of its expression. Using immunohistochemistry we demonstrated the presence of this MyHC isoform in pure fibres and also in hybrid fibres in co-expression with other MyHC. PMID:20416608

Picard, Brigitte; Cassar-Malek, Isabelle

2009-05-01

203

Self-Organization of Myosin II in Reconstituted Actomyosin Bundles  

PubMed Central

Cells assemble a variety of bundled actomyosin structures in the cytoskeleton for activities such as cell-shape regulation, force production, and cytokinesis. Although these linear structures exhibit varied architecture, two common organizational themes are a punctate distribution of myosin II and distinct patterns of actin polarity. The mechanisms that cells use to assemble and maintain these organizational features are poorly understood. To study these, we reconstituted actomyosin bundles in vitro that contained only actin filaments and myosin II. Upon addition of ATP, the bundles contracted and the uniformly distributed myosin spontaneously reorganized into discrete clusters. We developed a mathematical model in which the motion of myosin II filaments is governed by the polarities of the actin filaments with which they interact. The model showed that the assembly of myosins into clusters is driven by their tendency to migrate to locations with zero net actin filament polarity. With no fitting parameters, the predicted distribution of myosin cluster separations was in close agreement with our experiments, including a ?3/2 power law decay for intermediate length scales. Thus, without an organizing template or accessory proteins, a minimal bundle of actin and myosin has the inherent capacity to self-organize into a heterogeneous banded structure. PMID:22995499

Stachowiak, Matthew R.; McCall, Patrick M.; Thoresen, Todd; Balcioglu, Hayri E.; Kasiewicz, Lisa; Gardel, Margaret L.; O'Shaughnessy, Ben

2012-01-01

204

Stochastic dynamics and mechanosensitivity of myosin II minifilaments  

NASA Astrophysics Data System (ADS)

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

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

2014-09-01

205

Stochastic dynamics and mechanosensitivity of myosin II minifilaments  

E-print Network

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

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

2014-04-06

206

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

SciTech Connect

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.

Minoda, Hiroki [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan) [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Okabe, Tatsuhiro; Inayoshi, Yuhri [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan)] [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan); Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032 (Japan)] [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032 (Japan); Katayama, Eisaku [Graduate School of Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)] [Graduate School of Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Wakabayashi, Takeyuki [Department of Biosciences, School of Science and Engineering, Teikyo University, Utsunomiya, Tochigiken 320-8551 (Japan)] [Department of Biosciences, School of Science and Engineering, Teikyo University, Utsunomiya, Tochigiken 320-8551 (Japan); Akimoto, Tsuyoshi [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan)] [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan); Sugi, Haruo, E-mail: sugi@kyf.biglobe.ne.jp [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan)] [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan)

2011-02-25

207

Biochemistry 1993,32, 9859-9865 9859 Kinetic Characterization of Reductively Methylated Myosin Subfragment 1t  

E-print Network

Biochemistry 1993,32, 9859-9865 9859 Kinetic Characterization of Reductively Methylated Myosin,1993;Revised Manuscript Received June 11, 1993' ABSTRACT: Reductive methylation of myosin-S1 converts 97% of lysine residues in native myosin-S1 to dimethyllysine without detectable modification of other amino acid

Rayment, Ivan

208

Functional roles for myosin 1c in cellular signaling pathways  

PubMed Central

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

Bond, Lisa M.; Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2013-01-01

209

Synaptic plasticity in the MyosinVa mutant mouse  

E-print Network

The trafficking of essential proteins into spines is an important aspect of synaptic plasticity. MyosinVa, an actin-based motor protein, has been implicated in the synaptic delivery of AMPARs during LTP [1]. However an ...

Tunca, Cansu, 1977-

2009-01-01

210

Fujita-Becker et al. N-terminal region of myosin-2 Functional Characterization of the Amino-Terminal Region of Myosin-2  

E-print Network

Fujita-Becker et al. N-terminal region of myosin-2 1 Functional Characterization of the Amino-Terminal Region of Myosin-2 Setsuko Fujita-Becker , Georgios Tsiavaliaris§ , Reiko Ohkura¶ , Takashi Shimada, Komaba 3-8-1, Tokyo 153-8902, Japan. Running Title: N-terminal region of myosin-2 Address correspondence

Manstein, Dietmar J.

211

Partial cDNA Sequence Analysis of Myosin Va from Rainbow Trout (Oncorhynchus mykiss) and Its Relationship to Myosin V Isoforms from Other Vertebrates  

Microsoft Academic Search

Partial cDNA sequences of myosin V from rainbow trout Oncorhynchus mykiss were analyzed and showed high similarity to MVa from other vertebrates. Phylogenetic analysis has shown that events resulting in the formation of paralogous copies of myosin Va, Vb, and Vc occurred before the divergence of vertebrates into different classes. Expres- sion analysis of myosin Va, Vb, and Vc in

Katia Gisele Oliveira Rancura; Cesar Martins; Michelli Rivero Montano; Robson M. Carvalho; Alexandre Azevedo

2008-01-01

212

Developmental regulation of myosin gene expression in mouse cardiac muscle  

Microsoft Academic Search

Expression of the two isoforms of cardiac myosin heavy chain (MHC), MHCot and MHC\\/3, in mammals is regulated postnatally by a variety of stim- uli, including serum hormone levels. Less is known about the factors that regulate myosin gene expression in rapidly growing cardiac muscle in embryos. Using isoform-specific 35S-labeled cRNA probes correspond- ing to the two MHC genes and

Gary E. Lyons; Stefano Schiaflino; David Sassoon; Paul Barton; Margaret Buckingham

1990-01-01

213

Myosin types during the development of embryonic chicken fast and slow muscles.  

PubMed Central

We have studied the myosin types present in developing fast and slow muscles of the chicken embryo. Myosin light chains were characterized by their mobility on sodium dodecyl sulfate/polyacrylamide gels; myosin heavy chains were identified by their reaction with antibodies specific for adult fast or adult slow myosin heavy chains. During development, the pectoralis muscle, a fast muscle in the adult, contains heavy chains and two of the three light chains characteristic of adult fast muscle myosin. However, the anterior latissimus dorsi muscle, a slow muscle in the adult, also contains fast myosin light and heavy chains during early development. Only after the time of innervation does this muscle begin synthesizing predominantly the slow myosin heavy and light chains. We hypothesize that the synthesis of fast myosin in both early fast and slow muscles is the result of the endogenous program for muscle development; initiation of the synthesis of slow myosin, however, is dependent upon exogenous factors. Images PMID:412189

Rubinstein, N A; Pepe, F A; Holtzer, H

1977-01-01

214

Non-sarcomeric mode of myosin II organization in the fibroblast lamellum  

PubMed Central

The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed. PMID:8227130

1993-01-01

215

Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation  

Microsoft Academic Search

The phosphorylation of regulatory myosin light chains by the Ca2÷\\/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B., and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endo-

Zoe M. Goeckeler; Robert B. Wysolmerski

1995-01-01

216

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin  

Microsoft Academic Search

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of\\u000a shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers\\u000a with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition\\u000a of 20 ?M blebbistatin decreased tensions by

Melanie Stewart; Kathy Franks-Skiba; Roger Cooke

2009-01-01

217

Received 30 May 2012 | Accepted 19 Dec 2012 | Published 29 Jan 2013 Interplay between myosin IIA-mediated  

E-print Network

myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and preserves overall podosome integrity. Conversely, myosin IIA contracts the actin network filaments podosome size and protrusion. We demonstrate a previously unrecognized interplay between actin and myosin

Cai, Long

218

Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts  

SciTech Connect

V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V{sub 1} domain responsible for ATP hydrolysis and integral V{sub 0} domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.

Yao, GuanFeng [Department of Orthopedics, The Second Affiliated Hospital, ShanTou University Medical College, ShanTou, GuangDong 515041 (China); Feng, HaoTian [Department of Surgery, The University of Western Australia (Australia); Cai, YanLing [Department of Orthopedics, The Second Affiliated Hospital, ShanTou University Medical College, ShanTou, GuangDong 515041 (China); Qi, WeiLi [Department of Orthopedics, The Second Affiliated Hospital, ShanTou University Medical College, ShanTou, GuangDong 515041 (China); Kong, KangMei [Department of Orthopedics, The Second Affiliated Hospital, ShanTou University Medical College, ShanTou, GuangDong 515041 (China)]. E-mail: kangmeikong@21cn.com

2007-06-15

219

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

SciTech Connect

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

H Shi; G Blobel

2011-12-31

220

Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle.  

PubMed

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ?20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges. PMID:24285364

Duggal, Divya; Nagwekar, Janhavi; Rich, Ryan; Midde, Krishna; Fudala, Rafal; Gryczynski, Ignacy; Borejdo, Julian

2014-02-15

221

Identification of myosin heavy chain isoforms in skeletal muscle of four Southern African wild ruminants.  

PubMed

The aim was to separate and characterize the myosin heavy chain (MHC) isoforms of four southern African wild ruminants, namely Blesbuck (Damaliscus dorcas phillipsi), Kudu (Tragelaphus strepsiceros), Black Wildebeest (Connochaetes gnou) and Blue Wildebeest (Connochaetes taurinus). Longissimus dorsi muscle samples were subjected to SDS-PAGE and Western blot analyses using antibodies raised against MHC isoforms. The specificity of these antibodies was assessed using immunohistochemistry combined with ATPase histochemistry, Three MHC isoforms were separated and the bands were identified from fastest to slowest migrating as MHC I, MHC IIx and MHC IIa. The mobility of the MHC isoforms was similar for all four species, including that of bovine, but differed from human muscle. Kudu muscle exhibited the lowest proportion of MHC I and the highest proportion of MHC IIx, whereas Blesbuck muscle had the least MHC IIx. The two Wildebeest species were intermediate in isoform content. In conclusion, when new species are studied, existing electrophoretic protocols may need to be modified to achieve quantifiable separation and isoform migration pattern must be verified in order to reach correct interpretations. Furthermore, antibody specificity may differ between techniques as well as species and needs confirmation. PMID:17631404

Kohn, Tertius A; Hoffman, Louw C; Myburgh, Kathryn H

2007-10-01

222

Force generation by Myosin II Filaments in Compliant Networks  

E-print Network

Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) networks to generate contractile forces in cells. How their properties control force production in environments with varying stiffness is poorly understood. Here, we incorporated literature values for properties of myosin II isoforms into a cross-bridge model. Similar actin gliding speeds and force-velocity curves expected from previous experiments were observed. Motor force output on an elastic load was regulated by two timescales--that of their attachment to F-actin, which varied sharply with the ensemble size, motor duty ratio, and external load, and that of force build up, which scaled with ensemble stall force, gliding speed, and load stiffness. While such regulation did not require force-dependent kinetics, the myosin catch bond produced positive feedback between attachment time and force to trigger switch-like transitions from short attachments and small forces to high force-generating runs at threshold parameter values. Parameters representing skeletal muscle myosin, non-muscle myosin IIB, and non-muscle myosin IIA revealed distinct regimes of behavior respectively: (1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness, (2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force build-up times that exceed physiological timescales, and (3) small assemblies of low-duty ratio motors operating at intermediate speeds may respond sharply to changes in mechanical context--at low forces or stiffness, they serve as low affinity cross-links but they can transition to effective force production via the positive feedback mechanism described above. These results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment.

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

2014-04-12

223

Arabidopsis Myosin XI: A Motor Rules the Tracks.  

PubMed

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

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

2014-11-01

224

The Mechanism of Cu+ Transport ATPases  

PubMed Central

Cu+-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu+. In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu+, delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu+-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59–64) shows that a kinked transmembrane segment forms a “platform” exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the “entrance” of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu+-ATPase CopA do not affect ATPase activity in the presence of Cu+ free in solution. However, Cu+ bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu+ to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu+ transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu+ chaperone. This docking likely places the Cu+ proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu+ with the pump is transient because Cu+ is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation. PMID:23184962

Padilla-Benavides, Teresita; McCann, Courtney J.; Argüello, José M.

2013-01-01

225

Elastic lever arm model for myosin V  

E-print Network

We present a mechanochemical model for myosin V, a two-headed processive motor protein. We derive the properties of a dimer from those of an individual head, which we model both with a 4-state cycle (detached, attached with ADP.Pi, attached with ADP and attached without nucleotide) and alternatively with a 5-state cycle (where the power stroke is not tightly coupled to the phosphate release). In each state the lever arm leaves the head at a different, but fixed, angle. The lever arm itself is described as an elastic rod. The chemical cycles of both heads are coordinated exclusively by the mechanical connection between the two lever arms. The model explains head coordination by showing that the lead head only binds to actin after the power stroke in the trail head and that it only undergoes its power stroke after the trail head unbinds from actin. Both models (4- and 5-state) reproduce the observed hand-over-hand motion and fit the measured force-velocity relations. The main difference between the two models concerns the load dependence of the run length, which is much weaker in the 5-state model. We show how systematic processivity measurement under varying conditions could be used to distinguish between both models and to determine the kinetic parameters.

Andrej Vilfan

2005-03-14

226

Elastic lever arm model for myosin V  

E-print Network

We present a mechanochemical model for myosin V, a two-headed processive motor protein. We derive the properties of a dimer from those of an individual head, which we model both with a 4-state cycle (detached, attached with ADP.Pi, attached with ADP and attached without nucleotide) and alternatively with a 5-state cycle (where the power stroke is not tightly coupled to the phosphate release). In each state the lever arm leaves the head at a different, but fixed, angle. The lever arm itself is described as an elastic rod. The chemical cycles of both heads are coordinated exclusively by the mechanical connection between the two lever arms. The model explains head coordination by showing that the lead head only binds to actin after the power stroke in the trail head and that it only undergoes its power stroke after the trail head unbinds from actin. Both models (4- and 5-state) reproduce the observed hand-over-hand motion and fit the measured force-velocity relations. The main difference between the two models c...

Vilfan, A

2005-01-01

227

Polymorphisme de l'actylcholinestrase et de la myosine au cours du dveloppement des muscles rapide  

E-print Network

of acetylcholinesterase and myosin during the development of slow and fast muscle fibers after neonatal denervation, particularly at the level of acetylcholinesterase (AChE) molecular forms and myosin slow (LCs) and fast (LCf

Boyer, Edmond

228

Obscurin regulates the organization of myosin into A bands.  

PubMed

Obscurin is a giant sarcomeric protein composed of adhesion modules and signaling domains. It surrounds myofibrils at the level of the Z disk and the M line. To study the role of obscurin during myofibrillogenesis, we used adenovirus-mediated gene delivery to overexpress part of its COOH terminus in primary cultures of postnatal day 1 (P1) skeletal myotubes. Examination of the subcellular distribution of a number of sarcomeric proteins revealed that the organization of myosin into A bands was dramatically reduced. Myosin assembled into A bands normally in mock- or control-infected P1 myotubes. Overexpression of the COOH terminus of obscurin did not affect the organization of other sarcomeric markers, including actin, alpha-actinin, titin, and myomesin. Assembly of myomesin into nascent M lines in treated myotubes suggests that these structures can form independently of A bands. Immunoblot analysis indicated that there was a small ( approximately 20%) but consistent decrease in the amount of myosin expressed in cells infected with the COOH terminus of obscurin. Coimmunoprecipitation experiments in which we used adult skeletal muscle homogenates demonstrated that obscurin exists in a complex with myosin. Thus our findings suggest that the COOH-terminal region of obscurin interacts with sarcomeric myosin and may play a critical role in its ability to assemble into A bands in striated muscle. PMID:15013951

Kontrogianni-Konstantopoulos, Aikaterini; Catino, Dawn H; Strong, John C; Randall, William R; Bloch, Robert J

2004-07-01

229

Myosin light chain kinase phosphorylation in tracheal smooth muscle  

SciTech Connect

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in {sup 32}P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E. (Univ. of Texas Southwestern Medical Center, Dallas (USA))

1990-09-25

230

Structural Basis for Myosin V Discrimination Between Distinct Cargoes  

SciTech Connect

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

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

2006-01-01

231

Atomic force microscopy of thermally treated myosin filaments.  

PubMed

Heat-induced morphological change in myosin filaments was observed using atomic force microscope. The thickness of fixed native myosin filament was estimated to be 95 +/- 5 nm. When myosin filaments in 0.1 M NaCl at pH 6.0 were heated at 40, 55, and 70 degrees C for 10 min, the particulate structure appeared spirally on the surface of the filament at 40 degrees C, and the thickness of the filament was 75 +/- 10 nm. When myosin filaments were treated at 55 degrees C, several filaments were formed associated with side-by-side interaction through projected myosin heads to form a strand. The surface of the strand looked knobby. The thickness of thermally denatured filaments at 55 degrees C was 48 +/- 5 nm, and that of strands was about 80-110 nm, indicating the involvement of several filaments in a strand. The strands became to be rope-like at 70 degrees C, and the individual filaments in a strand were not distinguishable. PMID:15913330

Iwasaki, Tomohito; Washio, Masahiro; Yamamoto, Katsuhiro

2005-06-01

232

Myosin VI and its cargo adaptors - linking endocytosis and autophagy  

PubMed Central

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

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

2013-01-01

233

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes  

Microsoft Academic Search

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes.

Aziz El-Amraoui; Jean-Sébastien Schonn; Polonca Küssel-Andermann; Stéphane Blanchard; Claire Desnos; Jean-Pierre Henry; Uwe Wolfrum; François Darchen; Christine Petit

2002-01-01

234

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

PubMed Central

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

1992-01-01

235

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.  

PubMed

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+. PMID:141940

Tuena de Gómez Puyou, M; Gómez Puyou, A; Salmóm, M

1977-07-01

236

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?  

NASA Technical Reports Server (NTRS)

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

Hochstein, L. I.

1996-01-01

237

BIOLOGICAL SYSTEMS A STUDY OF THE SELF-ASSOCIATION OF MYOSIN  

E-print Network

BIOLOGICAL SYSTEMS A STUDY OF THE SELF-ASSOCIATION OF MYOSIN BY INTENSITY FLUCTUATION SPECTROSCOPY. Nous concluonsque la myosine est dans un équilibre monomère-dimèreen solu- tion et aussi que les deux-associationof myosin has been reported by Godfreyand Harrington (1970). We, Herbert (1970) and Herbert and Carlson

Paris-Sud XI, Université de

238

Yeast UCS proteins promote actomyosin interactions and limit myosin turnover in cells  

E-print Network

Yeast UCS proteins promote actomyosin interactions and limit myosin turnover in cells Matthew Lord for the conserved family of UCS proteins: helping to fold myosin motor proteins and stimulating the motor function of folded myosins. We examined both func- tions in yeast. The fission yeast UCS protein (Rng3p) concentrates

239

Actin-Myosin Viscoelastic Flow in the Keratocyte Lamellipod Boris Rubinstein,  

E-print Network

Actin-Myosin Viscoelastic Flow in the Keratocyte Lamellipod Boris Rubinstein, Maxime F. Fournier by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin

Mogilner, Alex

240

Nucleotides Increase the Internal Flexibility of Filaments of Dephosphorylated Acanthamoeba Myosin II*  

E-print Network

Nucleotides Increase the Internal Flexibility of Filaments of Dephosphorylated Acanthamoeba Myosin activity of Acan- thamoeba myosin II minifilaments is dependent both on Mg2 concentration and on the state for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend

Rau, Don C.

241

Conservation within the myosin motor domain: implications for structure and function  

E-print Network

Conservation within the myosin motor domain: implications for structure and function M Jamie TV Cope1, James Whisstock2, Ivan Rayment3 and John Kendrick-Jones1* Background: Myosins are motors that use energy supplied by ATP to travel along actin filaments. The structure of myosin is known

Rayment, Ivan

242

Molecular Dynamics Analysis of Structural Factors Influencing Back Door Pi Release in Myosin  

E-print Network

Molecular Dynamics Analysis of Structural Factors Influencing Back Door Pi Release in Myosin J the myosin active site for phosphate (Pi) generated by adenosine 5#-triphosphate hydrolysis. We used for 18 O-exchange with Pi at the active site, and Pi release being the rate-limiting step in the myosin

Rayment, Ivan

243

Membrane Tension, Myosin Force, and Actin Turnover Maintain Actin Treadmill in the Nerve Growth Cone  

E-print Network

Membrane Tension, Myosin Force, and Actin Turnover Maintain Actin Treadmill in the Nerve Growth treadmill. We show that actin retrograde flow is primarily generated by myosin contractile forces, but when myosin is inhibited, leading-edge membrane tension increases and drives the flow. By comparing

Mogilner, Alex

244

Coupling between Normal Modes Drives Protein Conformational Dynamics: Illustrations Using Allosteric Transitions in Myosin II  

E-print Network

Allosteric Transitions in Myosin II Wenjun Zheng * and D. Thirumalai * Physics Department, University illustrate the new concepts using myosin II motor protein as an example. The biological implications of mode, and the force-generating converter and lever arm in myosin isoforms are discussed. We evaluate the robustness

Thirumalai, Devarajan

245

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical  

E-print Network

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)-- seem predictable for low myosin activity in cells

Discher, Dennis

246

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

E-print Network

Dynamics of Myosin-Driven Skeletal Muscle Contraction: I. Steady-State Force Generation Ganhui Lan, a mechanistic explanation of the collective myosin force generation is lacking. We present a theoretical model of muscle contraction based on the conformational movements of individual myosins and experimentally

Sun, Sean

247

Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro  

Microsoft Academic Search

Monoclonal antibodies (McAbs) against the myosin heavy chain (MHC) of adult chicken pectoralis muscle have been tested for reactivity with pectoralis myosin at selected stages of chick development in vivo and in vitro. Three such McAbs, MF 20 and MF 14, which bind to light meromyosin, and MF 30, which binds to myosin subfragment two ($2), were used to assay

DAVID BADER; TOMOH MASAKI; DONALD A. FISCHMAN

1982-01-01

248

Specificity of blebbistatin, an inhibitor of myosin II JOHN LIMOUZE1  

E-print Network

Specificity of blebbistatin, an inhibitor of myosin II JOHN LIMOUZE1 , AARON F. STRAIGHT2 , TIMOTHY; accepted in revised form 12 March 2004 Key words: myosin, blebbistatin, inhibitors Abstract Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined

Straight, Aaron

249

Dynamics of Myosin-V Processivity Ganhui Lan* and Sean X. Sun*y  

E-print Network

Dynamics of Myosin-V Processivity Ganhui Lan* and Sean X. Sun*y *Department of Mechanical ABSTRACT Myosin-V is an actin-associated processive molecular motor. Single molecule experiments revealed that myosin-V walks in a stepwise fashion with occasional backward steps. By combining the mechanical

Sun, Sean

250

The power stroke of myosin VI and the basis of reverse directionality  

E-print Network

The power stroke of myosin VI and the basis of reverse directionality Zev Bryant*, David Altman (sent for review October 24, 2006) Myosin VI supports movement toward the ( ) end of actin fila- ments, despite sharing extensive sequence and structural homol- ogy with ( )-end-directed myosins. A class

Spudich, James A.

251

Myosin-I Moves Actin Filaments on a Phospholipid Substrate: Implications for Membrane Targeting  

E-print Network

Myosin-I Moves Actin Filaments on a Phospholipid Substrate: Implications for Membrane Targeting, The Johns Hopkins Medical School, Baltimore, Maryland 21205 Abstract. Acanthamoeba myosin-I bound of 0.2 /Am/s. This movement required ATP and phosphoryla- tion of the myosin-I heavy chain. We prepared

252

During Multicellular Migration, Myosin II Serves a Structural Role Independent of its Motor Function  

E-print Network

During Multicellular Migration, Myosin II Serves a Structural Role Independent of its Motor have shown previously that cells lacking myosin II are impaired in multicellular motility. We now extend these results by determining whether myosin contractile function is necessary for normal

Chisholm, Rex L.

253

Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever  

E-print Network

Myosin VI Steps via a Hand-over- Hand Mechanism with Its Lever Arm Undergoing Fluctuations when of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085 Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center

Yildiz, Ahmet

254

INTRODUCTION Myosin II is an actin-based motor, which is important for many  

E-print Network

INTRODUCTION Myosin II is an actin-based motor, which is important for many aspects of motility in muscle and nonmuscle cells (Harrington and Rodgers, 1984). Myosin is a hetero-hexamer composed of two of serine and threonine residues located near the N terminus of the RLC regulates myosin activity. In smooth

Chisholm, Rex L.

255

Self-Organization of Myosin II in Reconstituted Actomyosin Bundles Matthew R. Stachowiak,  

E-print Network

Self-Organization of Myosin II in Reconstituted Actomyosin Bundles Matthew R. Stachowiak, Patrick M exhibit varied architecture, two common organi- zational themes are a punctate distribution of myosin II that contained only actin filaments and myosin II. Upon addition of ATP, the bundles contracted and the uniformly

Gardel, Margaret

256

A Kinetic Model Describing the Processivity of Myosin-V Karl I. Skau,*y  

E-print Network

A Kinetic Model Describing the Processivity of Myosin-V Karl I. Skau,*y Rebecca B. Hoyle of how myosin-V coordinates the biochemical reactions and mechanical motions of its two head elements a quantitative kinetic model of the myosin-V walk, consisting of five basic states augmented by two further

Turner, Matthew

257

The Organization of Myosin and Actin in Rapid Frozen Nerve Growth Cones  

E-print Network

The Organization of Myosin and Actin in Rapid Frozen Nerve Growth Cones P. C. Bridgman and M. E with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern

Dailey, Michael E.

258

Detailed Tuning of Structure and Intramolecular Communication Are Dispensable for Processive Motion of Myosin VI  

E-print Network

of Myosin VI Mary Williard Elting, Zev Bryant,§ Jung-Chi Liao,§ and James A. Spudich * Department, California ABSTRACT Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI

Spudich, James A.

259

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

E-print Network

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated

Yu, Yan

260

Electron microscopy of synthetic myosin filaments. Evidence for cross- bridge. Flexibility and copolymer formation  

PubMed Central

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture. PMID:1236853

1975-01-01

261

Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism.  

PubMed

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. PMID:25122766

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

262

FXYD Proteins Stabilize Na,K-ATPase  

PubMed Central

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (? subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (?1?1) to form ?1?1FXYD complexes. Compared with the control (?1?1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1 > FXYD2 ? FXYD4. Heating also inactivates E1 ? E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of ?1?1 or ?1?1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the ? subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat ?1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function. PMID:21228272

Mishra, Neeraj Kumar; Peleg, Yoav; Cirri, Erica; Belogus, Talya; Lifshitz, Yael; Voelker, Dennis R.; Apell, Hans-Juergen; Garty, Haim; Karlish, Steven J. D.

2011-01-01

263

Understanding myosin functions in plants: are we there yet?  

PubMed

Myosins are motor proteins that drive movements along actin filaments and have long been assumed to be responsible for cytoplasmic streaming in plant cells. This conjecture is now firmly established by genetic analysis in the reference species, Arabidopsis thaliana. This work and similar approaches in the moss, Physcomitrella patens, also established that myosin-driven movements are necessary for cell growth and polarity, organelle distribution and shape, and actin organization and dynamics. Identification of a mechanistic link between intracellular movements and cell expansion has proven more challenging, not the least because of the high level of apparent genetic redundancy among myosin family members. Recent progress in the creation of functional complementation constructs and identification of interaction partners promises a way out of this dilemma. PMID:24446546

Madison, Stephanie L; Nebenführ, Andreas

2013-12-01

264

Model for kinetics of myosin-V molecular motors.  

PubMed

A hand-over-hand model is presented for the processive movement of myosin-V based on previous biochemical experimental results and structural observations of nucleotide-dependent conformational changes of single-headed myosins. The model shows that the ADP-release rate of the trailing head is much higher than that of the leading head, thus giving a 1:1 mechanochemical coupling for the processive movement of the motor. It explains well the previous finding that some 36-nm steps consist of two substeps, while other 36-nm steps consist of no substeps. Using the model, the calculated kinetic behaviors of myosin-V such as the main and intermediate dwell time distributions, the load dependence of the average main and intermediate dwell time and the load dependence of occurrence frequency of the intermediate state under various nucleotide conditions show good quantitative agreement with previous experimental results. PMID:16386350

Xie, Ping; Dou, Shuo-Xing; Wang, Peng-Ye

2006-04-01

265

Coarse-Grained Simulation of Myosin-V Movement  

PubMed Central

We describe the development of a hierarchic modelling method applied to simulating the processive movement of the myosin-V molecular motor protein along an actin filament track. In the hierarchic model, three different levels of protein structure resolution are represented: secondary structure, domain, and protein, with the level of detail changing according to the degree of interaction among the molecules. The integrity of the system is maintained using a tree of spatially organised bounding volumes and distance constraints. Although applied to an actin-myosin system, the hierarchic framework is general enough so that it may easily be adapted to a number of other large biomolecular systems containing in the order of 100 proteins. We compared the simulation results with biophysical data, and despite the lack of atomic detail in our model, we find good agreement and can even suggest some refinements to the current model of myosin-V motion. PMID:22675402

Katsimitsoulia, Zoe; Taylor, William R.

2012-01-01

266

Myosin light chain phosphorylation and growth cone motility.  

PubMed

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition. PMID:12210102

Schmidt, John T; Morgan, Patricia; Dowell, Natalie; Leu, Byunghee

2002-09-01

267

Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1  

SciTech Connect

The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.

Bettache, N.; Bertrand, R.; Kassab, R. (Universite de Montpellier (France))

1990-09-25

268

The Mechanism of Assembly ofAcanthamoeba Myosin-II Minifilaments: Minifilaments Assemble by Three Successive Dimedzation Steps  

E-print Network

The Mechanism of Assembly ofAcanthamoeba Myosin-II Minifilaments: Minifilaments Assemble by Three ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight myosin-II is monomeric. At low ionic strength (myosin-II polymerizes into bipolar

269

Differences in myosin composition between human oro-facial, masticatory and limb muscles: enzyme-, immunohisto-and biochemical studies  

Microsoft Academic Search

Immunohistochemistry was used to determine the myosin composition of defined fibre types of three embryologically different adult muscles, the oro-facial, masseter and limb muscles. In addition, the myosin composition in whole muscle specimens was analysed with biochemical methods. Both similarities and differences between muscles in the content of myosin heavy chains and myosin light chains were found. Nevertheless, each muscle

P. Stål; P.-O. Eriksson; S. Schiaffino; G. S. Butler-Browne; L.-E. Thornell

1994-01-01

270

Assembly of avian skeletal muscle myosins: evidence that homodimers of the heavy chain subunit are the thermodynamically stable form  

Microsoft Academic Search

Using a double antibody sandwich ELISA we examined the heavy chain isoform composition of myosin molecules isolated from chicken pectoralis ma- jor muscle during different stages of development. At 2- and 40-d posthatch, when multiple myosin heavy chain isoforms are being synthesized, we detected no heterodimeric myosins, suggesting that myosins are homodimers of the heavy chain subunit. Chymotryptic rod fragments

Bruce Kerwin; Everett Bandman

1991-01-01

271

Qualitative Analysis of Skeletal Myosin as Substrate of Ca 2+- activated Neutral Protease: Comparison of Filamentous and Soluble, Native, and L2Deficient Myosin  

Microsoft Academic Search

Ca2+-activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions

S. M. PEMRICK; R. C. GREBENAU

272

Biosynthesis of the tonoplast H sup + -ATPase  

SciTech Connect

To determine whether the tonoplast H{sup +}-ATPase was differentially synthesized in oat seedlings, sections were labeled in vivo with ({sup 35}S)-methionine and ATPase subunits were immunoprecipitated. Subunits were detected in all portions of the seedling with the exception of the seed. The intracellular site of synthesis for two peripheral ATPase subunits was investigated. RNA encoding the 72 kDa (catalytic) subunit was found in membrane-bound polysomes. In contrast, message for the 60 kDa subunit was found on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences, suggesting the absence of cleaved precursors for the 72 or 60 kDa subunits.

Randall, S.K. (McGill Univ., Montreal, Quebec (Canada)); Sze, H. (Univ. of Maryland, College Park (USA))

1989-04-01

273

Myosins VIII and XI Play Distinct Roles in Reproduction and Transport of Tobacco Mosaic Virus  

PubMed Central

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection. PMID:25329993

Amari, Khalid; Di Donato, Martin; Dolja, Valerian V.; Heinlein, Manfred

2014-01-01

274

Myosin IB from Entamoeba histolytica is involved in phagocytosis of human erythrocytes.  

PubMed

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery in humans. The disease is prevalent worldwide. Infection with E. histolytica results in invasion of the intestine by the parasite, followed by tissue damage and inflammation. During this invasive process, parasites kill and phagocytose human epithelial cells, immune cells and erythrocytes. Expression of amoebic pathogenicity requires a dynamic cytoskeleton that allows movement, tissue penetration and changes in parasite morphology. Myosin IB is a member of the myosin I family of motor proteins. Studies conducted both with Dictyostelium discoideum, a non-pathogenic amoeba, and with the yeast Saccharomyces cerevisiae indicate the involvement of myosin IB in cellular processes including movement, phagocytosis and endocytosis. Recently, we isolated the gene encoding myosin IB from E. histolytica. Thus, we decided to analyze the role of myosin IB in pathogenesis of amoeba. Using a specific anti-myosin IB antibody, this protein was localized in cell regions including the pseudopod, vesicles and underneath the plasma membrane. When E. histolytica was activated for erythrophagocytosis, myosin IB was markedly recruited to both the phagocytic cup and around internalized phagosomes. To analyze the role of myosin IB in phagocytosis, a strain overexpressing the myosin IB gene was constructed. This strain synthesizes threefold more myosin IB than the wild-type strain. Challenge of the transfected cell line with erythrocytes showed that these amoebae were deficient in erythrophagocytosis mainly in the uptake step, suggesting a role for myosin IB in the pathogenic activity of a human parasite. PMID:10085254

Voigt, H; Olivo, J C; Sansonetti, P; Guillén, N

1999-04-01

275

Myosin I and adaptation of mechanical transduction by the inner ear.  

PubMed Central

Twenty years ago, the description of hair-cell stereocilia as actin-rich structures led to speculation that myosin molecules participated in mechanical transduction in the inner ear. In 1987, Howard and Hudspeth proposed specifically that a myosin I might mediate adaptation of the transduction current carried by hair cells, the sensory cells of the ear. We exploited the myosin literature to design tests of this hypothesis and to show that the responsible isoform is myosin 1c. The identification of this myosin as the adaptation motor would have been impossible without thorough experimentation on other myosins, particularly muscle myosins. The sliding-filament hypothesis for muscle contraction has thus led to a detailed understanding of the behaviour of hair cells. PMID:15647170

Gillespie, Peter G

2004-01-01

276

Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells  

PubMed Central

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMI?) is associated with endosomes and lysosomes. We show that the overproduction of MMI? or the production of nonfunctional truncated MMI? affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMI?, promote the dissociation of MMI? from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMI? might therefore be involved in membrane trafficking occurring between endosomes and lysosomes. PMID:10233157

Raposo, Graça; Cordonnier, Marie-Neige; Tenza, Danièle; Menichi, Bernadette; Dürrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

1999-01-01

277

Na+/K+-ATPase: Activity and inhibition  

NASA Astrophysics Data System (ADS)

The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

?olovi?, M.; Krsti?, D.; Krinulovi?, K.; Momi?, T.; Savi?, J.; Vuja?i?, A.; Vasi?, V.

2009-09-01

278

Engineering myosins for long-range transport on actin filaments  

NASA Astrophysics Data System (ADS)

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

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

2014-01-01

279

Characterization of a Myosin VII MyTH/FERM domain  

PubMed Central

A group of closely related myosins are characterized by the presence of at least one MyTH/FERM (myosin talin homology 4; band 4.1, ezrin, radixin, moesin) domain in their C-terminal tails. This domain interacts with a variety of binding partners, and mutations in either the MyTH4 or FERM domains of myosin VII and XV result in deafness, highlighting the functional importance of each domain. The N-terminal MyTH/FERM region of Dictyostelium myosin VII (M7) has been isolated as a first step toward gaining insight into the function of this domain and its interaction with binding partners. The M7 MyTH4/FERM domain (MF1) binds to both actin and microtubules in vitro, with dissociation constants of 13.7 and 1.7 ?M, respectively. Gel filtration and UV spectroscopy reveal that MF1 exists as a monomer in solution and forms a well-folded, compact conformation with a high degree of secondary structure. These results indicate that MF1 forms an integrated structural domain that serves to couple actin filaments and microtubules in specific regions of the cytoskeleton. PMID:21875595

Moen, Rebecca J.; Johnsrud, Daniel O.; Thomas, David D.; Titus, Margaret A.

2011-01-01

280

Structural kinetics of myosin by transient time-resolved FRET  

E-print Network

Structural kinetics of myosin by transient time-resolved FRET Yuri E. Nesmelova,1,2 , Roman V technique, ðTR�2 FRET (transient time-resolved FRET), which resolves protein structural states with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate

Thomas, David D.

281

Molecular mechanism regulating myosin and cardiac functions by ELC.  

PubMed

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

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

2014-07-18

282

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate and its Cr(H2O)4 and Co(NH3)4 complex derivatives are new fluorescent tools for labelling ATP binding sites of Na+/K+-ATPase.  

PubMed

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+-ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na+/K+-ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, Kd=200 microM) and the high (E1, Kd=22 microM) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Ki=(KdE1*KdE2)1/2, the experimentally measured inhibition constant of 66 microM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium(III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na+/K+-ATPase in a specific way. PMID:9728479

Linnertz, H; Lastres Becker, I; Krumscheid, R; Amler, E; Thoenges, D; Schoner, W

1997-01-01

283

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

Microsoft Academic Search

Abstract. Recent biochemical studies of p190, a cal- modulin,(CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of prop- erties with myosins (Espindola, E S., E. M. Esprea- rico, M. V. Coelho, A. R. Martins, E R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J.

Enilza M. Espreafico; Richard E. Cheney; Michela Matteoli; Alexandra A. C. Nascimento; Pietro V. De Camilli; Roy E. Larson; Mark S. Mooseker

1992-01-01

284

Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.  

PubMed

We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

Espinoza-Fonseca, L Michel; Colson, Brett A; Thomas, David D

2014-10-01

285

The mechanism of Torsin ATPase activation  

PubMed Central

Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin–activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism. PMID:25352667

Brown, Rebecca S. H.; Zhao, Chenguang; Chase, Anna R.; Wang, Jimin; Schlieker, Christian

2014-01-01

286

The mechanism of Torsin ATPase activation.  

PubMed

Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism. PMID:25352667

Brown, Rebecca S H; Zhao, Chenguang; Chase, Anna R; Wang, Jimin; Schlieker, Christian

2014-11-11

287

Flexible Light-Chain and Helical Structure of F-Actin Explain the Movement and Step Size of Myosin-VI  

E-print Network

Flexible Light-Chain and Helical Structure of F-Actin Explain the Movement and Step Size of Myosin of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland ABSTRACT Myosin-VI is a dimeric isoform of unconventional myosins. Single molecule experiments indicate that myosin-VI and myosin

Sun, Sean

288

Erythrocyte membrane ATPases in diabetes: effect of dikanut (Irvingia gabonensis).  

PubMed

The levels of the three ATPases found in the erythrocyte membrane of diabetic patients were significantly lower than normal subjects. The distribution of the enzymes was also different. Na+,K+-ATPase and Mg2+-ATPase reflected the status of blood glucose more than Ca2+-ATPase. The ratio between two of the ATPases was sensitive to glycemic response. When dikanut, a viscous preparation, was fed to diabetics for 4 weeks, blood glucose became normal and the activities of the three ATPases increased significantly. The ratio among the enzymes also approached that of normal subjects. A relationship was found between the blood glucose level and erythrocyte membrane ATPases which, if linked to insulin binding or level, may provide a rapid inexpensive assay in diabetes research. PMID:3026798

Adamson, I; Okafor, C; Abu-Bakare, A

1986-01-01

289

The Kinetics of Mechanically Coupled Myosins Exhibit Group Size-Dependent Regimes  

PubMed Central

Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2–3 ?m. Three distinct regimes were found: L < 0.3 ?m, sliding arrest; 0.3 ?m ? L ? 1 ?m, alternation between arrest and continuous sliding; L > 1 ?m, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono ? bi ? mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted. PMID:24047998

Hilbert, Lennart; Cumarasamy, Shivaram; Zitouni, Nedjma B.; Mackey, Michael C.; Lauzon, Anne-Marie

2013-01-01

290

Actin Structure-Dependent Stepping of Myosin 5a and 10 during Processive Movement  

PubMed Central

How myosin 10, an unconventional myosin, walks processively along actin is still controversial. Here, we used single molecule fluorescence techniques, TIRF and FIONA, to study the motility and the stepping mechanism of dimerized myosin 10 heavy-meromyosin-like fragment on both single actin filaments and two-dimensional F-actin rafts cross-linked by fascin or ?-actinin. As a control, we also tracked and analyzed the stepping behavior of the well characterized processive motor myosin 5a. We have shown that myosin 10 moves processively along both single actin filaments and F-actin rafts with a step size of 31 nm. Moreover, myosin 10 moves more processively on fascin-F-actin rafts than on ?-actinin-F-actin rafts, whereas myosin 5a shows no such selectivity. Finally, on fascin-F-actin rafts, myosin 10 has more frequent side steps to adjacent actin filaments than myosin 5a in the F-actin rafts. Together, these results reveal further single molecule features of myosin 10 on various actin structures, which may help to understand its cellular functions. PMID:24069366

Gunther, Laura K.; Sellers, James R.; Sakamoto, Takeshi

2013-01-01

291

Asymmetric Myosin Binding to the Thin Filament as Revealed by a Fluorescent Nanocircuit  

PubMed Central

The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the prepowerstroke orientation when bound near troponin, but is predominantly in the postpowerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament. PMID:23274408

Coffee Castro-Zena, Pilar G.; Root, Douglas D.

2013-01-01

292

Three-dimensional structure of the human myosin thick filament: clinical implications  

PubMed Central

High resolution information about the three-dimensional (3D) structure of myosin filaments has always been hard to obtain. Solving the 3D structure of myosin filaments is very important because mutations in human cardiac muscle myosin and its associated proteins (e.g. titin and myosin binding protein C) are known to be associated with a number of familial human cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand how normal heart muscle works and how it fails, as well as the effects of the known mutations on muscle contractility, it is essential to properly understand myosin filament 3D structure and properties in both healthy and diseased hearts. The aim of this review is firstly to provide a general overview of the 3D structure of myosin thick filaments, as studied so far in both vertebrates and invertebrate striated muscles. Knowledge of this 3D structure is the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins (titin or C-protein), can be studied in detail. This should, in turn, enable us to relate the structure of myosin thick filament to its function and to understanding the disease process. A long term objective of this research would be to assist the design of possible therapeutic solutions to genetic myosin-related human cardiomyopathies. PMID:24689030

AL-Khayat, Hind A.

2013-01-01

293

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

PubMed

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

Masuda, Tadashi

2013-09-01

294

Physical mapping of human myosin-IXB (MYO9B), the human orthologue of the rat myosin myr 5, to chromosome 19p13.1  

SciTech Connect

The gene for human myosin-IXB (MYO9B) was mapped to human chromosome 19p13.1 using fluorescence in situ hybridization. The rat myosin myr 5 gene orthologue was used for the genetic mapping of the human gene. 14 refs., 1 fig.

Baehler, M.; Kehrer, I.; Stoeffler, H.E. [Friedrich-Miescher-Laboratorium, Tuebingen (Germany)] [Friedrich-Miescher-Laboratorium, Tuebingen (Germany); Gorden, L.; Olsen, A.S. [Lawrence Livermore National Lab., CA (United States)] [Lawrence Livermore National Lab., CA (United States)

1997-07-01

295

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

PubMed Central

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

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

2006-01-01

296

Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR  

PubMed Central

Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR?/? mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR?/? intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine. PMID:23986201

Collaco, Anne M.; Geibel, Peter; Lee, Beth S.; Geibel, John P.

2013-01-01

297

Stress Generation by Actin-Myosin Networks and Bundles  

NASA Astrophysics Data System (ADS)

Forces and stresses generated by the action of myosin minifilaments are calculated in idealized computer-generated actin networks and bundles. The networks are generated as random collections of actin filaments in two dimensions, and bundles are obtained by constraining the filament orientations. The actin filaments are crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending of actin filaments, which accomodates minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself.

Carlsson, Anders; Dasanayake, Nilushi

2013-03-01

298

Stochastic force generation by small ensembles of myosin II motors  

E-print Network

Forces in the actin cytoskeleton are generated by small groups of non-processive myosin II motors for which stochastic effects are highly relevant. Using a crossbridge model with the assumptions of fast powerstroke kinetics and equal load sharing between equivalent states, we derive a one-step master equation for the activity of a finite-sized ensemble of mechanically coupled myosin II motors. For constant external load, this approach yields analytical results for duty ratio and force-velocity relation as a function of ensemble size. We find that stochastic effects cannot be neglected for ensemble sizes below 15. The one-step master equation can be used also for efficient computer simulations with linear elastic external load and reveals the sequence of build-up of force and ensemble rupture that is characteristic for reconstituted actomyosin contractility.

Thorsten Erdmann; Ulrich S. Schwarz

2012-02-14

299

Stochastic force generation by small ensembles of myosin II motors  

E-print Network

Forces in the actin cytoskeleton are generated by small groups of non-processive myosin II motors for which stochastic effects are highly relevant. Using a crossbridge model with the assumptions of fast powerstroke kinetics and equal load sharing between equivalent states, we derive a one-step master equation for the activity of a finite-sized ensemble of mechanically coupled myosin II motors. For constant external load, this approach yields analytical results for duty ratio and force-velocity relation as a function of ensemble size. We find that stochastic effects cannot be neglected for ensemble sizes below 15. The one-step master equation can be used also for efficient computer simulations with linear elastic external load and reveals the sequence of build-up of force and ensemble rupture that is characteristic for reconstituted actomyosin contractility.

Erdmann, Thorsten

2012-01-01

300

Stress generation by myosin minifilaments in actin bundles  

NASA Astrophysics Data System (ADS)

Forces and stresses generated by the action of myosin minifilaments are analyzed in idealized computer-generated actin bundles, and compared to results for isotropic actin networks. The bundles are generated as random collections of actin filaments in two dimensions with constrained orientations, crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending and stretching of actin filaments, which accomodate minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself.

Dasanayake, Nilushi L.; Carlsson, Anders E.

2013-06-01

301

Non-Muscle Myosin II Regulation of Lung Epithelial Morphology  

PubMed Central

Background The regulation of epithelial cell shape and orientation during lung branching morphogenesis is not clearly understood. Non-muscle myosins regulate cell size, morphology, and planar cell polarity. Here we test the hypothesis that non-muscle myosin II (NM II) regulates lung epithelial morphology in a spatially restricted manner. Results Epithelial cell orientation at airway tips in fetal mouse lungs underwent a significant transformation at E17. Treatment of E15 lung explants with the NM II inhibitor blebbistatin increased airway branching, epithelial cell size, and the degree of anisotropy in epithelial cells lining the airway stalks. In cultured MLE-12 lung epithelial cells, blebbistatin increased cell velocity, but left the migratory response to FGF-10 unchanged. Conclusions In the developing lung, NM II acts to constrain cell morphology and orientation, but may be suppressed at sites of branching and cell migration. The regulation of epithelial orientation may therefore undergo dynamic variations from E15 to E17. PMID:22972683

Plosa, Erin J.; Gooding, Kimberly A.; Zent, Roy; Prince, Lawrence S.

2012-01-01

302

New crystal structures of PII-type ATPases: excitement continues.  

PubMed

P-type ATPases are ATP-powered ion pumps, classified into five subfamilies (PI-PV). Of these, PII-type ATPases, including Ca2+-ATPase, Na+,K+-ATPase and gastric H+,K+-ATPase, among others, have been the most intensively studied. Best understood structurally and biochemically is Ca2+-ATPase from sarcoplasmic reticulum of fast twitch skeletal muscle (sarco(endo)plasmic reticulum Ca2+-ATPase 1a, SERCA1a). Since publication of the first crystal structure in 2000, it has continuously been a source of excitement, as crystal structures for new reaction intermediates always show large structural changes. Crystal structures now exist for most of the reaction intermediates, almost covering the entire reaction cycle. This year the crystal structure of a missing link, the E1·Mg2+ state, finally appeared, bringing another surprise: bound sarcolipin (SLN). The current status of two other important PII-type ATPases, Na+,K+-ATPase and H+,K+-ATPase, is also briefly described. PMID:23871101

Toyoshima, Chikashi; Cornelius, Flemming

2013-08-01

303

A novel biosensor for mercuric ions based on motor proteins  

NASA Astrophysics Data System (ADS)

We explored the potential for use of the contractile proteins, actin and myosin, as biosensors of solutions containing mercury ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 ?m long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.

Martinez, R.; Kekic, Murat; Buljan, Vlado; Nicolau, Dan V.; dos Remedios, Cristobal G.

2004-03-01

304

STUDIES OF MYOSIN AND ITS PROTEOLYTIC FRAGMENTS BY LASER RAMAN SPECTROSCOPY  

E-print Network

STUDIES OF MYOSIN AND ITS PROTEOLYTIC FRAGMENTS BY LASER RAMAN SPECTROSCOPY E. B. CAREW Department bands in the Raman spectrum of myosin, at 1,304 cm-' and 1,270 cm-', are attributable to a) but not in that of subfragment-l (S-1), is assigned to the coiled-coil tail region of myosin; the second, seen in spectra of S-1

Stanley, H. Eugene

305

Myosin Drives Retrograde F-Actin Flow in Neuronal Growth Cones  

Microsoft Academic Search

Actin filaments assembled at the leading edge of neuronal growth cones are centripetally transported via retrograde F-actin flow, a process fundamental to growth cone guidance and other forms of directed cell motility. Here we investigated the role of myosins in retrograde flow, using two distinct modes of myosin inhibition: microinjection of NEM inactivated myosin S1 fragments, or treatment with 2,3-butanedione-2-monoxime,

C. H. Lin; E. M. Espreafico; M. S. Mooseker

1996-01-01

306

Inhibitors of myosin, but not actin, alter transport through Tradescantia plasmodesmata  

Microsoft Academic Search

Actin and myosin are components of plasmodesmata, the cytoplasmic channels between plant cells, but their role in regulating\\u000a these channels is unclear. Here, we investigated the role of myosin in regulating plasmodesmata in a well-studied, simple\\u000a system comprising single filaments of cells which form stamen hairs in Tradescantia virginiana flowers. Effects of myosin inhibitors were assessed by analysing cell-to-cell movement

Janine E. Radford; Rosemary G. White

2011-01-01

307

Immunolocalisation of myosin-V in the enteric nervous system of the rat  

Microsoft Academic Search

We show here the localisation of myosin-V in whole mount preparations of the mucous–submucous and the muscular layers of rat small intestine by using an affinity purified antibody specific to the tail domain of myosin-V. Myosin-V immunostaining was intense in the submucous and myenteric nervous plexuses, allowing the visualisation of neuronal cell bodies and fibres. Western blots of total muscle

A. C Drengk; J. K Kajiwara; S. B Garcia; V. S Carmo; R. E Larson; S Zucoloto; E. M Espreafico

2000-01-01

308

Structural and Functional Responses of Mammalian Thick Filaments to Alterations in Myosin Regulatory Light Chains  

Microsoft Academic Search

The ordered array of myosin heads, characteristic of relaxed striated muscle thick filaments, is reversibly disordered by phosphorylating myosin regulatory light chains, decreasing temperature and\\/or ionic strength, increasing pH, and depleting nucleotide. In the case of light chain phosphorylation, disorder, most likely due to a change in charge affecting the light chain amino-terminus, reflects increased myosin head mobility, thus increased

Rhea J. C Levine; Zhaohui Yang; Neal D Epstein; Lameh Fananapazir; James T Stull; H. Lee Sweeney

1998-01-01

309

A myosin inhibitor impairs auxin-induced cell division  

Microsoft Academic Search

Summary. The role of myosins for auxin-induced cell division was probed using the inhibitor 2,3-butanedione monoxime in the tobacco cell line VBI-0, where cell elongation and division are axially aligned under the control of auxin. A morphometric analysis revealed that cell division is blocked in a dose-dependent manner, whereas cell expansion continued. In addition, the polarity of terminal cells was

Carola Holweg; Anne Honsel; Peter Nick

2003-01-01

310

Myosin light chain kinase in microvascular endothelial barrier function  

PubMed Central

Microvascular barrier dysfunction is implicated in the initiation and progression of inflammation, posttraumatic complications, sepsis, ischaemia–reperfusion injury, atherosclerosis, and diabetes. Under physiological conditions, a precise equilibrium between endothelial cell–cell adhesion and actin–myosin-based centripetal tension tightly controls the semi-permeability of microvascular barriers. Myosin light chain kinase (MLCK) plays an important role in maintaining the equilibrium by phosphorylating myosin light chain (MLC), thereby inducing actomyosin contractility and weakening endothelial cell–cell adhesion. MLCK is activated by numerous physiological factors and inflammatory or angiogenic mediators, causing vascular hyperpermeability. In this review, we discuss experimental evidence supporting the crucial role of MLCK in the hyperpermeability response to key cell signalling events during inflammation. At the cellular level, in vitro studies of cultured endothelial monolayers treated with MLCK inhibitors or transfected with specific inhibiting peptides have demonstrated that induction of endothelial MLCK activity is necessary for hyperpermeability. Ex vivo studies of live microvessels, enabled by development of the isolated, perfused venule method, support the importance of MLCK in endothelial permeability regulation in an environment that more closely resembles in vivo tissues. Finally, the role of MLCK in vascular hyperpermeability has been confirmed with in vivo studies of animal disease models and the use of transgenic MLCK210 knockout mice. These approaches provide a more complete view of the role of MLCK in vascular barrier dysfunction. PMID:20479130

Shen, Qiang; Rigor, Robert R.; Pivetti, Christopher D.; Wu, Mack H.; Yuan, Sarah Y.

2010-01-01

311

Myosin Vs organize actin cables in fission yeast  

PubMed Central

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

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

2012-01-01

312

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

SciTech Connect

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

Not Available

1994-06-01

313

Arabidopsis Myosin XI-K Localizes to the Motile Endomembrane Vesicles Associated with F-actin  

PubMed Central

Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi) as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile endoplasmic reticulum (ER) subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell. PMID:22969781

Peremyslov, Valera V.; Klocko, Amy L.; Fowler, John E.; Dolja, Valerian V.

2012-01-01

314

Amino group modification of (Na + +K + )ATPase  

Microsoft Academic Search

The effects of three amino group reagents on the activity of (Na++K+)-ATPase3 and its component K+-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In

J. J. H. H. M. De Pont; S. E. Emst-De Vries; S. L. Bonting

1984-01-01

315

Kinesin ATPase: Rate-Limiting ADP Release  

NASA Astrophysics Data System (ADS)

The ATPase rate of kinesin isolated from bovine brain by the method of S. A. Kuznetsov and V. I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from ? 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.

Hackney, David D.

1988-09-01

316

Human myocardial Na,K-ATPase concentration in heart failure  

Microsoft Academic Search

The Na,K-ATPase is of major importance for active ion transport across the sarcolemma and thus for electrical as well as contractile function of the myocardium. Furthermore, it is receptor for digitalis glycosides. In human studies of the regulatory aspects of myocardial Na,K-ATPase concentration a major problem has been to obtain tissue samples. Methodological accomplishments in quantification of myocardial Na,K-ATPase using

Henning Bundgaard; Keld Kjeldsen

1996-01-01

317

Evolution of proton pumping ATPases: Rooting the tree of life  

Microsoft Academic Search

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction\\/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases\\/ATP synthases was found to

Johann Peter Gogarten; Lincoln Taiz

1992-01-01

318

Use of fluorescent techniques to study the in vitro movement of myosins.  

PubMed

Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories. There are two basic versions of the assay which involve different geometries. In the sliding actin in vitro motility assay, myosin molecules are bound to a coverslip surface in a simply constructed microscopic flow chamber. Fluorescently labeled actin filaments are added to the flow chamber in the presence of ATP, and the movement of these actin filaments powered by the surface-bound myosins is observed. This assay has been used widely for a variety of myosins including both processive and non-processive ones. From this assay, one can easily measure the rate at which myosin is translocating actin. The single-molecule motility assay uses an inverted geometry compared to the sliding actin in vitro motility assay. It is most useful for processive myosins. Here, actin filaments are affixed to the coverslip surface. Fluorescently labeled single molecules of myosins (usually ones with processive kinetics) are introduced, and the movement of single molecules along the actin filaments is observed. This assay typically uses total internal reflection fluorescent (TIRF) microscopy to reduce the background signal arising from myosins in solution. From this assay, one can measure the velocity of movement, the frequency of movement, and the run length. If sufficient photons can be collected, one can use Gaussian fitting of the point spread function to determine the position of the labeled myosin to within a few nanometers which allows for measurement of the step size and the stepping kinetics. Together, these two assays are powerful tools to elucidate myosin function. PMID:25095996

Toepfer, Christopher; Sellers, James R

2014-01-01

319

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age  

PubMed Central

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence. PMID:23558932

Souza, C.C.R.; Dombroski, T.C.D.; Machado, H.R.; Oliveira, R.S.; Rocha, L.B.; Rodrigues, A.R.A.; Neder, L.; Chimelli, L.; Correa, V.M.A.; Larson, R.E.; Martins, A.R.

2013-01-01

320

Consequences of unlocking the cardiac myosin molecule in human myocarditis and cardiomyopathies  

PubMed Central

Myocarditis, often initiated by viral infection, may progress to autoimmune inflammatory heart disease, dilated cardiomyopathy and heart failure. Although cardiac myosin is a dominant autoantigen in animal models of myocarditis and is released from the heart during viral myocarditis, the characterization, role and significance of anti-cardiac myosin autoantibodies is poorly defined. In our study, we define the human cardiac myosin epitopes in human myocarditis and cardiomyopathies and establish a mechanism to explain how anti-cardiac myosin autoantibodies may contribute to heart disease. We show that autoantibodies to cardiac myosin in sera from myocarditis and dilated cardiomyopathies in humans targeted primarily epitopes in the S2 hinge region of cardiac myosin. In addition, anti-cardiac myosin antibodies in sera or purified IgG from myocarditis and cardiomyopathy targeted the beta-adrenergic receptor and induced antibody-mediated cAMP-dependent protein kinase A (PKA) cell signaling activity in heart cells. Antibody-mediated PKA activity in sera was abrogated by absorption with anti-human IgG. Antibody-mediated cell signaling of PKA was blocked by antigen-specific inhibition by human cardiac myosin or the beta-adrenergic receptor but not the alpha adrenergic receptor or bovine serum albumin. Propranolol, a beta blocker and inhibitor of the beta-adrenergic receptor pathway also blocked the antibody-mediated signaling of the beta-adrenergic receptor and PKA. The data suggest that IgG antibody against human cardiac myosin reacts with the beta-adrenergic receptor and triggers PKA signaling in heart cells. In-summary, we have identified a new class of crossreactive autoantibodies against human cardiac myosin and the beta-adrenergic receptor in the heart. In addition, we have defined disease specific peptide epitopes in the human cardiac myosin rod S2 region in human myocarditis and cardiomyopathy as well as a mechanistic role of autoantibody in the pathogenesis of disease. PMID:18781470

MASCARO-BLANCO, ADITA; ALVAREZ, KATHY; YU, XICHUN; LINDENFELD, JOANN; OLANSKY, LEANN; LYONS, TIMOTHY; DUVALL, DAVID; HEUSER, JANET S.; GOSMANOVA, ALBINA; RUBENSTEIN, CARL J.; COOPER, LESLIE T.; KEM, DAVID C.; CUNNINGHAM, MADELEINE W.

2011-01-01

321

Expression and localization of myosin-1d in the developing nervous system.  

PubMed

Myosin-1d is a monomeric actin-based motor found in a wide range of tissues, but highly expressed in the nervous system. Previous microarray studies suggest that myosin-1d is found in oligodendrocytes where transcripts are upregulated during the maturation of these cells. Myosin-1d was also identified as a component of myelin-containing subcellular fractions in proteomic studies and mutations in MYO1D have been linked to autism. Despite the potential implications of these previous studies, there is little information on the expression and localization of myosin-1d in the developing nervous system. Therefore, we analyzed myosin-1d expression patterns in the peripheral and central nervous systems during postnatal development. In mouse sciatic nerve, myosin-1d is expressed along the axon and in the ensheathing myelin compartment. Analysis of mouse cerebellum prior to myelination at day 3 reveals that myosin-1d is present in the Purkinje cell layer, granule cell layer, and region of the cerebellar nuclei. Upon the onset of myelination, myosin-1d enrichment expands along axonal tracts, while still present in the Purkinje and granule cell layers. However, myosin-1d was undetectable in oligodendrocyte progenitor cells at early and late time points. We also show that myosin-1d interacts and is co-expressed with aspartoacylase, an enzyme that plays a key role in fatty acid synthesis throughout the nervous system. Together, these studies provide a foundation for understanding the role of myosin-1d in neurodevelopment and neurological disorders. PMID:22284616

Benesh, Andrew E; Fleming, Jonathan T; Chiang, Chin; Carter, Bruce D; Tyska, Matthew J

2012-02-27

322

The Effect of Heavy Chain Phosphorylation and Solution Conditions on the Assembly ofAcanthamoeba Myosin-II  

E-print Network

). Under phys- iological conditions, myosin-II polymerizes into bipolar fila- ments, thereby allowing myosin synthetic fila- ments depend upon the solution conditions during the assem- bly process (Josephs

323

Morphometry, ultrastructure, myosin isoforms, and metabolic capacities of the "mini muscles" favoured by selection for high activity in house mice  

E-print Network

Morphometry, ultrastructure, myosin isoforms, and metabolic capacities of the "mini muscles in the gastrocnemius muscle. In the gastrocnemius, the % myosin heavy chain (MHC) IIb was reduced by 50% in L3

Garland Jr., Theodore

324

Drosophila non-muscle myosin II bipolar filament formation: Importance of charged residues and specific domains for self-assembly .  

E-print Network

??Non-muscle myosin II generates contractile forces for processes such as cytokinesis, motility, and polarity. Contractility requires assembly of myosin molecules into bipolar mini-filaments through electrostatic… (more)

Ricketson, Derek Lee, 1980-

2009-01-01

325

Conformational and rheological changes in catfish myosin as affected by different acids during acid-induced unfolding and refolding.  

PubMed

Changes in the conformation of catfish (Ictalurus punctatus) myosin due to (i) anions, (ii) acid pH, and (iii) salt addition were determined using tryptophan fluorescence, hydrophobicity measurements, differential scanning calorimetry, and circular dichroism. The relationship between conformation and storage modulus (G') of acid-treated myosin was studied. Three acids, HCl, H2SO4, and H3PO4, were used for unfolding myosin at three acidic pH conditions, 1.5, 2.0, and 2.5. Unfolded myosin was refolded to pH 7.3. Denaturation and unfolding of myosin was significantly (p < 0.05) lower when salt (0.6 M NaCl) was present during acid unfolding than in the absence of salt. When salt was added before unfolding, the alpha-helix content of myosin treated at pH 1.5 was significantly lower than that treated at pH 2.5. When salt was added after refolding, the alpha-helix content of myosin was unaffected by different pH treatments. The G' of myosin increased with an increase in myosin denaturation. The G' of myosin was significantly (p < 0.05) higher when salt was added to myosin after refolding than before acid unfolding. Among the different anion treatments, the G' of acid-treated myosin decreased in the order Cl- approximately SO42- > PO43-. Among the different pH treatments, the G' of myosin treated at pH 1.5 was significantly (p < 0.05) higher than myosin treated at pH 2.5. The conditions that would result in maximum myosin denaturation and maximum G' were unfolding of myosin at pH 1.5 using Cl- (from HCl) followed by refolding at pH 7.3 and subsequent addition of 0.6 M NaCl. PMID:17439141

Raghavan, Sivakumar; Kristinsson, Hordur G

2007-05-16

326

Ecto-ATPase activity of vertebrate blood cells.  

PubMed

Ecto-ATPase activity was measured for red blood cells, white blood cells, and whole blood from a variety of vertebrates. A large range of red blood cell ecto-ATPase activity was observed; for example, at 10 degrees C, red blood cells from a catastomid fish (Catostomus macrocheilus) and a newt (Taricha rivularis) had activities of 56 +/- 9 and 25,000,000 +/- 14,000,000 pmol ATP per 10(6) red blood cells per hour, respectively (mean +/- SD). Several control experiments verified that the measured ATPase activity was not the result of intracellular ATPases released due to cell damage or lysis nor due to the release of intracellular nucleoside triphosphate or uptake of extracellular ATP. Red blood cell ecto-ATPase activity was relatively low within the teleosts, was high within the reptiles, and had the greatest range and single highest value within the amphibians. Within the endotherms, avian red blood cell ecto-ATPase activities were greater than mammalian red blood cell ecto-ATPase activities, which were the lowest for all vertebrates examined. The lowest ecto-ATPase activities measured were for human and skunk red blood cells, which had activities of 13 +/- 1 and 11 +/- 2 pmol ATP per 10(6) red blood cells per hour, respectively, at 35 degrees C. Ecto-ATPase activity was measured in white blood cells of several vertebrate species and appeared generally high and less variable than red blood cell ecto-ATPase activity. Measured whole blood ecto-ATPase activity showed a range of three orders of magnitude and correlated positively with red blood cell ecto-ATPase activities. Ecto-ATPase activity was also determined for red blood cells from fetal, 1-3 d old neonatal, and pregnant garter snakes (Thamnophis elegans); these activities were not significantly different from the activity of red blood cells from nonpregnant adult females. Overall, the data from the present study demonstrate a wide range of red blood cell and whole blood ecto-ATPase activities among vertebrates and include some of the highest ecto-ATPase activities reported to date. PMID:9361136

Bencic, D C; Yates, T J; Ingermann, R L

1997-01-01

327

Structural and Functional Impact of Site-Directed Methionine Oxidation in Myosin  

E-print Network

Structural and Functional Impact of Site-Directed Methionine Oxidation in Myosin Jennifer C. Klein oxidation in Dictyostelium (Dicty) myosin II using mutagenesis, in vitro oxidation, and site- directed spin-labeling for electron paramagnetic resonance (EPR). Protein oxidation by reactive oxygen and nitrogen species

Thomas, David D.

328

A model for processive movement of single-headed myosin-IX.  

PubMed

It is puzzling that in spite of its single-headed structure, myosin-IX can move processively along actin. Here, based on the experimental evidence that the strong binding of myosin to actin in rigor state induces structural changes to several local actin monomers, a Brownian ratchet model is proposed to describe this processive movement. In the model, the actin plays an active role in the motility of single-headed myosin, in contrast to the common belief that the actin acts only as a passive track for the motility of the myosin. The unidirectional movement is due to both the asymmetric periodic potential of the myosin interacting with actin and the forward Stokes force induced by the relative rotation of the neck domain to the motor domain, while the processivity is determined by the binding affinity of the myosin for actin in ATP state. This gives a good explanation to the high processivity of myosin-IX, which results from its high binding affinity for actin in ATP state due to the presence of unique loop 2 insertion or N-terminal extension. The experimental results on the motility of myosin-IX such as the step size, large forward/backward stepping ratio, run length, stall force, etc, are explained well. PMID:20627400

Xie, Ping

2010-09-01

329

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

PubMed Central

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

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

2012-01-01

330

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin  

E-print Network

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin Yana K: Received 24 July 2012 Available online 4 August 2012 Keywords: Myosin Actin Binding kinetics Muscle (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results

Rhode Island, University of

331

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle  

Microsoft Academic Search

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI

A. Termin; R. S. Staron; D. Pette

1989-01-01

332

Structure of the Actin-Myosin Complex and Its Implications for Muscle Contraction  

Microsoft Academic Search

Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP). A model for the rigor complex of F actin and the myosin head was obtained by combining the molecular structures of the individual proteins with the low-resolution electron density maps of the complex derived by cryo-electron microscopy and image analysis.

Ivan Rayment; Hazel M. Holden; Michael Whittaker; Christopher B. Yohn; Michael Lorenz; Kenneth C. Holmes; Ronald A. Milligan

1993-01-01

333

The role of myosin in vesicle transport during bovine chromaffin cell secretion.  

PubMed Central

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity. PMID:12225290

Neco, Patricia; Gil, Anabel; Del Mar Frances, Maria; Viniegra, Salvador; Gutierrez, Luis M

2002-01-01

334

Three-Dimensional Structure of Myosin Subfragment1: A Molecular Motor  

Microsoft Academic Search

Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described.

Ivan Rayment; Wojciech R. Rypniewski; Karen Schmidt-Base; Robert Smith; Diana R. Tomchick; Matthew M. Benning; Donald A. Winkelmann; Gary Wesenberg; Hazel M. Holden

1993-01-01

335

Protein Phosphatase 1 ? Paralogs Encode the Zebrafish Myosin Phosphatase Catalytic Subunit  

PubMed Central

Background The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. Methodology/Principal Findings We observed that in zebrafish two paralogous genes encoding PP1?, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. Conclusions/Significance Our work demonstrates that zebrafish have two genes encoding PP1?, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required. PMID:24040418

Jayashankar, Vaishali; Nguyen, Michael J.; Carr, Brandon W.; Zheng, Dale C.; Rosales, Joseph B.; Rosales, Joshua B.; Weiser, Douglas C.

2013-01-01

336

Myosin light chain isoform expression among single mammalian skeletal muscle fibers: species variations  

Microsoft Academic Search

Extensive heterogeneity in myosin heavy chain and light chain (MLC) isoform expression in skeletal muscle has been well documented in several mammalian species. The initial objective of this study was to determine the extent of heterogeneity in myosin isoform expression among single fibers in limb muscles of dogs, a species for which relatively little has been reported. Fibers were isolated

Sabahattin Bicer; Peter J. Reiser

2004-01-01

337

Structures of Smooth Muscle Myosin and Heavy Meromyosin in the Folded, Shutdown State  

Microsoft Academic Search

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle,

Stan A. Burgess; Shuizi Yu; Matt L. Walker; Rhoda J. Hawkins; Joseph M. Chalovich; Peter J. Knight

2007-01-01

338

Myosin Autoimmunity Is Not Essential for Cardiac Inflammation in Acute Chagas' Disease1  

E-print Network

Myosin Autoimmunity Is Not Essential for Cardiac Inflammation in Acute Chagas' Disease1 Juan S is an autoimmune disease induced by T. cruzi (6). In other words, T. cruzi infection induces autoimmune responses to acute myocarditis that is accompanied by autoimmunity to cardiac myosin in susceptible strains of mice

Engman, David M.

339

Identification and analysis of the myosin superfamily in Drosophila: a database approach  

Microsoft Academic Search

The recent sequencing of the genome of Drosophila melanogaster has provided a valuable resource for mining the database for genes of interest. We took advantage of this opportunity in an attempt to identify novel myosins in Drosophila and confirm the presence of the previously identified myosins from classes I, II, III, V, VI, and VII. The Drosophila database annotators predicted

R. A. Yamashita; J. R. Sellers; J. B. Anderson

2000-01-01

340

Eukaryotic V-ATPase: novel structural findings and functional insights.  

PubMed

The eukaryotic V-type adenosine triphosphatase (V-ATPase) is a multi-subunit membrane protein complex that is evolutionarily related to F-type adenosine triphosphate (ATP) synthases and A-ATP synthases. These ATPases/ATP synthases are functionally conserved and operate as rotary proton-pumping nano-motors, invented by Nature billions of years ago. In the first part of this review we will focus on recent structural findings of eukaryotic V-ATPases and discuss the role of different subunits in the function of the V-ATPase holocomplex. Despite structural and functional similarities between rotary ATPases, the eukaryotic V-ATPases are the most complex enzymes that have acquired some unconventional cellular functions during evolution. In particular, the novel roles of V-ATPases in the regulation of cellular receptors and their trafficking via endocytotic and exocytotic pathways were recently uncovered. In the second part of this review we will discuss these unique roles of V-ATPases in modulation of function of cellular receptors, involved in the development and progression of diseases such as cancer and diabetes as well as neurodegenerative and kidney disorders. Moreover, it was recently revealed that the V-ATPase itself functions as an evolutionarily conserved pH sensor and receptor for cytohesin-2/Arf-family GTP-binding proteins. Thus, in the third part of the review we will evaluate the structural basis for and functional insights into this novel concept, followed by the analysis of the potentially essential role of V-ATPase in the regulation of this signaling pathway in health and disease. Finally, future prospects for structural and functional studies of the eukaryotic V-ATPase will be discussed. PMID:24508215

Marshansky, Vladimir; Rubinstein, John L; Grüber, Gerhard

2014-06-01

341

Cell, Vol. 78, 733-737, September9, 1994,Copyright 1994by Cell Press Getting a Grip on Myosin Review  

E-print Network

Cell, Vol. 78, 733-737, September9, 1994,Copyright© 1994by Cell Press Getting a Grip on Myosin, and hence many people believe that myosin force transduction is well understood. How- ever, although it is firmly established that actin and myosin filaments slide past one another in a muscle sarcomere

Vale, Ronald D.

342

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR  

E-print Network

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent- filled cavity that extends to the nucleotide

Rayment, Ivan

343

1642 Biophysical Journal Volume 84 March 2003 16421650 A Simple Kinetic Model Describes the Processivity of Myosin-V  

E-print Network

the Processivity of Myosin-V Anatoly B. Kolomeisky* and Michael E. Fishery *Department of Chemistry, Rice of Maryland, College Park, Maryland 20742 ABSTRACT Myosin-V is a motor protein responsible for organelle study of myosin-V motility is presented following an approach used successfully to analyze the dynamics

344

The motor protein Myosin-X transports VE-cadherin along filopodia to allow the formation of  

E-print Network

The motor protein Myosin-X transports VE- cadherin along filopodia to allow the formation of early intrafilopodial actin filaments. We found that the actin motor protein myosin-X is co-localized and moves synchronously with filopodial VE- cadherin. Immunoprecipitation and pull-down assays confirmed that myosin

Paris-Sud XI, Université de

345

X-ray Structures of the Apo and MgATP-bound States of Dictyostelium discoideum Myosin Motor Domain*  

E-print Network

X-ray Structures of the Apo and MgATP-bound States of Dictyostelium discoideum Myosin Motor Domain, Wisconsin 53706-1544 Myosin is the most comprehensively studied molecu- lar motor that converts energy from of interactions between my- osin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin

Rayment, Ivan

346

Cell, Vol. 116, 737749, March 5, 2004, Copyright 2004 by Cell Press The Mechanism of Myosin VI Translocation  

E-print Network

Cell, Vol. 116, 737­749, March 5, 2004, Copyright 2004 by Cell Press The Mechanism of Myosin VI of Biochemistry The stepping mechanism for processive myosins hasStanford University School of Medicine been most thoroughly studied with myosin V. A popularStanford, California 94305 scheme to explain the stepping

Spudich, James A.

347

The Unconventional Myosin, Myo2p, Is a Calmodulin Target at Sites of Cell Growth in Saccharomycescerevisiae  

E-print Network

The Unconventional Myosin, Myo2p, Is a Calmodulin Target at Sites of Cell Growth, University ofWashington, Seattle, Washington98195 Abstract. Myo2p is an unconventional myosin re- quired unconven- tional myosin, Myo2p, is a putative motor protein implicated in transporting vesicles to the bud

Davis, Trisha N.

348

Rho Kinase's Role in Myosin Recruitment to the Equatorial Cortex of Mitotic Drosophila S2 Cells Is for  

E-print Network

Rho Kinase's Role in Myosin Recruitment to the Equatorial Cortex of Mitotic Drosophila S2 Cells Is for Myosin Regulatory Light Chain Phosphorylation Sara O. Dean, James A. Spudich* Department of Biochemistry, Stanford University, Stanford, California, United States of America Background. Myosin II recruitment

Spudich, James A.

349

Myosin V Colocalizes with Melanosomes and Subcortical Actin Bundles Not Associated with Stress Fibers in Human Epidermal Melanocytes  

Microsoft Academic Search

Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in

Jo Lambert; Jos Onderwater; Yves Vander Haeghen; Garnet Vancoillie; Henk K. Koerten; A. Mieke Mommaas; Jean Marie Naeyaert

1998-01-01

350

Ca2+-ATPases (SERCA): Energy Transduction and Heat Production in Transport ATPases  

Microsoft Academic Search

The sarcoplasmic reticulum Ca2+-ATPase is able to cleave ATP through two different catalytic routes. In one of them, a part of the chemical energy derived from ATP hydrolysis is used to transport Ca2+ across the membrane and part is dissipated as heat. In the second route, the hydrolysis of ATP is completed before Ca2+ transport and all the energy derived

L. Meis

2002-01-01

351

Structure of the S100A4/myosin-IIA complex  

PubMed Central

Background S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood. Results We determined the X-ray crystal structure of the S100A4?8C/MIIA1908-1923 peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4?8C binds the MIIA1908-1923 peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site. Conclusion The structure of the S100A4/MIIA1908-1923 peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908–1923 of the myosin-IIA heavy chain represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain. PMID:24252706

2013-01-01

352

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

PubMed

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

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

2014-09-16

353

Design principles governing the motility of myosin V  

E-print Network

The molecular motor myosin V exhibits a wide repertoire of pathways during the stepping process, which is intimately connected to its biological function. The best understood of these is hand-over-hand stepping by a swinging lever arm movement toward the plus-end of actin filaments, essential to its role as a cellular transporter. However, single-molecule experiments have also shown that the motor "foot stomps", with one hand detaching and rebinding to the same site, and backsteps under sufficient load. Explaining the complete taxonomy of myosin V's load-dependent stepping pathways, and the extent to which these are constrained by motor structure and mechanochemistry, are still open questions. Starting from a polymer model, we develop an analytical theory to understand the minimal physical properties that govern motor dynamics. In particular, we solve the first-passage problem of the head reaching the target binding site, investigating the competing effects of load pulling back at the motor, strain in the leading head that biases the diffusion in the direction of the target, and the possibility of preferential binding to the forward site due to the recovery stroke. The theory reproduces a variety of experimental data, including the power stroke and slow diffusive search regimes in the mean trajectory of the detached head, and the force dependence of the forward-to-backward step ratio, run length, and velocity. The analytical approach yields a formula for the stall force, identifying the relative contributions of the chemical cycle rates and mechanical features like the bending rigidities of the lever arms. Most importantly, by fully exploring the design space of the motor, we predict that myosin V is a robust motor whose dynamical behavior is not compromised by reasonable perturbations to the reaction cycle, and changes in the architecture of the lever arm.

Michael Hinczewski; Riina Tehver; D. Thirumalai

2013-10-24

354

Flexibility within the heads of muscle myosin-2 molecules.  

PubMed

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction. PMID:24333017

Billington, Neil; Revill, Derek J; Burgess, Stan A; Chantler, Peter D; Knight, Peter J

2014-02-20

355

Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b  

NASA Astrophysics Data System (ADS)

Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

Yamada, Ayako; Mamane, Alexandre; Lee-Tin-Wah, Jonathan; di Cicco, Aurélie; Prévost, Coline; Lévy, Daniel; Joanny, Jean-François; Coudrier, Evelyne; Bassereau, Patricia

2014-04-01

356

Stiffness of ? subunit of F(1)-ATPase.  

PubMed

F(1)-ATPase is a molecular motor in which the ? subunit rotates inside the ?(3)?(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the ? subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F(1) and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the ? subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F(1) from thermophilic Bacillus PS3: the internal part of the ? subunit embedded in the ?(3)?(3) ring, and the complex of the external part of the ? subunit and the ?(3)?(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the ? subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F(o) and F(1)-ATPase. PMID:20549499

Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

2010-11-01

357

On the mechanism of energy transduction in myosin subfragment 1.  

PubMed Central

It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Förster equation and that the existence of an intersite communication system can be deduced from piece-wise detection of interacting pairs of points. PMID:6585786

Botts, J; Takashi, R; Torgerson, P; Hozumi, T; Muhlrad, A; Mornet, D; Morales, M F

1984-01-01

358

Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells.  

PubMed

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F-actin-associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F-actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F-actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F-actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F-actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI-associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization-dependent motility in the moss cytoplasm, where myosin XI-associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI-associated vesicular structures and F-actin polymerization-driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F-actin in tip growing cells. PMID:23020796

Furt, Fabienne; Liu, Yen-Chun; Bibeau, Jeffrey P; Tüzel, Erkan; Vidali, Luis

2013-02-01

359

Identification of signals that facilitate isoform specific nucleolar localization of myosin IC  

SciTech Connect

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

Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

2013-05-01

360

Regulation of Intracellular Cholesterol Distribution by Na/K-ATPase*  

E-print Network

Regulation of Intracellular Cholesterol Distribution by Na/K-ATPase* Received for publication/K-ATPase 1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells

Brand, Paul H.

361

Purification and Properties of an ATPase from Sulfolobus solfataricus  

NASA Technical Reports Server (NTRS)

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

Hochstein, Lawrence I.; Stan-Lotter, Helga

1992-01-01

362

P4 ATPases: Flippases in Health and Disease  

PubMed Central

P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role. PMID:23579954

van der Mark, Vincent A.; Oude Elferink, Ronald P.J.; Paulusma, Coen C.

2013-01-01

363

Purification and properties of an ATPase from Sulfolobus solfataricus  

NASA Technical Reports Server (NTRS)

The paper reports properties of a sulfite-activated ATPase from Sulfolobus solfataricus, purified using ammonium sulfate precipitation, column chromatography on UltraGel and Sepharose 6B, and SDS-PAGE. The 92-fold purified enzyme had a relative molecular mass of 370,000. It could be dissociated into three subunits with respective molecular masses of 63,000, 48,000, and 24,000. The ATPase activity was found to be inhibitable by nitrate, N-ethylmaleimide (which bound predominantly to the largest subunit), and 4-chloro 7-nitrobenzofurazan, but not by azide, quercetin, or vanadate. While the ATPase from S. solfataricus shared a number of properties with the S. acidocaldarius ATPase, there were also significant differences suggesting the existence of several types of archaeal ATPases.

Hochstein, Lawrence I.; Stan-Lotter, Helga

1992-01-01

364

Mouse Nuclear Myosin I Knock-Out Shows Interchangeability and Redundancy of Myosin Isoforms in the Cell Nucleus  

PubMed Central

Background Nuclear myosin I (NM1) is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes. PMID:23593477

Venit, Tomas; Dzijak, Rastislav; Kalendova, Alzbeta; Kahle, Michal; Rohozkova, Jana; Schmidt, Volker; Rulicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Onder; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabe; Hozak, Pavel

2013-01-01

365

Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length  

PubMed Central

Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin “hinge” region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial “melting” of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was ~5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed. PMID:17316684

Suggs, Jennifer A.; Cammarato, Anthony; Kronert, William A.; Nikkhoy, Massoud; Dambacher, Corey M.; Megighian, Aram; Bernstein, Sanford I.

2007-01-01

366

Movement of Scallop Myosin on Nitella Actin Filaments: Regulation by Calcium Ronald D. Vale, Andrew G. Szent-Gyorgyi, and Michael P. Sheetz  

E-print Network

Movement of Scallop Myosin on Nitella Actin Filaments: Regulation by Calcium Ronald D. Vale, Andrew Biology Movement of scallop myosin on Nitella actin filaments: Regulation by calcium (motility to determine if Ca2' regulates scal- lop myosin movement on actin, we have measured motility of scallop myosin

Vale, Ronald D.

367

X-ray Structures of the MgADP, MgATPS, and MgAMPPNP Complexes of the Dictyostelium discoideum Myosin Motor Domain,  

E-print Network

Myosin Motor Domain, Andrew M. Gulick, Cary B. Bauer, James B. Thoden, and Ivan Rayment* Institute of the truncated myosin head from Dictyostelium discoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPS, and Mg to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower

Rayment, Ivan

368

Structural similarities of Na,K-ATPase and SERCA, the Ca(2+)-ATPase of the sarcoplasmic reticulum.  

PubMed Central

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca(2+)-ATPase) has recently been determined at 2.6 A (note 1 A = 0.1 nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647-655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca(2+)-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca(2+)-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions. PMID:11389677

Sweadner, K J; Donnet, C

2001-01-01

369

Molecular dynamics simulations of F1-ATPase.  

PubMed

F1-ATPase is a rotary motor enzyme. Despite many theoretical and experimental studies, the molecular mechanism of the motor rotation is still not fully understood. However, plenty of available data provide a clue as to how this molecular motor rotates: with nucleotide perturbations, the catalytically active ? subunit propagates its structural changes to the entire ?3?3 complex via both sides of the subunits, resulting that asymmetry is created in the ?3?3 hexamer ring. In the sequential reaction step, the structure of the asymmetrical ?3?3 complex changes from one state to the other due to the nucleotide perturbations, and the ? subunit axis follows the sequentially changing ?3?3 structure. Therefore, there are mainly two essential elements for motor rotation: the conformational change of the ? subunit and the asymmetrical structure of the ?3?3 subunit complex. Therefore, this chapter reports a series of studies focused on these two elements via combinational approaches of molecular dynamics (MD) simulations and experimental or other theoretical studies. In addition to the motor rotation factors, the combined study also revealed other important elements of F1-ATPase, such as torque transmission and the chemical reaction pathway, which is described in the later part of this chapter. All of these results provide insight into the rotational mechanism and deepen the understanding of this molecular motor. PMID:24446371

Ito, Yuko; Ikeguchi, Mitsunori

2014-01-01

370

Myosin Light Chain Kinase Signaling in Endothelial Barrier Dysfunction  

PubMed Central

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia–reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell–cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca++, protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability. PMID:22886693

Rigor, Robert R.; Shen, Qiang; Pivetti, Christopher D.; Wu, Mack H.; Yuan, Sarah Y.

2013-01-01

371

Myosin-X functions in polarized epithelial cells  

PubMed Central

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis. PMID:22419816

Liu, Katy C.; Jacobs, Damon T.; Dunn, Brian D.; Fanning, Alan S.; Cheney, Richard E.

2012-01-01

372

Mutations in myosin light chain kinase cause familial aortic dissections.  

PubMed

Mutations in smooth muscle cell (SMC)-specific isoforms of ?-actin and ?-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the ?-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections. PMID:21055718

Wang, Li; Guo, Dong-chuan; Cao, Jiumei; Gong, Limin; Kamm, Kristine E; Regalado, Ellen; Li, Li; Shete, Sanjay; He, Wei-Qi; Zhu, Min-Sheng; Offermanns, Stephan; Gilchrist, Dawna; Elefteriades, John; Stull, James T; Milewicz, Dianna M

2010-11-12

373

Mutations in Myosin Light Chain Kinase Cause Familial Aortic Dissections  

PubMed Central

Mutations in smooth muscle cell (SMC)-specific isoforms of ?-actin and ?-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the ?-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections. PMID:21055718

Wang, Li; Guo, Dong-chuan; Cao, Jiumei; Gong, Limin; Kamm, Kristine E.; Regalado, Ellen; Li, Li; Shete, Sanjay; He, Wei-Qi; Zhu, Min-Sheng; Offermanns, Stephan; Gilchrist, Dawna; Elefteriades, John; Stull, James T.; Milewicz, Dianna M.

2010-01-01

374

Adaptations in myosin heavy chain profile in chronically unloaded muscles  

NASA Technical Reports Server (NTRS)

In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

Talmadge, R. J.; Roy, R. R.; Bodine-Fowler, S. C.; Pierotti, D. J.; Edgerton, V. R.

1995-01-01

375

Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the Ca2+-ATPase of sarcoplasmic reticulum.  

PubMed Central

We have determined the DNA sequences of the genes encoding the three structural proteins of the Kdp-ATPase, an ATP-driven potassium transport system of Escherichia coli. Regions of the predicted amino acid sequence of KdpB, the phosphorylated protein of the system, are homologous to regions of the Ca2+-ATPase of rabbit sarcoplasmic reticulum. The phosphorylated aspartate residue of the latter is within a region of homology. PMID:6146979

Hesse, J E; Wieczorek, L; Altendorf, K; Reicin, A S; Dorus, E; Epstein, W

1984-01-01

376

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.  

PubMed

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin. PMID:160910

Kimura, I; Arai, K; Watanabe, S

1979-12-01

377

Myosins Are Differentially Expressed under Oxidative Stress in Chronic Streptozotocin-Induced Diabetic Rat Brains  

PubMed Central

Diabetes mellitus is a disease characterized by persistent hyperglycemia, which may lead to brain tissue damage due to oxidative stress and also contributes to neuronal death and changes in synaptic transmission. This study evaluated the effect of oxidative stress and the use of antioxidants supplementation on myosins expression levels in the brains of chronic diabetic rats induced by streptozotocin. Lipid peroxidation, antioxidant enzymes activities, and myosins-IIB and -Va expressions at transcriptional and translational levels were examined after 90 days induction. The chronic effect of the diabetes led to the upregulation of superoxide dismutase (SOD) and catalase (CAT) activities, and the downregulation of glutathione peroxidase (GPx), but there was no statistically significant increase in the malondialdehyde (MDA) levels. These alterations were accompanied by high myosin-IIB and low myosin-Va expressions. Although the antioxidant supplementation did not interfere on MDA levels, the oxidative stress caused by chronic hyperglycemia was reduced by increasing SOD and restoring CAT and GPx activities. Interestingly, after supplementation, diabetic rats recovered only myosin-Va protein levels, without interfering on myosins mRNA levels expressed in diabetic rat brains. Our results suggest that antioxidant supplementation reduces oxidative stress and also regulates the myosins protein expression, which should be beneficial to individuals with diabetes/chronic hyperglycemia. PMID:24982856

Calabria, Luciana Karen; Vieira da Costa, Alice; da Silva Oliveira, Renato Jose; Ramos Deconte, Simone; de Carvalho, Washington Joao; de Oliveira, Vanessa Neves; Rezende Alves de Oliveira, Luciana; Goulart, Luiz Ricardo; Espindola, Foued Salmen

2013-01-01

378

Involvement of myosin light-chain kinase in endothelial cell retraction  

SciTech Connect

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.

Wysolmerski, R.B.; Lagunoff, D. (Saint Louis Univ. School of Medicine, MO (USA))

1990-01-01

379

Microtubule-dependent transport and organization of sarcomeric myosin during skeletal muscle differentiation  

PubMed Central

It has been proposed that microtubules (MTs) participate in skeletal muscle cell differentiation. However, it is still unclear how this happens. To examine whether MTs could participate directly in the organization of thick and thin filaments into sarcomeres, we observed the concomitant reorganization and dynamics of MTs with the behavior of sarcomeric actin and myosin by time-lapse confocal microscopy. Using green fluorescent protein (GFP)-EB1 protein to label MT plus ends, we determined that MTs become organized into antiparallel arrays along fusing myotubes. Their dynamics and orientation was found to be different across the thickness of the myotubes. We observed fast movements of Dsred-myosin along GFP-MTs. Comparison of GFP-EB1 and Dsred-myosin dynamics revealed that myosin moved toward MT plus ends. Immuno-electron microscopy experiments confirmed that myosin was actually associated with MTs in myotubes. Finally, we confirmed that MTs were required for the stabilization of myosin-containing elements prior to incorporation into mature sarcomeres. Collectively, our results strongly suggest that MTs become organized into a scaffold that provides directional cues for the positioning and organization of myosin filaments during sarcomere formation. PMID:16237460

Pizon, Veronique; Gerbal, Fabien; Diaz, Carmen Cifuentes; Karsenti, Eric

2005-01-01

380

A novel type of myosin implicated in signalling by rho family GTPases.  

PubMed Central

A novel widely expressed type of myosin (fifth unconventional myosin from rat: myr 5) from rat tissues, defining a ninth class of myosins, was identified. The predicted amino acid sequence of myr 5 exhibits several features not found previously in myosins. The myosin head domain contains a unique N-terminal extension and an insertion of 120 amino acids at a postulated myosin-actin contact site. Nevertheless, myr 5 is able to bind actin filaments in an ATP-regulated manner. The head domain is followed by four putative light chain binding sites. The tail domain of myr 5 contains a region which coordinates two atoms of zinc followed by a region that stimulates GTP hydrolysis of members of the ras-related rho subfamily of small G-proteins. Myr 5 therefore provides the first direct link between rho GTPases which have been implicated in the regulation of actin organization and the actin cytoskeleton. It is also the first unconventional myosin for which a tail binding partner(s), namely members of the rho family, has been identified. Images PMID:7882973

Reinhard, J; Scheel, A A; Diekmann, D; Hall, A; Ruppert, C; Bahler, M

1995-01-01

381

Identification and characterization of multiple novel Rab-myosin Va interactions  

PubMed Central

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

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

2013-01-01

382

Characterization and expression of a myosin heavy-chain isoform in juvenile walleye Sander vitreus.  

PubMed

In this study, myosin, the major component of myofibrillar protein in the skeletal muscle, was characterized and its expression was monitored during growth in juvenile walleye Sander vitreus. First, the coding region of myosin heavy chain (MyHC) from the fast skeletal muscle of walleye was amplified by long-distance PCR using a full-length cDNA. Phylogenetic analysis was used to determine the evolutionary relationship of this S. vitreus myosin sequence to other vertebrate myosin sequences. Next, it was established that the myosin isoform was most prevalent in the white muscle, compared with the red and cardiac muscle. Myosin expression was monitored over a series of experiments designed to influence growth. Specifically, change in MyHC mRNA was monitored after acute changes in feeding. Fish exposed to a one-week fasting period showed significant decreases in MyHC mRNA levels by the end of the fast. The effect of feeding was also examined more closely over a 24 h period after feeding, but results showed no significant change in myosin expression levels through this time period. Finally, fish with higher growth rates had higher MyHC mRNA and protein expression levels. This study indicates that MyHC mRNA expression is sensitive to the factors that may influence growth in juvenile S. vitreus. PMID:20738597

Dhillon, R S; Esbaugh, A J; Wang, Y S; Tufts, B L

2009-10-01

383

Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport  

PubMed Central

Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 ?m). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11]. PMID:25065759

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

2014-01-01

384

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

PubMed

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

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

2014-08-01

385

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

SciTech Connect

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

Schewkunow, Vitali [Friedrich-Alexander-University of Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Henkestrasse 91, Erlangen 91052 (Germany); Sharma, Karan P. [Boston BioProducts Inc., Worcester, MA 01604 (United States); Diez, Gerold; Klemm, Anna H. [Friedrich-Alexander-University of Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Henkestrasse 91, Erlangen 91052 (Germany); Sharma, Pal C. [Boston BioProducts Inc., Worcester, MA 01604 (United States); Goldmann, Wolfgang H. [Friedrich-Alexander-University of Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Henkestrasse 91, Erlangen 91052 (Germany)], E-mail: wgoldmann@biomed.uni-erlangen.de

2008-02-08

386

Three-dimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals  

PubMed Central

Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear- shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head. PMID:1869586

1991-01-01

387

Metal Fluoride Complexes of Na,K-ATPase  

PubMed Central

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. PMID:21708939

Cornelius, Flemming; Mahmmoud, Yasser A.; Toyoshima, Chikashi

2011-01-01

388

Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities  

NASA Technical Reports Server (NTRS)

The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

Caldwell, C.

1983-01-01

389

Knockout of the Na,K-ATPase ?2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction.  

PubMed

The ?2-isoform of the Na,K-ATPase (?2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing ?2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of ?2 to further define the tissue-specific role of ?2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of ?2 in the heart using ?-myosin heavy chain Cre. We have achieved a 90% knockout of ?2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of ?2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of ?2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of ?2 determines its unique roles in the regulation of cardiovascular function. PMID:23436327

Rindler, Tara N; Lasko, Valerie M; Nieman, Michelle L; Okada, Motoi; Lorenz, John N; Lingrel, Jerry B

2013-04-15

390

Characterization of native and reconstituted plasma membrane H + ATPase from the plasma membrane of Beta vulgaris  

Microsoft Academic Search

Summary Characteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase

Sharman D. O'Neill; Roger M. Spanswick

1984-01-01

391

Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy.  

PubMed

Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens. PMID:24478704

Hayek, Summer R; Lee, Samuel A; Parra, Karlett J

2014-01-01

392

On Myosin II dynamics in the presence of external loads  

E-print Network

We address the controversial hot question concerning the validity of the loose coupling versus the lever-arm theories in the actomyosin dynamics by re-interpreting and extending the phenomenological washboard potential model proposed by some of us in a previous paper. In this new model a Brownian motion harnessing thermal energy is assumed to co-exist with the deterministic swing of the lever-arm, to yield an excellent fit of the set of data obtained by some of us on the sliding of Myosin II heads on immobilized actin filaments under various load conditions. Our theoretical arguments are complemented by accurate numerical simulations, and the robustness of the model is tested via different choices of parameters and potential profiles.

A. Buonocore; L. Caputo; Y. Ishii; E. Pirozzi; T. Yanagida; L. M. Ricciardi

2005-04-18

393

Effect of oxygen free radicals on myosin in muscle fibres.  

PubMed

Experiments were performed on glycerol-extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin-labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.Vi-->AM.ADP in the ATP hydrolysis cycle. It was found that the essential -SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long-lived free radicals located partly on SH-1 sulphydryls. PMID:9637253

Könczöl, F; Lorinczy, D; Belagyi, J

1998-05-15

394

Myosin XIK is a major player in cytoplasm dynamics and is regulated by two amino acids in its tail.  

PubMed

It has recently been found that among the 17 Arabidopsis myosins, six (XIC, XIE, XIK, XI-I, MYA1, and MYA2) have a major role in the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum. Here, the same dominant negative tail fragments were also found to arrest the movement of Gogi bodies when transiently expressed in Arabidopsis plants. However, when a Golgi marker was transiently expressed in plants knocked out in these myosins, its movement was dramatically inhibited only in the xik mutant. In addition, a tail fragment of myosin XIK could inhibit the movement of several post-Golgi organelles, such as the trans-Golgi network, pre-vacuolar compartment, and endosomes, as well as total cytoplasmic streaming, suggesting that myosin XIK is a major player in cytoplasm kinetics. However, no co-localization of myosin tails with the arrested organelles was observed. Several deletion truncations of the myosin XIK tail were generated to corroborate function with localization. All deletion mutants possessing an inhibitory effect on organelle movement exhibited a diffuse cytoplasmic distribution. Point mutations in the tail of myosin XIK revealed that Arg1368 and Arg1443 are essential for its activity. These residues correspond to Lys1706 and Lys1779 from mouse myosin Va, which mediate the inhibitory head-tail interaction in this myosin. Therefore, such an interaction might underlie the dominant negative effect of truncated plant myosin tails and explain the mislocalization with target organelles. PMID:21914656

Avisar, Dror; Abu-Abied, Mohamad; Belausov, Eduard; Sadot, Einat

2012-01-01

395

Nonmuscle myosin II isoforms coassemble in living cells.  

PubMed

Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms. PMID:24814144

Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

2014-05-19

396

Sarcomere Lattice Geometry Influences Cooperative Myosin Binding in Muscle  

PubMed Central

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca2+ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca2+ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments. PMID:17630823

Tanner, Bertrand C. W; Daniel, Thomas L; Regnier, Michael

2007-01-01

397

Comparison of Rat Myosin from Fast and Slow Skeletal Muscle and the Effect of Disuse.  

National Technical Information Service (NTIS)

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 re...

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

1981-01-01

398

Insights into Human -Cardiac Myosin Function from Single Molecule and Single Cell Studies  

E-print Network

using single molecule and single cell approaches. Keywords Hypertrophic Cardiomyopathy. Single Molecule Analysis . Cardiac Myosin Introduction Hypertrophic cardiomyopathy (HCM) is the most common inherited contractile proteins [4]. Despite our understanding of the genetic underpinnings of cardiomyopathy

Spudich, James A.

399

Simulation model for combined motion of myosin cross-bridges agrees with experimental data.  

PubMed

The motivation for this work was to derive a theoretical model for the combined motion of a sample of muscle tissue with a small number (approximately 12) of myosin molecules. This was then compared to data collected at the University of North Texas Health Science center. A theoretical model of the motion of the myosin cross-bridges has been derived. The solution is a combination of solutions from the classical harmonic oscillator, Brownian motion, and Maxwell-Boltzmann statistics. The model illustrates the myosin behavior as a function of the number of myosin molecules, the temperature of the sample, and the spring constant. The results show that there is good agreement between the theoretical model and experimental data. PMID:24896359

Marandos, Peter; Midde, Krishna

2014-01-01

400

Regulation of Torsin ATPases by LAP1 and LULL1.  

PubMed

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder. PMID:23569223

Zhao, Chenguang; Brown, Rebecca S H; Chase, Anna R; Eisele, Markus R; Schlieker, Christian

2013-04-23

401

The Na-K-ATPase and Calcium-Signaling Microdomains  

NSDL National Science Digital Library

The Na-K-ATPase is an energy-transducing ion pump that converts the free energy of ATP into transmembrane ion gradients. It also serves as a functional receptor for cardiotonic steroids such as ouabain and digoxin. Binding of ouabain to the Na-K-ATPase can activate calcium signaling in a cell-specific manner. The exquisite calcium modulation via the Na-K-ATPase is achieved by the ability of the pump to integrate signals from numerous protein and non-protein molecules, including ion transporters, channels, protein kinases/phosphatases, as well as cellular Na+. This review focuses on the unique properties of the Na-K-ATPase and its role in the formation of different calcium-signaling microdomains.

Jiang Tian (University of Toledo Health Science Campus Physiology and Pharmacology); Zi-jian Xie (University of Toledo Health Science Campus)

2008-08-01

402

Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization  

Microsoft Academic Search

Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and ob- servation

Ahmet Yildiz; Joseph N. Forkey; Sean A. McKinney; Taekjip Ha; Yale E. Goldman; Paul R. Selvin

2003-01-01

403

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

PubMed Central

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

2013-01-01

404

Drosophila crinkled, Mutations of Which Disrupt Morphogenesis and Cause Lethality, Encodes Fly Myosin VIIA  

PubMed Central

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult “escapers” that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the ?100 and ?75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability. PMID:15579689

Kiehart, Daniel P.; Franke, Josef D.; Chee, Mark K.; Montague, R. A.; Chen, Tung-ling; Roote, John; Ashburner, Michael

2004-01-01

405

Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform  

PubMed Central

Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. USA. 86:6186-6190). The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC- cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC- cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin I- phenotype. PMID:2141028

1990-01-01

406

PAK1 regulates myosin II-B phosphorylation, filament assembly, localization and cell chemotaxis  

Microsoft Academic Search

Serine\\/threonine p21-activated kinase is an effector of Rac with a key role in the regulation of cytoskeletal organization. Non-muscle myosin II is a molecular motor, which is an important component of the cytoskeleton. Non-muscle myosin II-B plays a major role in cell motility and chemotaxis. We investigated the role of Rac and p21-activated kinase 1 (PAK1) in the regulation of

Liron Even-Faitelson; Michael Rosenberg; Shoshana Ravid

2005-01-01

407

Molecular and Phenotypic Effects of Heterozygous, Homozygous and Compound Heterozygote Myosin Heavy Chain Mutations  

Microsoft Academic Search

ABSTRACT Autosomal dominant familial hypertrophic cardiomyopathy,(FHC) has variable penetrance and phenotype. Heterozygous mutations in MYH7, encoding ?-myosin heavy chain, are the commonest causes of FHC, and we proposed that ‘enhanced’ mutant actin- myosin function is the causative molecular abnormality. We have studied individuals from families where members have two, one or no mutant MYH7alleles to examine for dose effects. In

Norman R. Alpert; Saidi A. Mohiddin; Dorothy Tripodi; Jacqueline Jacobson-Hatzell; Kelly Vaughn-Whitley; Christine Brosseau; David M. Warshaw; Lameh Fananapazir

2004-01-01

408

Function of FXYD Proteins, Regulators of Na, K-ATPase  

Microsoft Academic Search

   In this short review, we summarize our work on the role of members of the FXYD protein family as tissue-specific modulators\\u000a of Na, K-ATPase. FXYD1 or phospholemman, mainly expressed in heart and skeletal muscle increases the apparent affinity for\\u000a intracellular Na+ of Na, K-ATPase and may thus be important for appropriate muscle contractility. FXYD2 or ? subunit and FXYD4 or

Käthi Geering

2005-01-01

409

Measurement of Na +,K +ATPase Activity in Human Skeletal Muscle  

Microsoft Academic Search

There are few published measures of Na+,K+-ATPase activity in human skeletal muscle. This study investigated the suitability of the K+-stimulated 3-O-methylfluorescein phosphatase assay for measurement of Na+,K+-ATPase activity in human skeletal muscle. Factors investigated include enzyme kinetics, sample treatment, and ligand concentration. The addition of ouabain blocked maximal K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, confirming the specificity of the assay. Activity

Steve F. Fraser; Michael J. McKenna

1998-01-01

410

In vivo modification of Na +,K +ATPase activity in Drosophila  

Microsoft Academic Search

We have constructed and characterized transgenic Drosophila lines with modified Na+,K+-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type ? subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na+,K+-ATPase ? subunit hypomorphic mutant, 2206. In contrast, a mutant ? subunit (?D369N) leads to increased bang-sensitive paralysis and

Banghua Sun; Peizhang Xu; Weiya Wang; Paul M. Salvaterra

2001-01-01

411

Stabilization of Na,K–ATPase by ionic interactions  

Microsoft Academic Search

The effect of ions on the thermostability and unfolding of Na,K–ATPase from shark salt gland was studied and compared with that of Na,K–ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing

Elfrieda Fodor; Natalya U. Fedosova; Csilla Ferencz; Derek Marsh; Tibor Pali; Mikael Esmann

2008-01-01

412

Effect of Polygodial on the Mitochondrial ATPase of Saccharomyces cerevisiae  

Microsoft Academic Search

The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in Saccharomyces cerevisiae. In an acidification assay, polygodial completely suppressed the glucose-induced de- crease in external pH at 3.13 mg\\/ml, the same as the fungicidal concentration. Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p. Surprisingly, this ATPase was not directly inhibited by polygodial.

CHRISTOPHER S. LUNDE; ISAO KUBO

2000-01-01

413

Effect of Polygodial on the Mitochondrial ATPase of Saccharomyces cerevisiae  

PubMed Central

The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in Saccharomyces cerevisiae. In an acidification assay, polygodial completely suppressed the glucose-induced decrease in external pH at 3.13 ?g/ml, the same as the fungicidal concentration. Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p. Surprisingly, this ATPase was not directly inhibited by polygodial. In contrast, the two other membrane-bound ATPases in yeast were found to be susceptible to the compound. The mitochondrial ATPase was inhibited by polygodial in a dose-dependent manner at concentrations similar to the fungicidal concentration, whereas the vacuolar ATPase was only slightly inhibited. Cytoplasmic petite mutants, which lack mitochondrial DNA and are respiration deficient, were significantly less susceptible to polygodial than the wild type, as was shown in time-kill curves. A pet9 mutant which lacks a functional ADP-ATP translocator and is therefore respiration dependent was rapidly inhibited by polygodial. The results of these susceptibility assays link enzyme inhibition to physiological effect. Previous studies have reported that plasma membrane disruption is the mechanism of polygodial-induced cell death; however, these results support a more complex picture of its effect. A major target of polygodial in yeast is mitochondrial ATP synthase. Reduction of the ATP supply leads to a suppression of Pma1 ATPase activity and impairs adaptive responses to other facets of polygodial's cellular inhibition. PMID:10858359

Lunde, Christopher S.; Kubo, Isao

2000-01-01

414

Biochemical characterization of P-type copper ATPases.  

PubMed

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and inte