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Separation of large mammalian ventricular myosin differing in ATPase activity.  


To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity. PMID:17612641

Rupp, Heinz; Maisch, Bernhard



The Structural Basis of the Myosin ATPase Activity*  

E-print Network

. This protein comes in many shapes and sizes. Over 11 classes of myosin have been identified myosin II complexed with several nucleotides to- gether with the earlier studies on chicken skeletal myosin subfragment-1 (10­14). Structure of the Myosin Head The structure of chicken skeletal myosin

Rayment, Ivan


Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†  

PubMed Central

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo



Ca2+ activated myosin-ATPase in cardiac myofibrils of rainbow trout, freshwater turtle, and rat.  


The Ca(2+)-activated myosin-ATPase and its dependence on hypoxia were assessed in freshwater turtle, rainbow trout, and in some cases rat. At 20 degrees C and pH 7.3, the maximal ATPase activity was (mean +/- SEM): turtle 0.040 +/- 0.003, trout 0.090 +/- 0.005, and rat 0.12 +/- 0.004 mmol*min-1*g-1 myofibrillar dry weight. The turnover number was about three times lower for turtle than for trout. Trout is typically active at lower temperatures than turtle, and its myosin-ATPase activity was about three times lower at 10 degrees than at 20 degrees C. Addition of 12 mM phosphocreatine showed that the myosin-ATPase activity covered by myofibrillar creatine kinase was 22 +/- 2% for turtle, 14 +/- 2% for trout, and 69 +/- 5% for rat. At pH 6.8 relative to 7.3, the maximal M-ATPase activity was the same, whereas the Ca(2+)-sensitivity decreased, and more so for trout than for turtle. This difference disappeared, when trout myocardium was examined at 10 degrees C. P(i) (15 mM) affected neither maximal activity nor Ca(2+)-sensitivity. ADP, however, reduced maximal myosin-ATPase activity, and more so in trout than in turtle. In conclusion, the "slow"-type myosin, the low sensitivity of acidification and ADP, and the high creatine kinase/myosin-ATPase ratio in turtle relative to trout accord with the well-known ability of turtle myocardium to work during hypoxia. However, the difference in living temperature between turtle and trout obscures the situation (e.g. inclusion of rat data suggests that the creatine kinase/myosin-ATPase ratio is related to temperature. PMID:9262007

Degn, P; Gesser, H



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- ity, declines with age in rat muscle fibers. Am J Physiol Cell Physiol 283: C187­C192, 2002. First, and Biophysics, 2 Center on Aging, and 3 Department of Physical Medicine and Rehabilitation, University

Thomas, David D.


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.



Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle  

Microsoft Academic Search

The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with

L. Haagensen; D. H. Jensen; H. Gesser



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

PubMed Central

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

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



Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myofibrils from rainbow trout and freshwater turtle.  


The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase activity twice or more for both trout and turtle. As examined for trout myofibrils, the ATPase activity was reduced about four times by inhibiting the activity of myofibril-bound creatine kinase with iodoacetamide and this reduction was only partially counteracted, when the creatine kinase activity was restored by adding creatine kinase to the solution. Hence, the results suggest that myofibril-bound creatine kinase is needed to fully activate the myosin-ATPase activity in hearts from ectothermic vertebrates despite their low energy turn-over relative to endothermic species. PMID:18515165

Haagensen, L; Jensen, D H; Gesser, H



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



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


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

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



Characteristics of light chains of Chara myosin revealed by immunological investigation  

PubMed Central

Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca++-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively. PMID:22687741




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


Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca??-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively. PMID:22687741

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



Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.  

PubMed Central

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC. PMID:9062359

Fujita, H; Sugiura, S; Momomura, S; Omata, M; Sugi, H; Sutoh, K



The myosin ATPase mechanism does not require a conformationally sensitive aromatic residue  

Microsoft Academic Search

The effect of nucleotides or calcium ([Ca2+]free=1.0×10-4 m) or both on the near-u.v. absorption spectrum of myosins purified from two species of scallop,Aequipecten irradians andPlacopecten magellanicus, has been examined. No change in tyrosyl or tryptophanyl absorption was detected.

Peter D. Chantler; David J. Marsh; Stephen R. Martin



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

PubMed Central

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

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



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



Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.  


The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement. PMID:24465547

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



Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.  

PubMed Central

Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin. Images PMID:8090720

Jancso, A; Szent-Györgyi, A G



Myosins of secretory tissues  

PubMed Central

Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength. PMID:150427



Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on ?-myosin cross-bridge mechanics.  


Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of ?-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant ?-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM. PMID:25324513

Farman, Gerrie P; Muthu, Priya; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R



Drosophila melanogaster Myosin-18 Represents a Highly Divergent Motor with Actin Tethering Properties*  

PubMed Central

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 Kd 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.



Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis  

PubMed Central

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis. PMID:1386367



Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells.  

PubMed Central

Many neurohormones alter the force of cardiac contraction by variations in the intracellular Ca2+ concentration. alpha 1-Adrenergic and muscarinic stimulations, rather, modify the sensitivity of contractile proteins to Ca(2+)-calmodulin-myosin light-chain kinase (MLCK) complex induces a large increase in Ca2+ sensitivity (0.14 pCa unit) of these easily accessible myofilaments. This increase is further enhanced by up to 0.19 pCa unit when protein kinase C (PKC) is added together with MLCK. Similarly, the Ca2+ ATPase activity of skinned cells in suspension is increased in the presence of MLCK and further in the presence of both kinases. 32P-labelling and SDS/PAGE show that these changes are associated with light-chain 2 (LC2) phosphorylation together with phosphorylation of troponin I and troponin T when PKC is added. Although to a smaller extent than in smooth muscle, phosphorylation of cardiac myosin LC2 may be involved in the modulation of heart contractility. Images Fig. 4. Fig. 5. Fig. 6. PMID:1386218

Clement, O; Puceat, M; Walsh, M P; Vassort, G



The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.  

PubMed Central

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin. Images Fig. 1 Fig. 3 Fig. 4 PMID:8637864

Bobkov, A A; Bobkova, E A; Lin, S H; Reisler, E



Myosin Light Chain-activating Phosphorylation Sites Are Required for Oogenesis in Drosophila  

Microsoft Academic Search

The Drosophila spaghetti squash ( sqh ) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of verte- brate nonmuscle and smooth muscle myosin II has es- tablished that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in

Pascale Jordan; Roger Karess



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: [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)



Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions.  


Tropomyosin (Tm) bound to actin induces cooperative activation of actomyosin subfragment 1 (actin-S1) ATPase, observed as a sigmoid ATPase vs [S1] dependence. The activation is much steeper for gizzard muscle Tm (GTm) than for rabbit skeletal Tm (RSTm). To investigate if this greater cooperativity is due to increased communication between GTms along the thin filament, we studied effects of S1 binding on the state of actin-Tm using the fluorescence of pyrene-labeled Tm. Kinetic and equilibrium studies provided values for n, the apparent cooperative unit size [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273]. We report comparative studies of Tm-actin-S1 ATPase with values of n using GTm, RSTm, and 5aTm, a 1/7 shorter nonmuscle Tm from rat fibroblast cells [Pittenger, M. F., et al. (1994) Curr. Opin. Cell Biol., 6, 96]. 5aTm and GTm produce similar cooperative activation of actin-S1 ATPase and have similar n values that are 2-fold greater than RSTm, indicating a correlation between ATPase activation and n value. This appears to be due to the similarity of the C-terminal amino acid sequences of 5a and GTm which produce strong end-to-end interactions. The results are discussed in terms of a continuous flexible Tm strand on the actin filament. PMID:9354612

Lehrer, S S; Golitsina, N L; Geeves, M A



Thyroid hormone improves function and Ca2+ handling in pressure overload hypertrophy. Association with increased sarcoplasmic reticulum Ca2+-ATPase and alpha-myosin heavy chain in rat hearts.  

PubMed Central

We asked whether thyroid hormone (T4) would improve heart function in left ventricular hypertrophy (LVH) induced by pressure overload (aortic banding). After banding for 10-22 wk, rats were treated with T4 or saline for 10-14 d. Isovolumic LV pressure and cytosolic [Ca2+] (indo-1) were assessed in perfused hearts. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, and alpha- and beta-myosin heavy chain (MHC) proteins were assayed in homogenates of myocytes isolated from the same hearts. Of 14 banded hearts treated with saline, 8 had compensated LVH with normal function (LVHcomp), whereas 6 had abnormal contraction, relaxation, and calcium handling (LVHdecomp). In contrast, banded animals treated with T4 had no myocardial dysfunction; these hearts had increased contractility, and faster relaxation and cytosolic [Ca2+] decline compared with LVHcomp and LVHdecomp. Myocytes from banded hearts treated with T4 were hypertrophied but had increased concentrations of alpha-MHC and SERCA proteins, similar to physiological hypertrophy induced by exercise. Thus thyroid hormone improves LV function and calcium handling in pressure overload hypertrophy, and these beneficial effects are related to changes in myocyte gene expression. Induction of physiological hypertrophy by thyroid hormone-like signaling might be a therapeutic strategy for treating cardiac dysfunction in pathological hypertrophy and heart failure. PMID:9312172

Chang, K C; Figueredo, V M; Schreur, J H; Kariya, K; Weiner, M W; Simpson, P C; Camacho, S A



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



Characteristics of arterial myosin in experimental renal hypertension in the dog.  


We compared myosin samples isolated from iliac-femoral arteries of control and renal (stenosis) hypertensive dogs to determine the effects of increased blood pressure on the characteristics of the myosin. The ratio of 204-kd (SM-1) to 200-kd (SM-2) myosin heavy chains was approximately 1:0.75 for myosin from the iliac-femoral artery of normotensive dogs. This was not altered significantly in response to hypertension. Both SM-1 and SM-2 myosin heavy chains cross-reacted with antibody against smooth muscle myosin on Western blot analysis. In addition to these heavy chains, purified myosin from both groups showed a very faint protein band slightly below the 200-kd myosin heavy chain on electrophoresis on a highly porous sodium dodecyl sulfate-polyacrylamide gel. This protein band cross-reacted with antibody against nonmuscle myosin but not with smooth muscle myosin antibody. The 20- and 17-kd light chains of myosin isolated from normotensive and hypertensive dogs gave similar results on isoelectric focusing. Peptide maps of tryptic digests of heavy chains revealed both quantitative and qualitative differences. The Ca(2+)-activated myosin ATPase activity measured in high salt (0.5 mol/L KCl) was similar for myosin from both groups, whereas the potassium (ethylenedinitrilo)tetraacetic acid-stimulated ATPase of myosin from hypertensive animals was higher than that from normotensive animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8491497

Upadhya, A; Samuel, M; Cox, R H; Bagshaw, R J; Chacko, S



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.



Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed  

PubMed Central

SUMMARY The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds. PMID:22189767

Shaffer, Justin F.; Kier, William M.



Exercise training induces transitions of myosin isoform subunits within histochemically typed human muscle fibres  

Microsoft Academic Search

Fibre type composition based on histochemical myosin ATPase reaction was studied in cross section of biopsies from the vastus lateralis muscle of men. In addition, protein composition as well as peptide patterns of isolated myosin heavy chains were examined in batches of individually classified fibres from the same biopsies. High intensity endurance training during 8 weeks induces significant decreases by

Hugo Baumann; Monika Jäggi; Felix Soland; Hans Howald; Marcus C. Schaub



Enucleation of human erythroblasts involves non-muscle myosin IIB.  


Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts. PMID:22049517

Ubukawa, Kumi; Guo, Yong-Mei; Takahashi, Masayuki; Hirokawa, Makoto; Michishita, Yoshihiro; Nara, Miho; Tagawa, Hiroyuki; Takahashi, Naoto; Komatsuda, Atsushi; Nunomura, Wataru; Takakuwa, Yuichi; Sawada, Kenichi



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.



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.



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.



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


Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (? 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ? 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

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



Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.  


Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 ?m for mammalian class-1 myosins and greater than 90 ?m for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway. PMID:21680745

Chinthalapudi, Krishna; Taft, Manuel H; Martin, René; Heissler, Sarah M; Preller, Matthias; Hartmann, Falk K; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M; Manstein, Dietmar J



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



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; Hébert, Jean-Louis; Arsalane, Abdelilah; Moumen, Abdelouahab; Krokidis, Xénophon; Michel, Francine; Timbely, Oumar



Catalytic strategy used by the myosin motor to hydrolyze ATP  

PubMed Central

Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum–classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose ?-phosphate is not in the previously reported HP?O42? state, but in the H2P?O4? state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the ?-phosphate of ATP in a dissociated metaphosphate (P?O3?) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes. PMID:25006262

Kiani, Farooq Ahmad; Fischer, Stefan



Direct observation of the rotation of F1ATPase  

Microsoft Academic Search

Cells employ a variety of linear motors, such as myosin1-3, kinesin4 and RNA polymerase5, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum6 (a complex of about 100 protein molecules7). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest

Hiroyuki Noji; Ryohei Yasuda; Masasuke Yoshida; Kazuhiko Kinosita



Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.  


The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a highduty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.-Diensthuber, R. P., Tominaga, M., Preller, M., Hartmann, F. K., Orii, H., Chizhov, I., Oiwa, K., Tsiavaliaris, G. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor. PMID:25326536

Diensthuber, Ralph P; Tominaga, Motoki; Preller, Matthias; Hartmann, Falk K; Orii, Hidefumi; Chizhov, Igor; Oiwa, Kazuhiro; Tsiavaliaris, Georgios



Proteolysis of Myosin during Platelet Storage  

PubMed Central

Physical properties of actomyosin from either fresh or stored platelets have been compared. Actomyosin obtained from platelets after 3 days of storage contained myosin that was 60-80% degraded to myosin rod. No myosin rod was detected in fresh platelets. The platelet myosin rod is similar to the rod produced by limited proteolysis of skeletal muscle myosin. Images PMID:4825238

Abramowitz, Joel; Stracher, Alfred; Detwiler, Thomas C.



Direct observation of the mechanochemical coupling in myosin-V during processive movement  

PubMed Central

Myosin-Va transports intracellular cargos along actin filaments in cells1. This processive two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis2. The ability of myosin-Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded3-10. Mechanical studies at the multiple and single molecule level suggest that there is tight coupling (i.e. one ATP is hydrolyzed per power stroke), but this has not been directly demonstrated4,5,11. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin-Va and directly visualized the binding and dissociation of single fluorescently-labelled nucleotide molecules while simultaneously observing the stepping motion of the fluorescently labelled myosin-V as it moves along an actin filament. Here we show that preferential ADP dissociation from the trail head of myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin-V molecule retains at least one nucleotide (ADP in the lead head position) while moving. Thus we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with nanometer precision. PMID:18668042

Sakamoto, Takeshi; Webb, Martin R.; Forgacs, Eva; White, Howard D.; Sellers, James R.



Mammalian Myosin-18A, a Highly Divergent Myosin  

PubMed Central

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.



Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom  

PubMed Central

The control systems regulating muscle contraction in approximately 100 organisms have been categorized. Both myosin control and actin control operate simultaneously in the majority of invertebrates tested. These include insects, chelicerates, most crustaceans, annelids, priapulids, nematodes, and some sipunculids. Single myosin control is present in the muscles of molluscs, brachiopods, echinoderms, echiuroids, and nemertine worms. Single actin control was found in the fast muscles of decapods, in mysidacea, in a single sipunculid species, and in vertebrate striated muscles. Classification is based on functional tests that include measurements of the calcium dependence of the actomyosin ATPase activity in the presence and the absence of purified rabbit actin and myosin. In addition, isolated thin filaments and myosins were also analyzed. Molluscs lack actin control since troponin is not present in sufficient quantities. Even though the functional tests indicate the complete lack of myosin control in vertebrate striated muscle, it is difficult to exclude unambiguously the in vivo existence of this regulation. Both control systems have been found in animals from phyla which evolved early. We cannot ascribe any simple correlation between ATPase activity, muscle structure, and regulatory mechanisms. PMID:125778



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


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



UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast  

PubMed Central

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors. PMID:24244528

Stark, Benjamin C.; James, Michael L.; Pollard, Luther W.; Sirotkin, Vladimir; Lord, Matthew



Role of essential light chain EF hand domains in calcium binding and regulation of scallop myosin.  

PubMed Central

The specific Ca2+ binding site that triggers contraction of molluscan muscle requires the presence of an essential light chain (ELC) from a Ca2+ binding myosin. Of the four EF hand-like domains in molluscan ELCs, only domain III has an amino acid sequence predicted to be capable of binding Ca2+. In this report, we have used mutant ELCs to locate the Ca2+ binding site in scallop myosin and to probe the role of the ELC in regulation. Point mutations in domain III of scallop ELC have no effect on Ca2+ binding. Interestingly, scallop and rat cardiac ELC chimeras support Ca2+ binding only if domain I is scallop. These results are nevertheless in agreement with structural studies on a proteolytic fragment of scallop myosin, the regulatory domain. Furthermore, Ca2+ sensitivity of the scallop myosin ATPase requires scallop ELC domain I: ELCs containing cardiac domain I convert scallop myosin to an unregulated molecule whose activity is no longer repressed in the absence of Ca2+. Despite its unusual EF hand domain sequence, our data indicate that the unique and required contribution of molluscan ELCs to Ca2+ binding and regulation of molluscan myosins resides exclusively in domain I. Images Fig. 2 Fig. 3 PMID:7644472

Fromherz, S; Szent-Györgyi, A G



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.



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


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



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



Calcium-Pumping ATPases in Vesicles from Carrot Cells 1  

PubMed Central

Ca2+-ATPases keep cytoplasmic [Ca2+] low by pumping Ca2+ into intracellular compartments or out of the cell. The transport properties of Ca2+-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca2+ transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) partially inhibited oxalate-stimulated Ca2+ transport activity; however, it had no effect on calmodulin-stimulated Ca2+ uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca2+-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma32P]ATP resulted in the formation of a single acyl [32P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca2+, but independent of Mg2+. Its enhancement by La3+ is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca2+ transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca2+ transport, similar to their effect on the erythrocyte plasma membrane Ca2+-ATPase. These results would indicate that the calmodulin-stimulated Ca2+ transport originated in large part from a plasma membrane-type Ca2+ pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca2+-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested. ImagesFigure 5Figure 6 PMID:16668581

Hsieh, Wen-Ling; Pierce, Wayne S.; Sze, Heven



[Structural and functional bases of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of myometrium].  


Calix[4]arene C-97 (code is shown) is the macrocyclic compound which has lipophilic intramolecular higly-structured cavity formed by four aromatic cycles, one of which on the upper rim is modified by methylene bisphosphonic group. It was shown that calix[4]arene C-97 (100 microM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient I(0.5) being 83 +/- 7 microM. At the same time, this compound at 100 microM concentration significantly increases the effective hydrodynamic diameter of myosin subfragment-1, that may be indicative of intermolecular complexation between the calix[4]arene and myosin head. Computer simulation methods (docking, molecular dynamics, involving the Grid) have been used to clarify structural basis of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of the myometrium; participation of hydrophobic, electrostatic and pi-pi (stacking) interactions between calix[4]arene C-97 and amino acid residues of myosin subfragment-1, some of them being located near the active site of the ATPase has been found out. PMID:22679756

Labyntseva, R D; Bevza, A A; Bevza, O V; Cherenok, S O; Kal'chenko, V I; Kosterin, S O



Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity  

Microsoft Academic Search

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided

Lorenzo Alamo; Willy Wriggers; Antonio Pinto; Leiria Salazar; Fa-Qing Zhao; Roger Craig; Raúl Padrón



Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships  

PubMed Central

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms (Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from PMID:23189029

Syamaladevi, Divya P.; Sunitha, Margaret S; Kalaimathy, S.; Reddy, Chandrashekar C.; Iftekhar, Mohammed; Pasha, Shaik N.; Sowdhamini, R.



Phylogenetic analysis of new plant myosin sequences.  


We have sampled a large number of plant taxa, ranging from brown algae to angiosperms, for the presence of myosin sequences. Using phylogenetic analysis, we show that all but two of the new plant myosin sequences fall into two of three preexisting myosin classes. We identified two outlying sequences, which do not fall into any preexisting myosin class. Additionally, all genomic sequences encoding class XI myosins contain an intron in the region studied, suggesting that this genomic region has been conserved over at least 1 billion years of plant evolution. With these data, we can rapidly and consistently classify partial myosin sequences from plants. Our data show that plant myosins do not have clear orthologues in other kingdoms, providing interesting insights into the diversification of myosins. PMID:14562966

Bezanilla, Magdalena; Horton, Amy C; Sevener, Heather C; Quatrano, Ralph S



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

PubMed Central

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.



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.



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



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: PMID:24520162

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



Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast.  


Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity. PMID:16148042

Lord, Matthew; Laves, Ellen; Pollard, Thomas D



Structural kinetics of myosin by transient time-resolved FRET.  


For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. PMID:21245357

Nesmelov, Yuri E; Agafonov, Roman V; Negrashov, Igor V; Blakely, Sarah E; Titus, Margaret A; Thomas, David D



A small molecule species-specifically inhibits Fusarium myosin I.  


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

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



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)



Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila  

PubMed Central

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20. PMID:9412474

Jordan, Pascale; Karess, Roger



Filamin A-interacting protein (FILIP) is a region-specific modulator of myosin 2b and controls spine morphology and NMDA receptor accumulation  

PubMed Central

Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b. PMID:25220605

Yagi, Hideshi; Nagano, Takashi; Xie, Min-Jue; Ikeda, Hiroshi; Kuroda, Kazuki; Komada, Munekazu; Iguchi, Tokuichi; Tariqur, Rahman M.; Morikubo, Soichi; Noguchi, Koichi; Murase, Kazuyuki; Okabe, Masaru; Sato, Makoto



Myosin VI: cellular functions and motor properties.  

PubMed Central

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

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



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.



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



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



Oxidation-Initiated Myosin Subfragment Cross-Linking and Structural Instability Differences between White and Red Muscle Fiber Types.  


Both white and red muscles are commonly used in meat processing, and protein cross-linking, which may be affected by oxidants, is a key factor affecting the product quality. In this study, myofibrillar proteins (MPs) extracted from postrigor chicken Pectoralis major (PM, predominantly white) and Gastrocnemius (GN, predominantly red) muscles were subjected to a •OH-oxidizing system (10 ?M FeCl3 , 0.1 mM ascorbic acid, with 0, 5, 10, or 20 mM H2 O2 ) at pH 6.2, 4 °C for 18 h. The solubility of nonoxidized (control) PM MPs (63%) was higher than that of control GN MPs (41%). After oxidation with •OH generated at 5 mM H2 O2 , protein solubility decreased by 46% and 21% for PM and GN, respectively, due to aggregation. Chemical and electrophoretic analyses indicated H2 O2 -dose-dependent losses of sulfhydryls and the concomitant formation of disulfides which were more pronounced in PM protein samples. Oxidation favored cross-linking of myosin rod or tail in PM MPs compared to an equal susceptibility of myosin subfragment-1 (s-1) and rod to •OH in GN MPs. Both Ca- and K-ATPase activities in GN myosin were more sensitive to •OH than their PM counterparts, indicating a less stable s-1 region of GN myosin to oxidation. The uncoiling of rods from PM myosin was more rapid than that in GN myosin during heating. Oxidation induced cross-linking via disulfide bonds hindered the unfolding of rod, particularly in PM myosin. These data revealed the molecular events that underscore the necessity of meat processing and formulation control based on muscle fiber types. PMID:25604073

Liu, Changqi; Xiong, Youling L



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



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



Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts  

PubMed Central

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge. PMID:8449984



Cross-reactivity of termite myosin; a potential allergen  

Technology Transfer Automated Retrieval System (TEKTRAN)

Myosin and myosin isoforms are common food allergens in crustaceans; such as, shrimp, lobster, and crab. Allergy to Shellfish is a prevalent and potentially long lasting disorder that can severely affect health and quality of life. Myosin and myosin isoforms of dust mites and cockroaches are simil...


Human nonsyndromic hereditary deafness DFNA17 is due to a mutation in nonmuscle myosin MYH9.  


The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated. PMID:11023810

Lalwani, A K; Goldstein, J A; Kelley, M J; Luxford, W; Castelein, C M; Mhatre, A N



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)



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

SciTech Connect

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

Mykles, D.L.



Influence of myosin isoforms on tension cost and crossbridge kinetics in skinned rat cardiac muscle.  


1. In attempting to consolidate the role of ventricular isomyosins in regulating the contractility of the myocardium, actomyosin ATPase and crossbridge kinetics were obtained at 24 degrees C in chemically skinned isometrically contracting cardiac muscles containing V1 and V3 isomyosins. 2. The ATPase activity was measured at various levels of Ca2+ activation by the enzymatic coupling of ATP hydrolysis with the conversion of NADH to NAD+. The crossbridge kinetics were inferred from small-amplitude perturbations of muscle length and muscle tension, and characterized by the frequency-domain parameter fmin. 3. The ATPase rates of V1 and V3 muscles obtained at various levels of Ca2+ activation were plotted against the corresponding proportional tensions. The ATPase vs tension plots were linear with slopes of 4.92 nmol/min-1 per mm per mN and 1.98 nmol/min-1 per mm per mN, respectively for, V1 and V3 muscles. Individual calculations of ATPase-to-tension ratios (nmol/min-1 per mm per mN) gave corresponding averages of 4.98 +/- 0.12 (s.e.m., n = 12) and 2.16 +/- 0.12 (s.e.m., n = 10). The myosin isoform induced proportional change in tension cost was accompanied by a similar change in fmin (4.1 +/- 0.1 Hz and 1.95 +/- 0.03 Hz, means +/- s.e.m., for V1 and V3 muscles, respectively). 4. The observations and other published kinetic data are discussed in the context of models of crossbridge cycling. It is suggested that the tension economy of V3 muscle arises principally from an increase in the fraction of time, during the crossbridge cycle, when the crossbridge is exerting force. PMID:8582093

Rossmanith, G H; Hamilton, A M; Hoh, J F



Myosin tails and single ?-helical domains.  


The human genome contains 39 myosin genes, divided up into 12 different classes. The structure, cellular function and biochemical properties of many of these isoforms remain poorly characterized and there is still some controversy as to whether some myosin isoforms are monomers or dimers. Myosin isoforms 6 and 10 contain a stable single ?-helical (SAH) domain, situated just after the canonical lever. The SAH domain is stiff enough to be able to lengthen the lever allowing the myosin to take a larger step. In addition, atomic force microscopy and atomistic simulations show that SAH domains unfold at relatively low forces and have a high propensity to refold. These properties are likely to be important for protein function, enabling motors to carry cargo in dense actin networks, and other proteins to remain attached to binding partners in the crowded cell. PMID:25619246

Batchelor, Matthew; Wolny, Marcin; Dougan, Lorna; Paci, Emanuele; Knight, Peter J; Peckham, Michelle



Evidence for rotation of V1ATPase  

Microsoft Academic Search

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from

Hiromi Imamura; Masahiro Nakano; Hiroyuki Noji; Eiro Muneyuki; Shoji Ohkuma; Masasuke Yoshida; Ken Yokoyama



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

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



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.



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


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


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



Novel control of cardiac myofilament response to calcium by S-glutathionylation at specific sites of myosin binding protein C  

PubMed Central

Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C (cMyBP-C) and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cMyBP-C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol (DTT). Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to be functionally significant as controllers of myofilament Ca2+-responsiveness and dynamics. PMID:24312057

Patel, Bindiya G.; Wilder, Tanganyika; Solaro, R. John



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)



Purification of native myosin filaments from muscle.  

PubMed Central

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies. PMID:11606293

Hidalgo, C; Padrón, R; Horowitz, R; Zhao, F Q; Craig, R



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

SciTech Connect

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

Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)] [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States)] [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)] [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)



Structure and function of H + ATPase  

Microsoft Academic Search

(1) Extensive studies on proton-translocating ATPase (H+-ATPase) revealed that H+-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F1) of H+-ATPase showed that F1 is a hexagonal molecule with a central hole. The diameter of F1 is about 90 Å and its molecular weight is about 380,000.

Yasuo Kagawa; Nobuhito Sone; Hajime Hirata; Masasuke Yoshida



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.



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

NASA Technical Reports Server (NTRS)

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

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



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.



Myosin cleft closure determines the energetics of the actomyosin interaction  

PubMed Central

Formation of the strong binding interaction between actin and myosin is essential for force generation in muscle and in cytoskeletal motor systems. To clarify the role of the closure of myosin's actin-binding cleft in the actomyosin interaction, we performed rapid kinetic, spectroscopic, and calorimetric experiments and atomic-level energetic calculations on a variety of myosin isoforms for which atomic structures are available. Surprisingly, we found that the endothermic actin-binding profile of vertebrate skeletal muscle myosin subfragment-1 is unique among studied myosins. We show that the diverse propensity of myosins for cleft closure determines different energetic profiles as well as structural and kinetic pathways of actin binding. Depending on the type of myosin, strong actin binding may occur via induced-fit or conformational preselection mechanisms. However, cleft closure does not directly determine the kinetics and affinity of actin binding. We also show that cleft closure is enthalpically unfavorable, reflecting the development of an internal strain within myosin in order to adopt precise steric complementarity to the actin filament. We propose that cleft closure leads to an increase in the torsional strain of myosin's central ?-sheet that has been proposed to serve as an allosteric energy-transducing spring during force generation.—Takács, B., O'Neall-Hennessey, E., Hetényi, C., Kardos, J., Szent-Györgyi, A. G., Kovács, M. Myosin cleft closure determines the energetics of the actomyosin interaction. PMID:20837775

Takács, Balázs; O'Neall-Hennessey, Elizabeth; Hetényi, Csaba; Kardos, József; Szent-Györgyi, Andrew G.; Kovács, Mihály



Segregated assembly of muscle myosin expressed in nonmuscle cells.  

PubMed Central

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution. Images PMID:8298191

Moncman, C L; Rindt, H; Robbins, J; Winkelmann, D A



Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity  

PubMed Central

Summary Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free-head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens reveals that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal here has been to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction reveals intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC, and fitted to the reconstruction an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 crystal structure. The fitting suggests an intramolecular interaction between the cardiomyopathy loop of the free-head and its own S2 and two intermolecular interactions—between the cardio-loop of the free head and the ELC of the blocked head, and between the Leu-305 - Gln-327 “interaction loop” (loop I) of the free-head and the N-terminal fragment of the RLC of the blocked-head. These interactions, added to those previously described, would help to switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament. PMID:18951904

Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl



Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains.  


Serum levels of ventricular myosin heavy chains were quantitated in patients with acute myocardial infarction using a competitive radioimmunoassay involving monoclonal antibodies to the b-type myosin heavy chains of a human ventricle. Among the seven antibodies selected for their higher affinity for ventricular myosin heavy chains, only four antibodies detected significant and variable myosin amounts in the serum samples of nineteen patients with acute myocardial infarction; the same antibodies occasionally detected, if at all, low myosin amounts in the sera of patients with no clinical sign of myocardial infarction, and no myosin in the serum of the healthy control subjects. The peak levels of myosin release were observed 4.6 +/- 0.5 days (n = 13, P less than 0.01) after myocardial infarction and correlated rather well with the measured creatine kinase peak levels (the correlation coefficients were between 0.75 and 0.81, P less than 0.01, depending on the monoclonal antibody used for myosin determination). The time courses of myosin release varied according to the complexity of the heart attack observed. It was concluded that the titration of serum myosin was probably of little clinical value for therapeutic intervention during the acute phase of myocardial infarction; it could, however be an effective tool for retroactive detection of an infarct and for late estimation of infarct size. PMID:3938414

Leger, J O; Bouvagnet, P; Pau, B; Roncucci, R; Leger, J J



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



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



Genetic analysis of myosin assembly in Caenorhabditis elegans  

Microsoft Academic Search

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the\\u000a background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal\\u000a muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of\\u000a invertebrate muscles, and eukaryotic cells

Henry F. Epstein



Distribution of developmental myosin isoforms in isolated A-segments.  


Immunogold labelling was used to determine the distribution of myosin isoforms within the A-bands of developing chicken pectoralis muscles. Previous localization studies led to the suggestion that neonatal myosin is preferentially located in the centre of heterogeneous thick filaments that contain either embryonic or adult myosin in addition to neonatal myosin. To further explore the possibility that neonatal myosin may serve to nucleate thick filament assembly, a method was developed to isolate A-segments (arrays of myosin filaments) from myofibrils in the presence of MgATP. A-bands usually dissociate into thick and thin filaments in a relaxing buffer, but the inclusion of an antibody against M-line protein prevented separation of the thick filament array. Well-ordered A-segments, approximately 1.5 microns in length, were prepared from muscles 12, 29, 40 days, and approximately 1 year after hatching. After reaction with monoclonal antibodies specific for neonatal and adult myosins, the A-segments were labelled with gold-conjugated secondary antibodies prior to negative staining. An antibody which cross-reacts with embryonic myosin was used to localize that epitope in A-bands of myofibrils from day 1 and day 3 posthatch muscles. At ages where expression of neonatal myosin was high, extensive gold labelling of A-segments was observed in the electron microscope. However, no preferential distribution of antibodies was observed at any age, independent of whether embryonic or adult myosin was coexpressed with the neonatal myosin, suggesting that neonatal myosin is not segregated to any particular region in the A-bands of developing muscles. PMID:1491073

Gordon, D A; Lowey, S



Cargo recognition and cargo-mediated regulation of unconventional myosins.  


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



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.



Evolution of Plant P-Type ATPases  

PubMed Central

Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae) were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauri and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a non-vascular moss), Selaginella moellendorffii (a primitive vascular plant), and Arabidopsis thaliana (a model flowering plant). Each organism contained sequences for all five subfamilies of P-type ATPases. Whereas Na+ and H+ pumps seem to mutually exclude each other in flowering plants and animals, they co-exist in chlorophytes, which show representatives for two kinds of Na+ pumps (P2C and P2D ATPases) as well as a primitive H+-ATPase. Both Na+ and H+ pumps also co-exist in the moss P. patens, which has a P2D Na+-ATPase. In contrast to the primitive H+-ATPases in chlorophytes and P. patens, the H+-ATPases from vascular plants all have a large C-terminal regulatory domain as well as a conserved Arg in transmembrane segment 5 that is predicted to function as part of a backflow protection mechanism. Together these features are predicted to enable H+ pumps in vascular plants to create large electrochemical gradients that can be modulated in response to diverse physiological cues. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps. PMID:22629273

Pedersen, Christian N. S.; Axelsen, Kristian B.; Harper, Jeffrey F.; Palmgren, Michael G.



Myosin phosphatase target subunit: Many roles in cell function ?  

PubMed Central

Phosphorylation of myosin II is important in many aspects of cell function and involves a myosin kinase, e.g. myosin light chain kinase, and a myosin phosphatase (MP). MP is regulated by the myosin phosphatase target subunit (MYPT1). The domain structure, properties, and genetic analyses of MYPT1 and its isoforms are outlined. MYPT1 binds the catalytic subunit of type 1 phosphatase, ? isoform, and also acts as an interactive platform for many other proteins. A key reaction for MP is with phosphorylated myosin II and the first process shown to be regulated by MP was contractile activity of smooth muscle. In cell division and cell migration myosin II phosphorylation also plays a critical role and these are discussed. However, based on the wide range of partners for MYPT1 it is likely that MP is implicated with substrates other than myosin II. Open questions are whether the diverse functions of MP reflect different cellular locations and/or specific roles for the MYPT1 isoforms. PMID:18155661

Matsumura, Fumio; Hartshorne, David J.



Myosin XI is essential for tip growth in Physcomitrella patens.  


Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5' untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells. PMID:20525854

Vidali, Luis; Burkart, Graham M; Augustine, Robert C; Kerdavid, Erin; Tüzel, Erkan; Bezanilla, Magdalena



Role of Toxoplasma gondii Myosin A in Powering Parasite  

E-print Network

Role of Toxoplasma gondii Myosin A in Powering Parasite Gliding and Host Cell Invasion Markus Meissner,1 Dirk Schlu¨ter,2 Dominique Soldati1 * Obligate intracellular apicomplexan parasites rely of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells

Arnold, Jonathan


Molecular characterization of myosin phosphatase in endothelium.  


The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1?) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1? or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1? suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1?. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton. PMID:21678426

Kim, Kyung-Mi; Csortos, Csilla; Czikora, Istvan; Fulton, David; Umapathy, Nagavedi S; Olah, Gabor; Verin, Alexander D



On the kinetics that moves Myosin V  

E-print Network

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

Maes, Christian



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


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

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



Identification and Localization of Myosin Superfamily Members in Fish Retina and Retinal Pigmented Epithelium  

PubMed Central

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



Myosin isoform fiber type and fiber size in the tail of the Virginia opossum (Didelphis virginiana).  


Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. PMID:23152195

Hazimihalis, P J; Gorvet, M A; Butcher, M T



Signaling and Myosin-binding Protein C*  

PubMed Central

Myosin-binding protein C (MyBP-C) is a thick filament protein consisting of 1274 amino acid residues (149 kDa) that was identified by Starr and Offer over 30 years ago as a contaminant present in a preparation of purified myosin. Since then, numerous studies have defined the muscle-specific isoforms, the structure, and the importance of the proteins in normal striated muscle structure and function. Underlying the critical role the protein plays, it is now apparent that mutations in the cardiac isoform (cMyBP-C) are responsible for a substantial proportion (30–40%) of genotyped cases of familial hypertrophic cardiomyopathy. Although generally accepted that MyBP-C can interact with all three filament systems within the sarcomere (the thick, thin, and titin filaments), the exact nature of these interactions and the functional consequences of modified binding remain obscure. In addition to these structural considerations, cMyBP-C can serve as a point of convergence for signaling processes in the cardiomyocyte via post-translational modifications mediated by kinases that phosphorylate residues in the cardiac-specific isoform sequence. Thus, cMyBP-C is a critical nodal point that has both important structural and signaling roles and whose modifications are known to cause significant human cardiac disease. PMID:21257752

James, Jeanne; Robbins, Jeffrey



The emerging structure of vacuolar ATPases.  


Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research. PMID:16990452

Drory, Omri; Nelson, Nathan



Phenolic antioxidant scavenging of myosin radicals generated by hypervalent myoglobin.  


The scavenging activity of extracts of green tea (GTE), white grape (WGE), and rosemary (RE), all plant material with high phenolic content, and of the phenolic compounds 4-methylcatechol (4-MC), (+)-catechin, and carnosic acid toward long-lived myosin radicals generated by reaction with H2O2-activated myoglobin at room temperature (pH 7.5, I=1.0) was investigated by freeze-quench ESR spectroscopy. Myosin radicals were generated by incubating 16 ?M myosin, 800 ?M metmyoglobin, and 800 ?M H2O2 for 10 min, and the phenolic extracts were subsequently added (1% (w/w) phenolic compounds relative to myosin). GTE was able to scavenge myosin radicals and reduce the radical intensity by 65%. Furthermore, a low concentration of 4-MC (33 ?M) was found to increase the radical concentration when added to the myosin radicals, whereas a higher concentration of 4-MC and catechin (330 ?M) was found to scavenge myosin radicals and reduce the overall radical concentration by ?65%. PMID:23163579

Jongberg, Sisse; Lund, Marianne N; Østdal, Henrik; Skibsted, Leif H



Myosin I Overexpression Impairs Cell Migration  

PubMed Central

Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak, K.D., M.D. Peterson, M.C. Reedy, and M.A. Titus. 1995. J. Cell Biol. 131:1205–1221). Mutants that express a three- to sevenfold excess of myoB (myoB+ cells) were generated to further analyze the role of myosin I in these processes. The myoB+ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6–8-h delay in initiation of aggregation when placed under starvation conditions. The myoB+ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB+ cells are also deficient in their ability both to form distinct F-actin filled projections such as crowns and to become elongate and polarized. This defect can be attributed to the presence of at least threefold more myoB at the cortex of the myoB+ cells. In contrast, threefold overexpression of a truncated myoB that lacks the src homology 3 (SH3) domain (myoB/SH3? cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine (S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3? and S332A-myoB cells comparable to that found in the myoB+ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/ or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I. PMID:9024693

Novak, Kristine D.; Titus, Margaret A.



Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction  

PubMed Central

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca2+/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca2+/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle. PMID:16299103

Zhi, Gang; Ryder, Jeffrey W.; Huang, Jian; Ding, Peiguo; Chen, Yue; Zhao, Yingming; Kamm, Kristine E.; Stull, James T.



Accessibility of Myofilament Cysteines and Effects on ATPase Depend on the Activation State during Exposure to Oxidants  

PubMed Central

Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types. PMID:23894416

Gross, Sean M.; Lehman, Steven L.



Rotation of Escherichia coli F 1ATPase  

Microsoft Academic Search

By applying the same method used for F1-ATPase (TF1) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299–302), we observed ATP-driven rotation of a fluorescent actin filament attached to the ? subunit in Escherichia coli F1-ATPase. The torque value and the direction of the rotation were the same as those observed

Hiroyuki Noji; Katrin Häsler; Wolfgang Junge; Kazuhiko Kinosita; Masasuke Yoshida; Siegfried Engelbrecht



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.



Evidence for rotation of V1-ATPase  

PubMed Central

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft. PMID:12598655

Imamura, Hiromi; Nakano, Masahiro; Noji, Hiroyuki; Muneyuki, Eiro; Ohkuma, Shoji; Yoshida, Masasuke; Yokoyama, Ken



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-



Dual role of myosin II during Drosophila imaginal disc metamorphosis  

PubMed Central

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

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



Characterization of a myosin heavy chain gene from Brugia malayi.  


We have previously shown that an antigen recognized by antibodies in sera of several microfilaremic individuals from a Wuchereria bancrofti endemic area bears strong homology to an invertebrate muscle protein. We have cloned and sequenced the entire gene containing this antigen encoding fragment and present data that confirms that the antigen is myosin heavy chain (MHC). This gene, which we have named Bmmyo-1 extends over 11 kb and has the potential to encode a protein of 1957 amino acids. The coding sequence is interrupted by 14 introns, most of which are larger than those in the myosin gene of the free-living nematode, Caenorhabditis elegans. The protein encoded by this gene bears greatest homology (75.1% identity) to the C. elegans myosin isoform MHC-B, encoded by the unc-54 gene. MHC-B is the major body wall myosin in C. elegans. PMID:1741013

Werner, C; Rajan, T V



Myosin Heavy Chain Characteristics and Their Relationship to Exercise Performance  

E-print Network

There are a large variety of factors that affect performance in physical tasks. One factor is the muscle myosin heavy chain (MHC) composition of the muscle involved in the task. Differences in MHC can affect not just ...

Lane, Mike T.



Coordination of the two heads of myosin during muscle contraction  

Microsoft Academic Search

We have used luminescence resonance energy transfer between regulatory light chains (RLC) to detect structural changes within the dimeric myosin molecule in contracting muscle fibers. Fully functional scallop muscle fibers were prepared such that each myosin molecule contained a terbium-labeled (luminescent donor) RLC on one head and a rhodamine-labeled (acceptor) RLC on the other. Time-resolved luminescence energy transfer between the

Diane S. Lidke; David D. Thomas



Myosin thick filaments from adult rabbit skeletal muscles  

Microsoft Academic Search

Myosin subfragment 1 (S1) forms dimers in the presence of Mg2+ or MgADP or MgATP. The entire myosin molecule forms head–head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95°. Assuming that the length of the globular part of S1 is ?12 nm and that the S1\\/S2 joint (lever arm ?7

Jean-Emile Morel; Nathalie D’hahan; Patrick Bayol; Frédérique Cerqueira; Diana Rigault; Zalika Merah; Annette Gulik; Nathalie Guillo; Hanh Dam Hieu; Vincent Cabane; Matteo Ferrari; Gonzalo Figuera Picazo



Molecular engineering of a backwards-moving myosin motor  

Microsoft Academic Search

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized

Georgios Tsiavaliaris; Setsuko Fujita-Becker; Dietmar J. Manstein



Internal Motions in Myosin Head: Effect of ADP and ATP  

Microsoft Academic Search

Internal flexibility of myosin heads in glycerinated muscle fibres in the presence of MgADP plus orthovanadate and after addition of Ca-ATP was studied using an isothiocyanate-based spin label attached to the reactive sulfhydryl sites of myosin. The spin labels were immobilized on the microsecond time scale and exhibited significant orientational order in rigor. In AM+.ADP.Vistate a smaller fraction of ordered

Joseph Belagyi; Dénes Lo?rinczy



Structural changes accompanying phosphorylation of tarantula muscle myosin filaments  

Microsoft Academic Search

Electron microscopy has been used to study the structural changes that occur in the myosin illa- ments of tarantula striated muscle when they are phos- phorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATE EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea\\/glycerol gel electrophoresis and (32P)phosphate autoradiography. Negative staining reveals an ordered, helical

Roger Craig; Ratil Padr; John Kendrick-Jonesw



Fast and slow myosins as markers of muscle injury  

PubMed Central

Objective: The diagnosis of muscular lesions suffered by athletes is usually made by clinical criteria combined with imaging of the lesion (ultrasonography and/or magnetic resonance) and blood tests to detect the presence of non-specific muscle markers. This study was undertaken to evaluate injury to fast and slow-twitch fibres using specific muscle markers for these fibres. Methods: Blood samples were obtained from 51 non-sports people and 38 sportsmen with skeletal muscle injury. Western blood analysis was performed to determine fast and slow myosin and creatine kinase (CK) levels. Skeletal muscle damage was diagnosed by physical examination, ultrasonography and magnetic resonance and biochemical markers. Results: The imaging tests were found to be excellent for detecting and confirming grade II and III lesions. However, grade I lesions were often unconfirmed by these techniques. Grade I lesions have higher levels of fast myosin than slow myosin with a very small increase in CK levels. Grade II and III lesions have high values of both fast and slow myosin. Conclusions: The evaluation of fast and slow myosin in the blood 48 h after the lesion occurs is a useful aid for the detection of type I lesions in particular, since fast myosin is an exclusive skeletal muscle marker. The correct diagnosis of grade I lesions can prevent progression of the injury in athletes undergoing continual training sessions and competitions, thus aiding sports physicians in their decision making. PMID:18070807

Guerrero, M; Guiu-Comadevall, M; Cadefau, J A; Parra, J; Balius, R; Estruch, A; Rodas, G; Bedini, J L; Cussó, R



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



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


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



Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes.  


This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes. PMID:21371827

Francisco, C L; Jorge, A M; Dal-Pai-Silva, M; Carani, F R; Cabeço, L C; Silva, S R



Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes.  


In large mammals there is a correlation between microtubule network densification and contractile dysfunction in severe pressure-overload hypertrophy. In small mammals there is a similar correlation for the shift to beta-myosin heavy chain (MHC), a MHC isoform having a slower ATPase Vmax. In this study, murine left ventricular (LV) pressure overload invoked both mechanisms: microtubule network densification and beta-MHC expression. Cardiac beta-MHC was also augmented without altering tubulin levels by two load-independent means, chemical thyroidectomy and transgenesis. In hypertrophy, contractile function of the LV and its cardiocytes decreased proportionally; microtubule depolymerization restored normal cellular contraction. In hypothyroid mice having a complete shift from alpha-MHC to beta-MHC, contractile function of the LV and its cardiocytes also decreased, but microtubule depolymerization had no effect on cellular contraction. In transgenic mice having a cardiac beta-MHC increase similar to that in hypertrophy, contractile function of the LV and its cardiocytes was normal, and microtubule depolymerization had no effect. Thus, although both mechanisms may cause contractile dysfunction, for the extent of MHC isoform switching seen even in severe murine LV pressure-overload hypertrophy, microtubule network densification appears to have the more important role. PMID:12750067

Ishibashi, Yuji; Takahashi, Masaru; Isomatsu, Yukihisa; Qiao, Fei; Iijima, Yoshihiro; Shiraishi, Hirokazu; Simsic, Janet M; Baicu, Catalin F; Robbins, Jeffrey; Zile, Michael R; Cooper, George



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


To determine which myosin heavy chain (MHC) isoforms are expressed in canine skeletal muscles, different muscle samples of five mixed-breed dogs were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated MHC isoforms were identified by immunoblotting technique using a set of specific monoclonal antibodies. To compare the results of the electrophoretic and immunoblotting study, the pattern of MHC isoform expression and histochemical profiles of canine fibres were additionally demonstrated on serial muscle sections by immunohistochemistry and myofibrillar adenosine triphosphatase (mATPase) histochemistry. Not more than three MHC isoforms were demonstrated by SDS-PAGE in the analysed canine muscles. By the immunoblotting technique, the fastest migrating MHC band was identified as slow or MHC-I, the intermediate one as MHC-IIx and the slowest migrating band as MHC-IIa isoform. Since none of the three MHC bands and none of the analysed fibres were recognized by the antibody specific to MHC-IIb of rats, we concluded that MHC-IIb is not expressed in large skeletal muscles of dogs. Similarly, only three major fibre types, i.e. I, IIA and IIX, were revealed according to the pattern of MHC immunohistochemistry and mATPase reaction. Type IIA fibres were more alkali- and acid-stable than type IIX fibres after mATPase histochemistry; hence, the latter corresponded to type IIDog fibres. However, beside the three major fibre types, scarce hybrid fibres co-expressing two MHC isoforms (I/IIA and IIA/IIX) were demonstrated by immunohistochemistry. PMID:16113539

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



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.



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



The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans  

E-print Network

The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans Naimeh Taheri-Talesh1, Yi Xiong2¤, Berl R. Oakley1,2* 1Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States... of America, 2Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, United States of America Abstract Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest...

Taheri-Talesh, Naimeh; Xiong, Yi; Oakley, Berl R.



Rotational catalysis in proton pumping ATPases: from E. coli F-ATPase to mammalian V-ATPase.  


We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, F(O)F(1)). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among ? and ? subunits and the ? subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). PMID:22459334

Futai, Masamitsu; Nakanishi-Matsui, Mayumi; Okamoto, Haruko; Sekiya, Mizuki; Nakamoto, Robert K



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

PubMed Central

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. PMID:11516337

Reddy, Anireddy SN; Day, Irene S



The dynamic stator stalk of rotary ATPases  

PubMed Central

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases. PMID:22353718

Stewart, Alastair G.; Lee, Lawrence K.; Donohoe, Mhairi; Chaston, Jessica J.; Stock, Daniela



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



Arabidopsis myosin XI: a motor rules the tracks.  


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



Analysis of a cation-transporting ATPase of Plasmodium falciparum.  


We have cloned and characterised one gene, PfATPase4 which encodes a P-type ATPase containing all the primary sequence motifs characteristic of this class of transmembrane ion transporters, and also a fragment of a second P. falciparum P-type ATPase pseudogene (PfATPase5). Analysis of conserved domains and motifs of specific ATPases reveals that PfATPase4 is most analogous to Ca2+ ATPases of the endoplasmic reticulum. The PfATPase4 gene gives rise to a transcript of 8 kb shortly after erythrocyte invasion. Although this mRNA is not detected in later stages, the protein detected immunologically at 190 kDa persists throughout and is detected in free merozoites. Immunofluorescence microscopy reveals that the PfATPase4 protein is concentrated in discrete compartments at the periphery of the parasite. Detailed sequence and structural analyses of these and the other P-type ATPases of P. falciparum described previously, reveals that they comprise an unusual family in several respects. Firstly, the large number of non-homologous genes so far characterised reflects the complexities of ionic regulation in the diverse environments encountered by the parasite. Secondly, the plasmodial P-type ATPase family may be classified both at primary sequence and structural levels into two distinct groups-those typical of P-type ATPases (including PfATPase4) and those which are much more divergent. A third complexity is illustrated by the fact that one of the other members [1] here termed PfATPase6, has an even greater similarity to the sarcoplasmic reticulum Ca2+ ATPases than does PfATPase4, which raises questions about the possible functional relationship between these two members. PMID:8813672

Dyer, M; Jackson, M; McWhinney, C; Zhao, G; Mikkelsen, R



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))



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.



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



Domain Characterization of Rabbit Skeletal Muscle Myosin Light Chain Kinase  

PubMed Central

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal “tail,” of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235–319 and 165–173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core. PMID:1688558

Herring, B. Paul; Stull, James T.; Gallagher, Patricia J.



Chaperone-mediated reversible inhibition of the sarcomeric myosin power stroke.  


Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC-45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely. PMID:25240199

Nicholls, Paul; Bujalowski, Paul J; Epstein, Henry F; Boehning, Darren F; Barral, José M; Oberhauser, Andres F



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



Jaw-closing muscles of kangaroos express ?-cardiac myosin heavy chain  

Microsoft Academic Search

The masseter muscle of eutherian grazing mammals typically express ß or slow myosin heavy chain (MyHC). Myosins in the masseter of 4 species of kangaroos and a slow limb muscle of one of them were compared with their cardiac myosin by pyrophosphate and sodium dodecyl sulphate (SDS) gel electrophoresis, immunoblotting and immunohistochemistry. It was found that ventricular muscle contains three

Joseph F. Y. Hoh; Yoonah Kim; Louise G. Sieber; Wendy W. H. Zhong; Christine A. Lucas



Alternative Splicing in Class V Myosins Determines Association with Rab10*S?  

PubMed Central

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms. PMID:19008234

Roland, Joseph T.; Lapierre, Lynne A.; Goldenring, James R.



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

PubMed Central

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

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



An Internal Deletion Mutant of a Myosin Heavy Chain in Caenorhabditis elegans  

Microsoft Academic Search

Unc-54 I is the structural gene for a myosin heavy chain present in a major fraction of the total myosin of Caenorhabditis elegans. The allele e675, which possesses a normal amount of myosin but fails to assemble thick filaments, has been shown previously to contain a novel heavy chain of molecular weight 2 × 105, shorter by 104 than the

A. R. MacLeod; R. H. Waterston; S. Brenner



Sequence and phylogenetic analysis of squid myosin-V: A vesicle motor in nerve cells  

Microsoft Academic Search

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies (Tabb et al., 1998). In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V

Bradley J. Molyneaux; Mary K. Mulcahey; Phillip Stafford; George M. Langford



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

E-print Network

- generating element in the muscle is the motor protein myosin II (Huxley and Niedergerke, 1954; Huxley-dependent ADP release step can explain the dynamics of skeletal muscle contraction. Proteins of the myosinDynamics of Myosin-Driven Skeletal Muscle Contraction: I. Steady-State Force Generation Ganhui Lan

Sun, Sean


Myosin-II Tails Confer Unique Functions in Schizosaccharomyces pombe: Characterization of a Novel Myosin-II Tail  

PubMed Central

Schizosaccharomyces pombe has two myosin-IIs, Myo2p and Myp2p, which both concentrate in the cleavage furrow during cytokinesis. We studied the phenotype of mutant myosin-II strains to examine whether these myosins have overlapping functions in the cell. myo2+ is essential. myp2+ cannot rescue loss of myo2+ even at elevated levels of expression. myp2+ is required under specific nutritional conditions; thus myo2+ cannot rescue under these conditions. Studies with chimeras show that the tails rather than the structurally similar heads determine the gene-specific functions of myp2+ and myo2+. The Myo2p tail is a rod-shaped coiled-coil dimer that aggregates in low salt like other myosin-II tails. The Myp2p tail is monomeric in high salt and is insoluble in low salt. Biophysical properties of the full-length Myp2p tail and smaller subdomains indicate that two predicted coiled-coil regions fold back on themselves to form a rod-shaped antiparallel coiled coil. This suggests that Myp2p is the first type II myosin with only one head. The C-terminal two-thirds of Myp2p tail are essential for function in vivo and may interact with components of the salt response pathway. PMID:10637292

Bezanilla, Magdalena; Pollard, Thomas D.



Reversible association of myosin with the platelet cytoskeleton  

NASA Astrophysics Data System (ADS)

Platelets circulating in the human blood stream are smooth disk-shaped structures. The disks change within seconds of exposure to ADP or thrombin to irregular spheres bearing filopodia and pseudopodia. It is well-established that platelets also change shape (although more slowly) when chilled to 5°C1-5 and revert to disks on rewarming1,3. This cold-induced shape change may be due to the depolymerization of the submembranous microtubule ring. However, we found that chilling in the presence of Taxol, which stabilizes the microtubules, still results in shape change. Chilled platelets show an increase in the amount of myosin in the Triton-X insoluble residue or `cytoskeleton'6-9 which is correlated in time both with phosphorylation of the myosin regulatory light chain and with the induced shape change. We suggest here that the slow cold-induced change from disks to spheres is due primarily to a gradual activation of myosin.

Nachmias, Vivianne T.; Kavaler, Joshua; Jacubowitz, Sam



Actin cores of hair-cell stereocilia support myosin motility.  

PubMed Central

The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility. Images PMID:2236074

Shepherd, G M; Corey, D P; Block, S M



Mode of Cell Death Induction by Pharmacological Vacuolar H+-ATPase (V-ATPase) Inhibition*  

PubMed Central

The vacuolar H+-ATPase (V-ATPase), a multisubunit proton pump, has come into focus as an attractive target in cancer invasion. However, little is known about the role of V-ATPase in cell death, and especially the underlying mechanisms remain mostly unknown. We used the myxobacterial macrolide archazolid B, a potent inhibitor of the V-ATPase, as an experimental drug as well as a chemical tool to decipher V-ATPase-related cell death signaling. We found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations which was executed by the mitochondrial pathway. Prior to apoptosis induction archazolid led to the activation of a cellular stress response including activation of the hypoxia-inducible factor-1? (HIF1?) and autophagy. Autophagy, which was demonstrated by degradation of p62 or fusion of autophagosomes with lysosomes, was induced at low concentrations of archazolid that not yet increase pH in lysosomes. HIF1? was induced due to energy stress shown by a decline of the ATP level and followed by a shutdown of energy-consuming processes. As silencing HIF1? increases apoptosis, the cellular stress response was suggested to be a survival mechanism. We conclude that archazolid leads to energy stress which activates adaptive mechanisms like autophagy mediated by HIF1? and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in cancer therapy caught up at the interplay of apoptosis, autophagy, and cellular/metabolic stress. PMID:23168408

von Schwarzenberg, Karin; Wiedmann, Romina M.; Oak, Prajakta; Schulz, Sabine; Zischka, Hans; Wanner, Gerhard; Efferth, Thomas; Trauner, Dirk; Vollmar, Angelika M.



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

E-print Network

the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Crystal cysteines into the sequence of a "cysteine- light" myosin motor at position 499 on the lower cleft and the attached actin filament. Crystal structures of the motor domain of myosin show the lever arm domain in two

Spudich, James A.


Myosin Filament Assembly Requires a Cluster of Four Positive Residues Located in the Rod Domain  

PubMed Central

Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins. PMID:22728135

Thompson, Robert C.; Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie



Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens  

PubMed Central

Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation. PMID:9518724

Lutz, Gordon J; Cuizon, Denise B; Ryan, Allen F; Lieber, Richard L



The slow sarco\\/endoplasmic reticulum Ca2+ ATPase declines independently of slow myosin in soleus muscle of diabetic rats  

Microsoft Academic Search

- duced diabetes of rats (stz-rats), the soleus muscle showed peripheral neuropathy and the SER - CA2a level decreased in type I (slow-oxidative) fibers compared to the control muscles, whereas the expression of the corresponding slow MyHC1 did not change. No difference was found at the mRNA and protein levels of SERCA and MyHC isoforms in the whole soleus, except

Gábor Rácz; András Szabó; Ágota Vér



Myosin Binding Protein C, a Phosphorylation-Dependent Force Regulator in Muscle That Controls the Attachment of Myosin Heads by Its Interaction With Myosin S2  

Microsoft Academic Search

Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on b-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to

Gudrun Kunst; Kai R. Kress; Mathias Gruen; Dietmar Uttenweiler; Mathias Gautel; Rainer H. A. Fink


Engineering controllable bidirectional molecular motors based on myosin  

NASA Astrophysics Data System (ADS)

Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.

Chen, Lu; Nakamura, Muneaki; Schindler, Tony D.; Parker, David; Bryant, Zev



Engineering myosins for long-range transport on actin filaments.  


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

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



Myosin-V stepping kinetics: A molecular model for processivity  

E-print Network

-enhanced laser trap to measure the stepping rate of myosin-V molecules purified from brain. This allowed us the amplitude of the thermal diffusion. For some beads, it was also calibrated by measurement of the bead rise time in response to sudden trap displacement and by the 3-dB corner frequency in the diffusion power

Spudich, James A.


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 on the submillisecond timescale during the tran- sient phase of a biochemical reaction. ðTR�2FRET is accomplished

Thomas, David D.


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

PubMed Central

Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree. Conclusion Dictyostelium contains thirteen myosins together with 6–8 MLCs (myosin light chain) to assist in a variety of actin-based processes in the cell. Although they are homologous to myosins of higher eukaryotes, the myosins of Dictyostelium should be considered with care as models for specific functions of vertebrate myosins. PMID:16857047

Kollmar, Martin



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

PubMed Central

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

Štrbenc, Malan; Smerdu, Vika; Poga?nik, Azra; Fazarinc, Gregor



Stabilization of the ADP/Metaphosphate Intermediate during ATP Hydrolysis in Pre-power Stroke Myosin  

PubMed Central

It has been proposed recently that ATP hydrolysis in ATPase enzymes proceeds via an initial intermediate in which the dissociated ?-phosphate of ATP is bound in the protein as a metaphosphate (P?O3?). A combined quantum/classical analysis of this dissociated nucleotide state inside myosin provides a quantitative understanding of how the enzyme stabilizes this unusual metaphosphate. Indeed, in vacuum, the energy of the ADP3?·P?O3?·Mg2+ complex is much higher than that of the undissociated ATP4?. The protein brings it to a surprisingly low value. Energy decomposition reveals how much each interaction in the protein stabilizes the metaphosphate state; backbone peptides of the P-loop contribute 50% of the stabilization energy, and the side chain of Lys-185+ contributes 25%. This can be explained by the fact that these groups make strong favorable interactions with the ?- and ?-phosphates, thus favoring the charge distribution of the metaphosphate state over that of the ATP state. Further stabilization (16%) is achieved by a hydrogen bond between the backbone C=O of Ser-237 (on loop Switch-1) and a water molecule perfectly positioned to attack the P?O3? in the subsequent hydrolysis step. The planar and singly negative P?O3? is a much better target for the subsequent nucleophilic attack by a negatively charged OH? than the tetrahedral and doubly negative P?O42? group of ATP. Therefore, we argue that the present mechanism of metaphosphate stabilization is common to the large family of nucleotide-hydrolyzing enzymes. Methodologically, this work presents a computational approach that allows us to obtain a truly quantitative conception of enzymatic strategy. PMID:24165121

Kiani, Farooq Ahmad; Fischer, Stefan



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.



Regulation of Luminal Acidification by the V-ATPase  

PubMed Central

Specialized cells in the body express high levels of V-ATPase in their plasma membrane and respond to hormonal and nonhormonal cues to regulate extracellular acidification. Mutations in or loss of some V-ATPase subunits cause several disorders, including renal distal tubular acidosis and male infertility. This review focuses on the regulation of V-ATPase-dependent luminal acidification in renal intercalated cells and epididymal clear cells, which are key players in these physiological processes. PMID:23997191

Brown, Dennis



Origin of apparent negative cooperativity of F 1ATPase  

Microsoft Academic Search

In order to get insight into the origin of apparent negative cooperativity observed for F1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F1-ATPase, ?(W463F)3?(Y341W)3? and ?(K175A\\/T176A\\/W463F)3?(Y341W)3?. For ?(W463F)3?(Y341W)3?, apparent Km's of ATPase kinetics (4.0 and 233 ?M) did not agree with apparent Km's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM,

Sakurako Ono; Kiyotaka Y. Hara; Jun Hirao; Tadashi Matsui; Hiroyuki Noji; Masasuke Yoshida; Eiro Muneyuki



Cloning of a new cation ATPase from Plasmodium falciparum: conservation of critical amino acids involved in calcium binding in mammalian organellar Ca(2+)-ATPases.  


In order to study molecules that may be involved in pH gradient formation in Plasmodium, we have identified a novel cation-translocating ATPase (P-type ATPase) gene from P. falciparum (Pf). We report the full-length nucleotide and deduced amino acid (aa) sequences of this gene that we called PfATPase4. The PfATPase4 protein shares features with the different members of eukaryotic P-type ATPases, such as a similar transmembrane (TM) organization and aa identity in functionally important regions. Interestingly, the PfATPase4 protein possesses conserved aa involved in calcium binding in mammalian organellar Ca(2+)-ATPases. PMID:7789797

Trottein, F; Thompson, J; Cowman, A F



Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin  

PubMed Central

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane. PMID:23382463

Tomatis, Vanesa M.; Papadopulos, Andreas; Malintan, Nancy T.; Martin, Sally; Wallis, Tristan; Gormal, Rachel S.; Kendrick-Jones, John; Buss, Folma



Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.  


Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane. PMID:23382463

Tomatis, Vanesa M; Papadopulos, Andreas; Malintan, Nancy T; Martin, Sally; Wallis, Tristan; Gormal, Rachel S; Kendrick-Jones, John; Buss, Folma; Meunier, Frédéric A



Ecto-ATPase activity of vertebrate blood cells.  


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



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



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.



Biphasic myosin II light chain activation during clot retraction.  


Clot retraction is an essential step during primary haemostasis, thereby promoting thrombus stability and wound healing. Integrin ?IIb?3 plays a critical role in clot retraction, by inducing acto-myosin interactions that allow platelet cytoskeleton reorganisation. However, the signalling pathways that lead to clot retraction are still misunderstood. In this study, we report the first data on the kinetics of myosin II light chain (MLC) phosphorylation during clot retraction. We found an early phosphorylation peak followed by a second peak. By using specific inhibitors of kinases and small G proteins, we showed that MLC kinase (MLCK), RhoA/ROCK, and Rac-1 were involved in clot retraction and in the early MLC phosphorylation peak. Only Rac-1 and actin polymerisation, controlled by outside-in signalling, were crucial to the second MLC phosphorylation peak. PMID:23965920

Egot, Marion; Kauskot, Alexandre; Lasne, Dominique; Gaussem, Pascale; Bachelot-Loza, Christilla



Selective purification of the thiol peptides of myosin  

PubMed Central

1. A method for selective purification of thiol peptides is described. Thiol groups in a protein are treated with radioactive cystine by disulphide–thiol interchange. The labelled cystine peptides in a digest can then be fractionated for peptide `maps'. Performic acid oxidation of paper strips containing the radioactive peptides followed by further ionophoresis yields the purified cysteic acid peptides. 2. The thiol peptides in a peptic digest of cystine-exchanged myosin were purified in this way, and their amino acid sequences were determined. 3. The conclusion that myosin contains at least 16, and probably between 20 and 22, unique thiol sequences indicates that the molecule consists of two chemically equivalent components. PMID:5660634

Weeds, A. G.; Hartley, B. S.



Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis  

Microsoft Academic Search

Myosin-II is required for cytokinesis in Schizosaccharomyces pombe[1–3], but unlike other unicellular organisms, S. pombe has two structurally distinct myosin-IIs, Myo2p and Myp2p, which are required under different conditions [4]. Disruption of myo2+ is lethal, whereas disruption of myp2+ leads to defects in cytokinesis when nutrients are limiting and to cold-sensitivity in 1 M KCl. In dividing cells, both myosin-IIs

Magdalena Bezanilla; Jeanne M. Wilson; Thomas D. Pollard



Reverse actin sliding triggers strong myosin binding that moves tropomyosin  

SciTech Connect

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)



Chemomechanical Coupling and Motor Cycles of Myosin V  

PubMed Central

The molecular motor myosin V has been studied extensively both in bulk and single molecule experiments. Based on the chemical states of the motor, we construct a systematic network theory that includes experimental observations about the stepping behavior of myosin V. We utilize constraints arising from nonequilibrium thermodynamics to determine motor parameters and demonstrate that the motor behavior is governed by three chemomechanical motor cycles. The competition between these cycles can be understood via the influence of external load forces onto the chemical transition rates for the binding of adenosine triphosphate and adenosine diphosphate. In addition, we also investigate the functional dependence of the mechanical stepping rates on these forces. For substall forces, the dominant pathway of the motor network is profoundly different from the one for superstall forces, which leads to a stepping behavior that is in agreement with the experimental observations. Our theory provides a unified description of the experimental data as obtained for myosin V in single motor experiments. PMID:21463588

Bierbaum, Veronika; Lipowsky, Reinhard



Proper expression of myosin genes in transgenic nematodes.  


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

Fire, A; Waterston, R H



Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle.  

PubMed Central

Recombinant DNA clones containing sequences for two different types of myosin heavy chain (HC) genes from chicken embryonic skeletal muscle were constructed and analyzed. Specificity of the clones for myosin HC was demonstrated by hybrid-arrested translation, by hybridization to a 7.0-kb mRNA, and by comparison of DNA sequences with known amino acid sequences of rabbit skeletal muscle myosin HC. Restriction enzyme and electron-microscopic heteroduplex analysis showed the presence of two distinct but homologous cDNA sequences. Hybrid melting curves indicated that both types of sequences represent fast myosin HC sequences. Images PMID:6265916

Umeda, P K; Sinha, A M; Jakovcic, S; Merten, S; Hsu, H J; Subramanian, K N; Zak, R; Rabinowitz, M



Evolutionary traces decode molecular mechanism behind fast pace of myosin XI  

PubMed Central

Background Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in high-velocity cytoplasmic streaming in various plant cells from algae to angiosperms. The velocities at which they process are ten times faster than its closest class V homologues. Results To provide sequence determinants and structural rationale for the molecular mechanism of this fast pace myosin, we have compared the sequences from myosin class V and XI through Evolutionary Trace (ET) analysis. The current study identifies class-specific residues of myosin XI spread over the actin binding site, ATP binding site and light chain binding neck region. Sequences for ET analysis were accumulated from six plant genomes, using literature based text search and sequence searches, followed by triple validation viz. CDD search, string-based searches and phylogenetic clustering. We have identified nine myosin XI genes in sorghum and seven in grape by sequence searches. Both the plants possess one gene product each belonging to myosin type VIII as well. During this process, we have re-defined the gene boundaries for three sorghum myosin XI genes using fgenesh program. Conclusion Molecular modelling and subsequent analysis of putative interactions involving these class-specific residues suggest a structural basis for the molecular mechanism behind high velocity of plant myosin XI. We propose a model of a more flexible switch I region that contributes to faster ADP release leading to high velocity movement of the algal myosin XI. PMID:21942950



Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation  

NASA Astrophysics Data System (ADS)

Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 ?m and 4.0 ?m. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

Nucciotti, V.; Stringari, C.; Sacconi, L.; Vanzi, F.; Linari, M.; Piazzesi, G.; Lombardi, V.; Pavone, F. S.



Mutants altering coordinate synthesis of specific myosins during nematode muscle development.  


Mutations in the unc-52 gene on linkage group II retard the construction of body-wall muscle sarcomeres during larval development in the nematode Caenorhabditis elegans. Unc-52 mutants show decreased accumulation of myosin heavy chains relative to other polypeptides during larval development, correlating with the structural retardation. Pulse radiolabeling experiments show that decreased synthesis of specific body-wall myosin heavy chains that are encoded by the unc-54 gene on linkage group I is responsible for the defective myosin accumulation. In the wild type, a constant ratio of the synthesis of the unc-54-coded myosin B to myosin A, about 2:1, is maintained during the larval stages in which the synthesis of both myosins increases exponentially and rapid sarcomere growth and addition ensues. During the first 26 hr of larval development, before any structural or behavioral effects of unc-52 mutations are apparent, the synthesis of myosin heavy chains is also normal. By 38 hr, decreased synthesis of myosin B is detected in the unc-52 mutant SU200, when sarcomere growth slows considerably. The effects of mutation in the unc-52 locus are trans acting upon the synthesis of unc-54-coded myosin in a specific set of muscle cells during a defined period of larval development. PMID:6928685

Zengel, J M; Epstein, H F



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



The mechanism of rotating proton pumping ATPases.  


Two proton pumps, the F-ATPase (ATP synthase, FoF1) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F1 or V1) and a membrane intrinsic (Fo or Vo) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F1 sector was found to rotate at surprisingly high speeds (>400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the beta subunits, or between the beta and gamma subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F1-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation. PMID:20170625

Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Nakamoto, Robert K; Futai, Masamitsu



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



Evolution of Copper Transporting ATPases in Eukaryotic Organisms  

PubMed Central

Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution. PMID:23024604

Gupta, Arnab; Lutsenko, Svetlana



Evolution of copper transporting ATPases in eukaryotic organisms.  


Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution. PMID:23024604

Gupta, Arnab; Lutsenko, Svetlana



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.


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



The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans  

PubMed Central

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product?=?MyoB) and a myosin V homolog, myoE (product?=?MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape. PMID:22359575

Taheri-Talesh, Naimeh; Xiong, Yi; Oakley, Berl R.



Glycine 699 is pivotal for the motor activity of skeletal muscle myosin  

PubMed Central

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms. PMID:8769415



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

PubMed Central

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



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.; Corrêa, V.M.A.; Larson, R.E.; Martins, A.R.



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




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

E-print Network

-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis contraction by activating Rho kinase (Rok) and Citron kinase, which can phosphorylate the myosin II regulatory

Spudich, James A.


Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin  

Microsoft Academic Search

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light mero- myosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin- antibody complexes has localized the antibody binding site to LMM at




Effect of cholera toxin on ATPase activities in rabbit small intestinal mucosa.  


Cholera toxin induced the appearance of ATPase activity in rabbit small intestinal mucosa. This enzyme significantly differed from other ATPases, including Na(+),K(+)-ATPase and HCO3(-)-ATPase in the small intestinal epithelium of rabbits, by some properties, in particular, by relation to divalent and monovalent cations and anions, pH optimum, substrate specificity, and inhibitory analysis. PMID:23658913

Shubin, V S; Yurkiev, V A



Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction  

E-print Network

Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction, and isometric contraction. Experiments were per- formed on fiber bundles perfused con- tinuously with an ATP during isometric contraction, at least in the probed region of the myosin head. These motions could arise

Thomas, David D.



E-print Network

spectra were taken on a Spex Ramalog 4 system (Spex Industries, Inc., Metuchen, NJ) using an Ar+ laserSTUDIES OF MYOSIN AND ITS PROTEOLYTIC FRAGMENTS BY LASER RAMAN SPECTROSCOPY E. B. CAREW Department) 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


In vitro actin filament sliding velocities produced by mixtures of different types of myosin.  

PubMed Central

Using in vitro motility assays, we examined the sliding velocity of actin filaments generated by pairwise mixings of six different types of actively cycling myosins. In isolation, the six myosins translocated actin filaments at differing velocities. We found that only small proportions of a more slowly translating myosin type could significantly inhibit the sliding velocity generated by a myosin type that translocated filaments rapidly. In other experiments, the addition of noncycling, unphosphorylated smooth and nonmuscle myosin to actively translating myosin also inhibited the rapid sliding velocity, but to a significantly reduced extent. The data were analyzed in terms of a model derived from the original working cross-bridge model of A.F. Huxley. We found that the inhibition of rapidly translating myosins by slowly cycling was primarily dependent upon only a single parameter, the cross-bridge detachment rate at the end of the working powerstroke. In contrast, the inhibition induced by the presence of noncycling, unphosphorylated myosins required a change in another parameter, the transition rate from the weakly attached actomyosin state to the strongly attached state at the beginning of the cross-bridge power stroke. PMID:9083681

Cuda, G; Pate, E; Cooke, R; Sellers, J R



Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments*?  

PubMed Central

Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion. PMID:25429108

Desai, Rama; Geeves, Michael A.; Kad, Neil M.



The case for a common ancestor: kinesin and myosin motor proteins and G proteins  

E-print Network

The case for a common ancestor: kinesin and myosin motor proteins and G proteins F. JON KULL1 , 2 surprising structural and functional similarities between the motor domains of kinesin and myosin. Common, the evolutionary relationships between these proteins, even among the motor proteins, has not been obvious, since

Vale, Ronald D.


Myosin VIII regulates protonemal patterning and developmental timing in the moss Physcomitrella patens.  


Plants have two classes of myosins. While recent work has focused on class XI myosins showing that myosin XI is responsible for organelle motility and cytoplasmic streaming, much less is known about the role of myosin VIII in plant growth and development. We have used a combination of RNAi and insertional knockouts to probe myosin VIII function in the moss Physcomitrella patens. We isolated ?myo8ABCDE plants demonstrating that myosin VIII is not required for plant viability. However, myosin VIII mutants are smaller than wild-type plants in part due to a defect in cell size. Additionally, ?myo8ABCDE plants produce more side branches and form gametophores much earlier than wild-type plants. In the absence of nutrient media, ?myo8ABCDE plants exhibit significant protonemal patterning defects, including highly curved protonemal filaments, morphologically defective side branches, as well as an increase in the number of branches. Exogenous auxin partially rescues protonemal defects in ?myo8ABCDE plants grown in the absence of nutrients. This result, together with defects in protonemal branching, smaller caulonemal cells, and accelerated development in the ?myo8ABCDE plants, suggests that myosin VIII is involved in hormone homeostasis in P. patens. PMID:21873296

Wu, Shu-Zon; Ritchie, Julie A; Pan, Ai-Hong; Quatrano, Ralph S; Bezanilla, Magdalena



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.



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



Biosynthesis of the Tonoplast H+-ATPase from Oats 1  

PubMed Central

To determine whether the tonoplast-type H+-ATPase was differentially synthesized in various parts of the oat seedling, sections of 4-day-old oat (Avena sativa L. var Lang) seedlings were labeled in vivo with [35S]methionine and ATPase subunits were precipitated with polyclonal antisera. ATPase subunits were detected in all portions of the seedling with the exception of the seed. Lesser amounts of the 60 and 72 kilodalton polypeptides of the ATPase were found in apical regions (0-5 millimeter) than in maturing regions (10-15, or 20-25 millimeter from the tip) of the roots or shoots. To initiate a study of the biosynthesis of the ATPase, the intracellular site of synthesis for two peripheral ATPase subunits was investigated. Poly(A) RNA from either free or membrane-bound polysomes was isolated and translated in vitro. Message encoding the 72 kilodalton (catalytic) subunit was found predominantly in mRNA isolated from membrane-bound polysomes. In contrast, the message for the 60 kilodalton (putative regulatory) subunit was found predominantly on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences (limit of resolution, approximately 1 kilodalton), suggesting the absence of cleaved precursors for the 72 or 60 kilodalton subunits. These data suggest a complex mechanism for the synthesis and assembly of the tonoplast ATPase. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16666699

Randall, Stephen K.; Sze, Heven



Inhibition of plant vacuolar H(+)-ATPase by diethylpyrocarbonate.  


Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate. PMID:11418093

Chu, C L; Hsiao, Y Y; Chen, C H; Van, R C; Lin, W J; Pan, R L



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


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



Effects of adaptation to sea water, 170% sea water and to fresh water on activities and subcellular distribution of branchial Na + ?K + ATPase, low- and high affinity Ca ++ ATPase, and ouabain-insensitive ATPase in Gillichthys mirabilis  

Microsoft Academic Search

1.Branchial activities of Na+-K+-ATPase, ouabain-insensitive ATPase, (Mg++-ATPase) and Ca++-ATPase were measured inGillichthys mirabilis after adaptation to salinities ranging from 170% SW to FW. Stabilities of these activities against freezing and deoxycholate solubilization and the temperature-dependence of activity rates were also investigated. Subcellular distribution and some kinetic properties of these activities, and of SDH were compared in branchial tissues of fish

Byron A. Doneen



Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer.  


Mechanotransduction encompasses the role of mechanical forces in controlling cell behavior by activating signal transduction pathways. Most forces at a cellular level are caused by myosin II, which contracts and cross-links actin. Myosin II-dependent forces are transmitted through the actin cytoskeleton to molecular endpoints that promote specific cellular outcomes, e.g., cell proliferation, adhesion, or migration. For example, most adhesive and migratory phenomena are mechanically linked by a molecular clutch comprised of mechanosensitive scaffolds. Myosin II activation and mechanosensitive molecular mechanisms are finely tuned and spatiotemporally integrated to coordinate morphogenetic events during development. Mechanical events dependent on myosin II also participate in tumor cell proliferation, invasion, and metastatic dissemination. Specifically, tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for proliferation and/or dissemination. These observations position myosin II-dependent force generation and mechanotransduction at the crossroads between normal development and cancer. PMID:23934154

Aguilar-Cuenca, Rocío; Juanes-García, Alba; Vicente-Manzanares, Miguel



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


Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0M NaCl at 25°C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution. PMID:25445683

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



Flexibility within the heads of muscle myosin-2 molecules.  


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



Flexibility within the Heads of Muscle Myosin-2 Molecules?  

PubMed Central

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.



Proteasomal ATPase-Associated Factor 1 Negatively Regulates Proteasome Activity by Interacting with Proteasomal ATPases  

PubMed Central

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome. PMID:15831487

Park, Yoon; Hwang, Yong-Pil; Lee, Jong-Sik; Seo, Sang-Hyun; Yoon, Sungjoo Kim; Yoon, Jong-Bok



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.



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

PubMed Central

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

Betapudi, Venkaiah



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: [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)



Na,K-ATPase: radiation inactivation studies.  


Na,K-ATPase from duck salt gland and ox brain in the membrane-bound or solubilized form was studied by the radiation inactivation technique using ATP, CTP, GTP or p-NPP as substrates. The values of radiation inactivation size (RIS) were compared with the target size (TS) for the alpha-subunit of the enzyme obtained by an independent method as well as with analytical centrifugation data obtained for C12E8-solubilized enzyme. It was concluded that during ATP (CTP) hydrolysis the enzyme operates as an oligomeric structure; the complex formation requires the presence of K+ and adenosine triphosphate binding to the sites with a low affinity for the nucleotide. Specially designed experiments revealed that the degree of enzyme oligomerization increases with an increase in the microviscosity of the membrane lipid environment. PMID:2167088

Boldyrev, A A; Lopina, O D; Fedosova, N U



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

PubMed Central

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

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



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)



Pause and rotation of F1-ATPase during catalysis  

PubMed Central

F1-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central ? subunit relative to the surrounding ?3?3 ring. Here, we show that the rotation of F1-ATPase spontaneously lapses into long (?30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F1-ATPase previously known as the ADP-Mg inhibited form in which F1-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the ? subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position. PMID:11707579

Hirono-Hara, Yoko; Noji, Hiroyuki; Nishiura, Masaya; Muneyuki, Eiro; Hara, Kiyotaka Y.; Yasuda, Ryohei; Kinosita, Kazuhiko; Yoshida, Masasuke



Regulation of Torsin ATPases by LAP1 and LULL1  

PubMed Central

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



Construction, expression and characterization of a plasmid-encoded Na(+)-specific ATPase hybrid consisting of Propionigenium modestum F0-ATPase and Escherichia coli F1-ATPase.  


The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon subunits of the H(+)-dependent E. coli ATPase. The transformants showed Na(+)-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na+ and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed. PMID:8020498

Kaim, G; Dimroth, P



Myosin II is a negative regulator of oligodendrocyte morphological differentiation  

PubMed Central

During their development as myelinating cells oligodendrocyte progenitors (OPC) undergo dramatic changes in the organization of their cytoskeleton. These changes involve an increase in cell branching and lamella extension, which are important for the ability of oligodendrocytes to myelinate multiple axons in the CNS. We have previously shown that the levels of the actin-associated motor protein non-muscle myosin II (NMII) decrease as oligodendrocyte differentiate and that inhibition of NMII activity increases branching and myelination, suggesting that NMII is a negative regulator of oligodendrocyte differentiation. In agreement with this interpretation, we have found that overexpression of NMII prevents oligodendrocyte branching and differentiation, and that OPC maturation is accelerated in NMII knockout mice as shown by a significant increase in the percentage of mature MBP+ cells. Although several pathways have been implicated in oligodendrocyte morphogenesis, their specific contribution to the regulation of NMII activity has not been directly examined. We tested the hypothesis that the activity of NMII in OPC is controlled by Fyn kinase via downregulation of RhoA-ROCK-NMII phosphorylation. We found that treatment with PP2 or knockdown of Fyn using siRNA, prevents the decrease in myosin phosphorylation normally observed during OPC differentiation, and that the inhibition of branching induced by overexpression of constitutively active RhoA can be reversed by treatment with Y27632 or blebbistatin. Taken together our results demonstrate that Fyn kinase downregulates NMII activity thus promoting oligodendrocyte morphological differentiation. PMID:22437915

Wang, Haibo; Rusielewicz, Tomasz; Tewari, Ambika; Leitman, Ellen M.; Einhenber, Steven; Melendez-Vasquez, Carmen V.



Anillin promotes astral microtubule-directed cortical myosin polarization  

PubMed Central

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex. PMID:21737681

Tse, Yu Chung; Piekny, Alisa; Glotzer, Michael



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.



Characterization of an ecto-ATPase of Tritrichomonas foetus.  


In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity. PMID:11750998

Jesus, José B; Lopes, Angela H C S; Meyer-Fernandes, José R



Giardia lamblia: biochemical characterization of an ecto-ATPase activity.  


In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78+/-0.08 nmol Pi x h(-1)x10(-6) cells). The ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53+/-0.07 mM. ATP was the best substrate for this enzyme. The apparent K(m) for ATP was 0.21+/-0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg(2+)-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A(1) (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5 AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl(2). Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology. PMID:18413274

de Sá Pinheiro, Ana Acacia; Cosentino-Gomes, Daniela; Lanfredi-Rangel, Adriana; Ferraro, Rodrigo Barbosa; De Souza, Wanderley; Meyer-Fernandes, José Roberto



Stimulation of F 1ATPase activity by sodium dodecyl sulfate  

Microsoft Academic Search

F1-ATPase is a rotary molecular motor in which the ? subunit rotates inside the cylinder made of ?3?3 subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F1-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP. Maximal stimulation was obtained at 0.003% (w\\/v) SDS, the initial

Mohammad Delawar Hossain; Shou Furuike; Yasuhiro Onoue; Kengo Adachi; Masasuke Yoshida; Kazuhiko Kinosita Jr.



Temperature-sensitive reaction intermediate of F1ATPase  

Microsoft Academic Search

F1-ATPase is a rotary molecular motor that makes 120° stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F1-ATPase was discovered at a temperature below 4°C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly

Rikiya Watanabe; Ryota Iino; Katsuya Shimabukuro; Masasuke Yoshida; Hiroyuki Noji



Neutral Phospholipids Stimulate Na,K-ATPase Activity  

PubMed Central

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of ?TM8–10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human ?1?1 or ?1?1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of ?1?1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments ?TM8–10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid. PMID:23430748

Haviv, Haim; Habeck, Michael; Kanai, Ryuta; Toyoshima, Chikashi; Karlish, Steven J. D.



Copper Transporting P-Type ATPases and Human Disease  

Microsoft Academic Search

Copper transporting P-type ATPases, designated ATP7A and ATP7B, play an essential role in mammalian copper balance. Impaired intestinal transport of copper, resulting from mutations in the ATP7A gene, lead to Menkes disease in humans. Defects in a similar gene, the copper transporting ATPase ATP7B, result in Wilson disease. This ATP7B transporter has two functions: transport of copper into the plasma

Diane W. Cox; Steven D. P. Moore



Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft.  

PubMed Central

We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement. Images PMID:8862525

Ruppel, K M; Spudich, J A



Infrared spectroscopic studies on the V-ATPase.  


V-ATPase is an ATP-driven rotary motor that vectorially transports ions. Together with F-ATPase, a homologous protein, several models on the ion transport have been proposed, but their molecular mechanisms are yet unknown. V-ATPase from Enterococcus hirae forms a large supramolecular protein complex (total molecular weight: ~700,000) and physiologically transports Na? and Li? across a hydrophobic lipid bilayer. Stabilization of these cations in the binding site has been discussed on the basis of X-ray crystal structures of a membrane-embedded domain, the K-ring (Na? and Li? bound forms). Sodium or lithium ion binding-induced difference FTIR spectra of the intact E. hirae V-ATPase have been measured in aqueous solution at physiological temperature. The results suggest that sodium or lithium ion binding induces the deprotonation of Glu139, a hydrogen-bonding change in the tyrosine residue and rigid ?-helical structures. Identical difference FTIR spectra between the entire V-ATPase complex and K-ring strongly suggest that protein interaction with the I subunit does not cause large structural changes in the K-ring. This result supports the previously proposed Na? transport mechanism by V-ATPase stating that a flip-flop movement of a carboxylate group of Glu139 without large conformational changes in the K-ring accelerates the replacement of a Na? ion in the binding site. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems. PMID:25111748

Kandori, Hideki; Furutani, Yuji; Murata, Takeshi



Biochemical characterization of P-type copper ATPases  

PubMed Central

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 interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper. PMID:25242165

Inesi, Giuseppe; Pilankatta, Rajendra; Tadini-Buoninsegni, Francesco



Biochemical characterization of P-type copper ATPases.  


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 interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper. PMID:25242165

Inesi, Giuseppe; Pilankatta, Rajendra; Tadini-Buoninsegni, Francesco



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

NASA Astrophysics Data System (ADS)

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

Nie, Wei


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


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



Role for Myosin II in Regulating Positioning of Salmonella-Containing Vacuoles and Intracellular Replication?  

PubMed Central

Salmonella enterica serovar Typhimurium grows within host cells in a permissive compartment termed the Salmonella-containing vacuole (SCV). These bacteria use two distinct type III secretion systems (T3SS) to deliver virulence proteins (effectors) into cells. Effectors secreted by the Salmonella pathogenicity island 1 (SPI-1)-encoded T3SS mediate invasion and early SCV maturation steps, while those secreted by the SPI-2 T3SS affect the SCV at later stages postinfection. Some SPI-2 effectors modulate microtubule motor activity on the SCV. Here, we show that the actin-based motor myosin II also affects SCV dynamics during infection. Following invasion, myosin II is required for SCV positioning near the nucleus of host cells. Later, myosin II counteracts the activities of the SPI-2 effectors PipB2 and SseJ to maintain SCV positioning and stability, respectively. Myosin II activity was required for maximal bacterial growth in macrophages. Rho kinase activity was required for SCV positioning. The effector SopB, a known activator of Rho GTPases, was found to be required for SCV positioning, and transfection of cells with SopB was sufficient to induce myosin II phosphorylation. These studies reveal a novel role for myosin II in controlling SCV dynamics during infection and suggest that SopB activates myosin II. PMID:18411289

Wasylnka, Julie A.; Bakowski, Malina A.; Szeto, Jason; Ohlson, Maikke B.; Trimble, William S.; Miller, Samuel I.; Brumell, John H.



Pharmacological activation of myosin II paralogs to correct cell mechanics defects.  


Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. PMID:25605895

Surcel, Alexandra; Ng, Win Pin; West-Foyle, Hoku; Zhu, Qingfeng; Ren, Yixin; Avery, Lindsay B; Krenc, Agata K; Meyers, David J; Rock, Ronald S; Anders, Robert A; Freel Meyers, Caren L; Robinson, Douglas N



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

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



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

PubMed Central

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

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



Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation  

PubMed Central

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation. PMID:20308429

Pasapera, Ana M.; Schneider, Ian C.; Rericha, Erin; Schlaepfer, David D.



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

PubMed Central

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175?kDa heavy chain (175?kDa myosin) and the other of 170?kDa heavy chain (170?kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175?kDa and 170?kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175?kDa myosin heavy chain showed that the 175?kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175?kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175?kDa myosin. Furthermore, this myosin was co-fractionated with GFP–ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175?kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells. PMID:19039101

Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo



Evolutionary appearance of the plasma membrane H (+) -ATPase containing a penultimate threonine in the bryophyte.  


The plasma membrane H (+) -ATPase provides the driving force for solute transport via an electrochemical gradient of H (+) across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H (+) -ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H (+) -ATPase (pT H (+) -ATPase) and non-pT H (+) -ATPase as in the green algae, and that pT H (+) -ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H (+) -ATPase genes, designated PpHA (Physcomitrella patens H (+) -ATPase). Six isoforms are the pT H (+) -ATPase; a remaining isoform is non-pT H (+) -ATPase. An apparent 95-kD protein was recognized by anti-H (+) -ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H (+) -ATPase. Furthermore, we could not detect the pT H (+) -ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H (+) -ATPase most likely appeared for the first time in bryophyte. PMID:22836495

Okumura, Masaki; Takahashi, Koji; Inoue, Shin-Ichiro; Kinoshita, Toshinori



Motor and tail homology 1 (Th1) domains antagonistically control myosin-1 dynamics.  


Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane. PMID:24507605

Mazerik, Jessica N; Kraft, Lewis J; Kenworthy, Anne K; Tyska, Matthew J



Structure of the light chain-binding domain of myosin V  

PubMed Central

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

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



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.



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:



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


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



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


The main goal of this study was to find out, whether the appearance of fibres without evident myosin heavy chain (MyHC) transcript expression (negative fibres) implies the existence of additional MyHC transcripts in human muscle fibres. Fourteen different skeletal muscles were analysed also to verify how MyHC transcript expression matches histochemical phenotypes of fibres. For this purpose, the expression of beta-slow, 2a and 2x MyHC transcripts, demonstrated by in situ hybridisation technique, was analysed within type I, IIC, IIA, IIAX and IIX fibres, determined according to the activity of myofibrillar ATPase. Additionally, MyHC isoform expression was immunohistochemically demonstrated and metabolic profiles of negative fibres were estimated. From a total of 4444 muscle fibres analysed, only 0.8% of fibres were negative, among them type I prevailed, the remainder were type IIA and IIX fibres. The majority of fibres expressed only beta, 2a and 2x MyHC transcripts and they mostly matched type I, IIA and IIX fibres respectively, but two minor hybrid fibre groups (beta/2a and 2ax) exhibited variable histochemical phenotype. The infrequency, the prevailing oxidative-glycolytic metabolic profile of negative type I fibres and frequent co-appearance with transitional type IIC fibres imply that the negative fibres rather result from fibre type transition than express an additional slow or even 2b MyHC transcripts. The appearance of hybrid and mismatched fibres additionally indicates that fibre type transition occurs also in presumably normal skeletal muscles, what enables the muscles to tune even with minimal changes in mechanical demands. PMID:12222825

Smerdu, V; Erzen, I



Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments  

PubMed Central

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli. PMID:3693404



Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy  

PubMed Central

We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates ? phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure. PMID:24156065

Rivard, Maxime; Couture, Charles-André; Miri, Amir K.; Laliberté, Mathieu; Bertrand-Grenier, Antony; Mongeau, Luc; Légaré, François



75 FR 1798 - Prospective Grant of Exclusive License: Development of V-ATPase Inhibitor Compounds for the...  

Federal Register 2010, 2011, 2012, 2013, 2014

...osteoporosis, and Alzheimer's diseases. Briefly, vacuolar type (H+) ATPase (V-ATPase) has been described as ``a universal proton pump of eukaryotes''. V-ATPase is responsible for maintaining internal acidity and is important in myriad of...



F1-ATPase of Escherichia coli  

PubMed Central

F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ?, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·? binding and dissociation, we show that formation of the extended, inhibitory conformation of ? (?X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ?X state, and post-hydrolysis conditions stabilize it. We also show that ? inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ? is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ? N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ? suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position. PMID:23400782

Shah, Naman B.; Hutcheon, Marcus L.; Haarer, Brian K.; Duncan, Thomas M.



Kinetic characterization of Na,K-ATPase inhibition by Eosin  

PubMed Central

Eosin is a probe for the Na pump nucleotide site. In contrast to previous studies examining eosin effects on Na only ATPase, we examined Na,K ATPase and K activated pNPPase activity in red blood cell membranes and purified renal Na,K ATPase. At saturating ATP (3mM) the eosin IC50 for Na pump inhibition was 19uM. Increasing ATP concentrations (0.2 – 2.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixed-type inhibitor of ATPase activity. To test if eosin can bind to the high affinity ATP site, purified Na,K ATPase was labeled with 20 uM FITC. With increasing eosin concentrations (0.1 uM – 10 uM) the incorporation of FITC into the ATP site significantly decreases suggesting that eosin prevents FITC reaction at the high affinity ATP site. Eosin was a more potent inhibitor of K activated phosphatase activity than of Na,K ATPase activity. At 5mM pNPP the eosin IC50 for Na pump inhibition was 3.8 ± 0.23 uM. Increasing pNPP concentrations (0.45 – 14.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixed-type inhibitor of pNPPase activity. These results can be fit by a model in which eosin and ATP bind only to the nucleotide site; in some pump conformations, this site is rigid and the binding is mutually exclusive and in other conformations, the site is flexible and able to accommodate both eosin and ATP (or pNPP). Interestingly, eosin inhibition of pNPPase became competitive after the addition of C12E8 (0.1%) but the inhibition of ATPase remained mixed. PMID:17331759

Ogan, Jeffrey T.; Reifenberger, Matthew S.; Milanick, Mark A.; Gatto, Craig



Analysis of gene duplication repeats in the myosin rod.  


The helical coiled-coil region of the myosin rod in the nematode Caenorhabditis elegans is a repetitive sequence 1094 amino acids long which contains 39 repeats of a 28-residue pattern. The repeats are extremely significant when compared with the statistical distributions expected, first for random sequences, and then for sequences with a typical seven-residue coiled-coil periodicity. New and improved statistical tests are used. The repeats are stronger in the first 350 residues of the rod (fragment S-2) than in the remainder. The corresponding DNA sequence of the unc-54 gene shows the same features, but they are less significant when judged by the number of identical bases than are the amino acid similarities, as measured by Dayhoff scores. The rod sequence shows strong evidence for a longer repeat unit of 196 residues, which may be related to the cross-bridge spacing of 143 A in muscle. PMID:6620380

McLachlan, A D



Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni?  

PubMed Central

Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action. PMID:19104008

Gnanasekar, Munirathinam; Salunkhe, Ashok M.; Mallia, A. Krishna; He, Yi Xun; Kalyanasundaram, Ramaswamy



Simulation model for combined motion of myosin cross-bridges agrees with experimental data.  


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



The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy.  


No matter how many times one explores the structure of the myosin molecule, there is always something new to discover. Here, I describe the myosin mesa, a structural feature of the motor domain that has the characteristics of a binding domain for another protein, possibly myosin-binding protein C (MyBP-C). Interestingly, many well-known hypertrophic cardiomyopathy (HCM) mutations lie along this surface and may affect the putative interactions proposed here. A potential unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy is discussed here. It involves increased power output of the cardiac muscle as a result of HCM mutations causing the release of inhibition by myosin binding protein C. PMID:25619247

Spudich, James A



Cloning of the Genes Encoding Two Murine and Human Cochlear Unconventional Type I Myosins  

Microsoft Academic Search

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue

Fabien Crozet; Aziz El Amraoui; Stéphane Blanchard; Marc Lenoir; Chantal Ripoll; Philippe Vago; Christian Hamel; Cécile Fizames; Fabienne Levi-Acobas; Danièle Depétris; Dominique Weil; Rémy Pujol; Christine Petit



Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula  

PubMed Central

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ?62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (Cf) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa?1. This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa?1. The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm?1) is smaller than that of frog (2.78 ± 0.30 pN nm?1). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog. PMID:22310308

Park-Holohan, S; Linari, M; Reconditi, M; Fusi, L; Brunello, E; Irving, M; Dolfi, M; Lombardi, V; West, T G; Curtin, N A; Woledge, R C; Piazzesi, G



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



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

PubMed Central

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

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



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



Atomic Structure of Scallop Myosin Subfragment S1 Complexed with MgADP  

Microsoft Academic Search

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 Å resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre–power stroke or near-rigor state structures; moreover, in contrast to these structures, the

Anne Houdusse; Vassilios N. Kalabokis; Daniel Himmel; Andrew G. Szent-Györgyi; Carolyn Cohen



Mutants Altering Coordinate Synthesis of Specific Myosins during Nematode Muscle Development  

Microsoft Academic Search

Mutations in the unc-52 gene on linkage group II retard the construction of body-wall muscle sarcomeres during larval development in the nematode Caenorhabditis elegans. Unc-52 mutants show decreased accumulation of myosin heavy chains relative to other polypeptides during larval development, correlating with the structural retardation. Pulse radiolabeling experiments show that decreased synthesis of specific body-wall myosin heavy chains that are

Janice M. Zengel; Henry F. Epstein



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



Class XIII myosins from the green alga Acetabularia : driving force in organelle transport and tip growth?  

Microsoft Academic Search

The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily\\u000a (Cope et al., 1996, Structure 4: 969–987). Here we report a complete analysis of their gene structure and their corresponding transcripts\\u000a Aclmyol and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found.

Oliver Vugrek; Heiko Sawitzky; Diedrik Menzel



A new model for the surface arrangement of myosin molecules in tarantula thick filaments  

Microsoft Academic Search

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 Angstrom have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head.\\u000aWe

Gerald Offer; Peter J. Knight; Stanley A. Burgess; Lorenzo Alamo; Raúl Padrón



Ouabain Binding Site in a Functioning Na+/K+ ATPase*  

PubMed Central

The Na+/K+ ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na+/K+ ATPase sequence. These five normally functional LBT-Na+/K+ ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na+/K+ ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na+/K+ ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies. PMID:21911500

Sandtner, Walter; Egwolf, Bernhard; Khalili-Araghi, Fatemeh; Sánchez-Rodríguez, Jorge E.; Roux, Benoit; Bezanilla, Francisco; Holmgren, Miguel



Ouabain binding site in a functioning Na+/K+ ATPase.  


The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies. PMID:21911500

Sandtner, Walter; Egwolf, Bernhard; Khalili-Araghi, Fatemeh; Sánchez-Rodríguez, Jorge E; Roux, Benoit; Bezanilla, Francisco; Holmgren, Miguel



Hormonal regulation of Na -K -ATPase in cultured epithelial cells  

SciTech Connect

Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

Johnson, J.P.; Jones, D.; Wiesmann, W.P.



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


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

Sarshad, Aishe A; Percipalle, Piergiorgio



P-cadherin counteracts myosin II-B function: implications in melanoma progression  

PubMed Central

Background Malignant transformation of melanocytes is frequently attended by a switch in cadherin expression profile as shown for E- and N-cadherin. For P-cadherin, downregulation in metastasizing melanoma has been demonstrated, and over-expression of P-cadherin in melanoma cell lines has been shown to inhibit invasion. The strong invasive and metastatic nature of cutaneous melanoma implies a deregulated interplay between intercellular adhesion and migration-related molecules Results In this study we performed a microarray analysis to compare the mRNA expression profile of an invasive BLM melanoma cell line (BLM LIE) and the non-invasive P-cadherin over-expression variant (BLM P-cad). Results indicate that nonmuscle myosin II-B is downregulated in BLM P-cad. Moreover, myosin II-B plays a major role in melanoma migration and invasiveness by retracting the tail during the migratory cycle, as shown by the localization of myosin II-B stress fibers relative to Golgi and the higher levels of phosphorylated myosin light chain. Analysis of P-cadherin and myosin II-B in nodular melanoma sections and in a panel of melanoma cell lines further confirmed that there is an inverse relationship between both molecules. Conclusions Therefore, we conclude that P-cadherin counteracts the expression and function of myosin II-B, resulting in the suppression of the invasive and migratory behaviour of BLM melanoma cells PMID:20860798



Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.  

PubMed Central

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors. Images PMID:1421576

Giuliano, K A; Kolega, J; DeBiasio, R L; Taylor, D L



Supervillin binding to myosin II and synergism with anillin are required for cytokinesis  

PubMed Central

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis. PMID:24088567

Smith, Tara C.; Fridy, Peter C.; Li, Yinyin; Basil, Shruti; Arjun, Sneha; Friesen, Ryan M.; Leszyk, John; Chait, Brian T.; Rout, Michael P.; Luna, Elizabeth J.



Changes in expression of nonmuscle myosin heavy chain isoforms during muscle and nonmuscle tissue development.  


Anti-human platelet myosin antibodies and two anti-peptide antibodies, anti-peptide IIA and anti-peptide IIB, which recognize macrophage-type (MIIA) and brain-type (MIIB) isoforms of nonmuscle myosin heavy chain, respectively, were used to study expression of nonmuscle myosin isoforms in various tissues of mice during development. Tissue-specific changes in the relative isoform concentrations were observed by performing immunoblots of crude myosin extracts from nonmuscle and muscle tissues. In fetal and neonatal mouse tissues, the anti-peptide IIB antibodies stained a single band, called MIIB2, while the anti-peptide IIA and anti-platelet myosin antibodies stained a band that migrated faster than MIIB2. In brain, a slower moving band, MIIB1, started to appear at 2 weeks after birth, and in the adult cerebellum it was at least as abundant as MIIB2. In thymus, MIIB2 decreased selectively shortly after birth, while in liver both MIIB2 and MIIA rapidly disappeared, but the isoform(s) detected by anti-platelet myosin antibodies (MIIApla) remained constant. The MIIB2 and MIIA as well as MIIApla found in striated muscles from fetal and neonatal mice decreased to levels that were below the limit of detection by 3 weeks of age. In cryosections of skeletal and cardiac muscles, MIIB2 was localized within the muscle cells, while MIIA and MIIApla were primarily in the blood vessels and capillaries. PMID:8482409

Murakami, N; Trenkner, E; Elzinga, M



Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.  


Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions. PMID:21718700

Calábria, Luciana Karen; Peixoto, Pablo Marco Veras; Passos Lima, Andreia Barcelos; Peixoto, Leonardo Gomes; de Moraes, Viviane Rodrigues Alves; Teixeira, Renata Roland; Dos Santos, Claudia Tavares; E Silva, Letícia Oliveira; da Silva, Maria de Fátima Rodrigues; dos Santos, Ana Alice Diniz; Garcia-Cairasco, Norberto; Martins, Antônio Roberto; Espreafico, Enilza Maria; Espindola, Foued Salmen



Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing.  


Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss. PMID:24105371

Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B



Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.  


During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²?CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s?¹ MLCK?¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s?¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s?¹) would be much faster than with pSMM (<0.1-0.2 s?¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle. PMID:24144337

Hong, Feng; Facemyer, Kevin C; Carter, Michael S; Jackson, Del R; Haldeman, Brian D; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P; Cremo, Christine R; Baker, Josh E



Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail  

PubMed Central

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. PMID:3040773



Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.  


We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. PMID:3040773

Hammer, J A; Bowers, B; Paterson, B M; Korn, E D



Rotary catalysis of the stator ring of F(1)-ATPase.  


F(1)-ATPase is a rotary motor protein in which 3 catalytic ?-subunits in a stator ?(3)?(3) ring undergo unidirectional and cooperative conformational changes to rotate the rotor ?-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the ?-subunit as a "dictator" completely controlling the chemical and conformational states of the 3 catalytic ?-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 ?-subunits undergo cyclic conformational changes even in the absence of the rotor ?-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F(1)-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). PMID:22465022

Iino, Ryota; Noji, Hiroyuki



Response of Na(+)-dependent ATPase activities to the contaminant ammonia nitrogen in Tapes philippinarum: possible atpase involvement in ammonium transport.  


In vivo and in vitro experiments elicited different responses to ammonia nitrogen (ammonia-N) of gill and mantle Na,K-ATPase and ouabain-insensitive Na-ATPase activities in the Philippine clam Tapes philippinarum. Short-term (120 h) exposed clams to sublethal ammonia-N (NH(3)+NH (4) (+) ) concentrations (1.5 and 3.0 mg/L ammonia-N) showed enhanced gill and mantle ouabain-insensitive ATPase activity and decreased mantle Na,K-ATPase activity with respect to unexposed clams, while gill Na,K-ATPase was unaffected. In vitro experiments showed that NH (4) (+) could efficiently replace Na(+) in ouabain-insensitive ATPase activation and K(+), but not Na(+), in Na, K-ATPase activation. Simple saturation kinetics was constantly followed with similar K (0.5) values to that of the substituted cation. The same maximal ouabain-insensitive ATPase activation was obtained at 80 mM Na(+) or NH (4) (+) in the gills and at 50 mM Na(+) or NH (4) (+ ) in the mantle and that of Na,K-ATPase at 10 mM K(+) or NH (4) (+) in the presence of 100 mM Na(+) in both tissues. The two coexistent ATPase activities maintained their typical response to ouabain also when stimulated by NH (4) (+) : when activated by Na(+)+K(+) or by Na(+)+NH (4) (+) the ATPase activity was completely suppressed by 10(-3 )M ouabain, whereas the Na(+)- or NH (4) (+) -stimulated ATPase activity was unaffected by up to 10(-2 )M ouabain. The whole of the data suggests a possible involvement of the two ATPase activities in NH (4) (+) transmembrane transport. PMID:18175160

Pagliarani, Alessandra; Bandiera, Patrizia; Ventrella, Vittoria; Trombetti, Fabiana; Manuzzi, Maria Pia; Pirini, Maurizio; Borgatti, Anna Rosa



Hypoxic responses of Na+/K+ ATPase in trout hepatocytes.  


Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production. PMID:15879061

Bogdanova, A; Grenacher, B; Nikinmaa, M; Gassmann, M



A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization  

Microsoft Academic Search

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a

Christine Hettmann; Angelika Herm; Ariane Geiter; Bernd Frank; Eva Schwarz; Thierry Soldati; Dominique Soldati



Comparative study on acid-induced gelation of myosin from Atlantic cod ( Gardus morhua) and burbot ( Lota lota)  

Microsoft Academic Search

Physicochemical and rheological properties of myosin from Atlantic cod and burbot during acid-induced gelation at room temperature (22–23°C) by d-gluconic acid-?-lactone (GDL) were monitored. Turbidity and particle size of both myosins increased and salt soluble content decreased when pH decreased, suggesting the formation of protein aggregates caused by acidification. The formation of disulphide bonds in myosin gelation was induced by

Siriporn Riebroy; Soottawat Benjakul; Wonnop Visessanguan; Ulf Erikson; Turid Rustad




Microsoft Academic Search

The expression of the V-type proton ATPase (H + - ATPase) was examined in the gill of the freshwater rainbow trout (Oncorhynchus mykiss) using immunocytochemistry in concert with laser scanning confocal or electron microscopy. A synthetic peptide consisting of the carboxy- terminal region of the 31 kDa subunit of the bovine renal H + -ATPase was used to generate an





EPA Science Inventory

The effects of triethyltin (TET) on ATPase activities in brain and liver homogenates and subcellular fractions were compared in neonatal and adult rats. n 5 day old rats, relative sensitivities to TET inhibition were: brain and liver mitochondrial ATPase >> rain Na+/K+ ATPase > b...


Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride  

E-print Network

Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride Zulfiqar activity of Escherichia coli ATP synthase by magnesium fluoride (MgFx) was studied. Wild-type F1-ATPase synthesis mechanism; Magnesium fluoride; ATPase inhibition; Transition state analog 1. Introduction ATP

Zulfiqar Ahmad


On the localization of sarcotubular ATPase activity in mammalian skeletal muscle  

Microsoft Academic Search

ATPase activity of the sarcoplasmic reticulum has been demonstrated at the level of the light microscope. Although this membrane system is usually viewed as ultrastructural in its dimensions, it was possible to identify sarcotubular enzymic activity in frozen sections. In skeletal muscle fibers of the rat diaphragm, sarcotubular ATPase can be distinguishedin situ from ATPases associated with mitochondria and myofibrils.

Geraldine F. Gauthier



Independent Evolution of Heavy Metal-Associated Domains in Copper Chaperones and Copper-Transporting ATPases  

E-print Network

Independent Evolution of Heavy Metal-Associated Domains in Copper Chaperones and Copper and structure to the Cu- binding heavy metal-associated (HMA) domains of Cu- transporting ATPases (Cu to the Cu-binding heavy metal-associated (HMA) domains of Cu-transporting ATPases (Cu- ATPases) whose genes

Jordan, King


SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity  

E-print Network

SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ that the SWI2/SNF2 chromatin-remodeling ATPases SPLAYED (SYD) and BRAHMA (BRM) are redundantly required for flower patterning and for the activation of AP3 and AG. The SWI2/SNF2 ATPases are recruited

Plotkin, Joshua B.


[Nonhyperbolic kinetics of Na,K-ATPase--a new viewpoint].  


The kinetics of the 130 kDa monomer obtained by treatment of duck salt gland Na,K-ATPase with C12E8 was compared with that of the membrane-bound enzyme. The shapes of the substrate-velocity curves for the membrane-bound and solubilized forms were quite different: a hyperbolic one for the monomeric Na,K-ATPase and a nonhyperbolic one for the native enzyme. A reaction scheme for ATP hydrolysis based on a comparative analysis of kinetic properties of these two forms is proposed. Experimental evidence in favour of this hypothesis is presented. PMID:1653044

Fedosova, N U; Boldyrev, A A



Non-weight bearing-induced muscle weakness: the role of myosin quantity and quality in MHC type II fibers  

PubMed Central

We tested the hypothesis that non-weight bearing-induced muscle weakness (i.e., specific force) results from decreases in myosin protein quantity (i.e., myosin content per half-sarcomere and the ratio of myosin to actin) and quality (i.e., force per half-sarcomere and population of myosin heads in the strong-binding state during muscle contraction) in single myosin heavy chain (MHC) type II fibers. Fisher-344 rats were assigned to weight-bearing control (Con) or non-weight bearing (NWB). The NWB rats were hindlimb unloaded for 2 wk. Diameter, force, and MHC content were determined in permeabilized single fibers from the semimembranosus muscle. MHC isoform and the ratio of MHC to actin in each fiber were determined by gel electrophoresis and silver staining techniques. The structural distribution of myosin from spin-labeled fiber bundles during maximal isometric contraction was evaluated using electron paramagnetic resonance spectroscopy. Specific force (peak force per cross-sectional area) in MHC type IIB and IIXB fibers from NWB was significantly reduced by 38% and 18%, respectively. MHC content per half-sarcomere was significantly reduced by 21%. Two weeks of hindlimb unloading resulted in a reduced force per half-sarcomere of 52% and fraction of myosin strong-binding during contraction of 34%. The results suggest that reduced myosin and actin content (quantity) and myosin quality concomitantly contribute to non-weight bearing-related muscle weakness. PMID:24829495

Kim, Jong-Hee



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

SciTech Connect

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States); Patel, Yashomati M. [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States)], E-mail:



Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.  


X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 ?m at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 ?m but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments. PMID:24344169

Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella



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

PubMed Central

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 (S2), were used to assay the appearance and accumulation of specific MHC epitopes with: (a) indirect, solid phase radioimmune assay (RIA), (b) immunoautoradiography, (c) immunofluorescence microscopy. McAb MF 20 bound strongly and equivalently to MHC at all stages of embryonic development in vivo. In contrast, the MF 30 epitope was barely detectable at 12 d of incubation but its concentration rose rapidly just before hatching. No detectable binding of MF 14 to pectoralis myosin could be measured during myogenesis in vivo until 1 wk after hatching. Immunofluorescence studies revealed that all three epitopes accumulate in the same myocytes of the developing pectoralis muscle. Since all three McAbs bound with high activity to native and denatured forms of myosin, it is unlikely that differential antibody reactivity can be explained by conformational changes in myosin during development in vivo. When myogenesis in vitro was monitored using the same McAbs, MF 20 bound to the MHC at all stages tested while reactivity of MF 30 and MF 14 with myosin from cultured muscle was never observed. Thus, this study demonstrates three different immunochemical states of the MHC during development in vivo of chick pectoralis muscle and the absence of later occurring immunochemical transitions in the MHC of cultured embryonic muscle. PMID:6185504



Single Myosin Lever-Arm Orientation in a Muscle Fiber Detected with Photoactivatable GFP†  

PubMed Central

Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of ~70° to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence an important research objective is to detect in situ the lever arm orientation. Single molecule measurements are preferred because they clarify ambiguities unavoidable with ensemble measurements, however, detecting single molecules in the condensed tissue medium where myosin concentration exceeds 100 µM is challenging. A photoactivatable green fluorescent protein (PAGFP) tagged myosin light chain (MLC) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy and PAGFP was sparsely photoactivated such that polarized fluorescence identified single probe orientation. Several physiological states of the muscle fiber were characterized revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight or bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where myosin concentration is low suggests molecular crowding in the A-band promotes occupancy of the straight myosin conformation (Burghardt et al., 2007, Biophys. J. 93, 2226). The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke. PMID:19127992

Burghardt, Thomas P.; Li, Jinhui; Ajtai, Katalin



Absence of Platelet Phenotype in Mice Lacking the Motor Protein Myosin Va  

PubMed Central

Background The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. Methodology/Principal Findings Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a?/? platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, ?-granule and lysosomal secretion, integrin ?IIb?3 activation, Ca2+ signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a?/? platelets showed no significant functional defects in these responses, or in the numbers of dense and ?-granules expressed. Conclusion Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and ?-granules or other functional responses in platelets. PMID:23349704

Hers, Ingeborg; Poole, Alastair W.



Slow myosin ATP turnover in the super-relaxed state in tarantula muscle.  


We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2'-/3'-O-(N'-methylanthraniloyl)adenosine-5'-O-triphosphate, as monitored by the decrease in fluorescence when 2'-/3'-O-(N'-methylanthraniloyl)adenosine-5'-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (?30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2'-/3'-O-(N'-methylanthraniloyl)adenosine-5'-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the "interacting-heads motif," observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations. PMID:21763701

Naber, Nariman; Cooke, Roger; Pate, Edward



Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells  

PubMed Central

We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin- G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells. PMID:62755



A minimum catalytic unit of F 1ATPase shows non-cooperative ATPase activity inherent in a single catalytic site with a K m 70 ?M  

Microsoft Academic Search

F1-ATPase has three interacting catalytic sites and shows complicated kinetics. Here, we report reconstitution of a complex, most likely composed of one ? subunit and one ? subunit, with a single catalytic site from thermophilic Bacillus PS3 F1-ATPase on the solid surface. The complex has an ATPase activity which obeys a simple non-cooperative kinetics with a Km(ATP) of 70 ?M

Koji Saika; Masasuke Yoshida



Ecto-F1-ATPase and MHC Class I Close Association on cell membranes Pierre Vantourout1,2  

E-print Network

precipitates from immunofluorescence-negative cells, suggesting that ATPase epitopes are masked. Removal of 2-ATPase epitopes on MHC-I+ cell lines. Ecto- F1-ATPase is detected by immunofluorescence on primary IFN- and/or TNF- treatment induces a dose-dependent disappearance of F1-ATPase epitopes. Finally

Boyer, Edmond


Modelling the effect of myosin X motors on filopodia growth  

NASA Astrophysics Data System (ADS)

We present a numerical simulation study of the dynamics of filopodial growth in the presence of active transport by myosin X motors. We employ both a microscopic agent-based model, which captures the stochasticity of the growth process, and a continuum mean-field theory which neglects fluctuations. We show that in the absence of motors, filopodia growth is overestimated by the continuum mean-field theory. Thus fluctuations slow down the growth, especially when the protrusions are driven by a small number (10 or less) of F-actin fibres, and when the force opposing growth (coming from membrane elasticity) is large enough. We also show that, with typical parameter values for eukaryotic cells, motors are unlikely to provide an actin transport mechanism which enhances filopodial size significantly, unless the G-actin concentration within the filopodium greatly exceeds that of the cytosol bulk. We explain these observations in terms of order-of-magnitude estimates of diffusion-induced and advection-induced growth of a bundle of Brownian ratchets.

Wolff, K.; Barrett-Freeman, C.; Evans, M. R.; Goryachev, A. B.; Marenduzzo, D.



Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa  

SciTech Connect

Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K



Mechanical modulation of catalytic power on F1ATPase  

Microsoft Academic Search

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F1-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate

Rikiya Watanabe; Daichi Okuno; Shouichi Sakakihara; Katsuya Shimabukuro; Ryota Iino; Masasuke Yoshida; Hiroyuki Noji



Elucidation of the Na+, K+-ATPase digitalis binding site.  


Despite controversy over their use and the potential for toxic side effects, cardiac glycosides have remained an important clinical component for the treatment for congestive heart failure (CHF) and supraventricular arrhythmias since the effects of Digitalis purpurea were first described in 1785. While there is a wealth of information available with regard to the effects of these drugs on their pharmacological receptor, the Na(+), K(+)-ATPase, the exact molecular mechanism of digitalis binding and inhibition of the enzyme has remained elusive. In particular, the absence of structural knowledge about Na(+), K(+)-ATPase has thwarted the development of improved therapeutic agents with larger therapeutic indices via rational drug design approaches. Here, we propose a binding mode for digoxin and several analogues to the Na(+), K(+)-ATPase. A 3D-structural model of the extracellular loop regions of the catalytic alpha1-subunit of the digitalis-sensitive sheep Na(+), K(+)-ATPase was constructed from the crystal structure of an E(1)Ca(2+) conformation of the SERCA1a and a consensus orientation for digitalis binding was inferred from the in silico docking of a series of steroid-based cardiotonic compounds. Analyses of species-specific enzyme affinities for ouabain were also used to validate the model and, for the first time, propose a detailed model of the digitalis binding site. PMID:15886034

Keenan, Susan M; DeLisle, Robert K; Welsh, William J; Paula, Stefan; Ball, William J



A functional calcium-transporting ATPase encoded by chlorella viruses  

PubMed Central

Calcium-transporting ATPases (Ca2+ pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca2+ pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca2+ pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca2+ pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a 32P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca2+, thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca2+-transporting ATPase encoded by a virus. PMID:20573858

Bonza, Maria Cristina; Martin, Holger; Kang, Ming; Lewis, Gentry; Greiner, Timo; Giacometti, Sonia; Van Etten, James L.; De Michelis, Maria Ida; Thiel, Gerhard; Moroni, Anna



Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans  

PubMed Central

Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M) to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration). Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001) decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases. PMID:25364660

Kumar, Prabhanshu; Maurya, Pawan Kumar



Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.  


Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases. PMID:11325721

Rice, W J; Young, H S; Martin, D W; Sachs, J R; Stokes, D L



Selective delipidation of the plasma membrane by surfactants: Enrichment of sterols and activation of ATPase  

SciTech Connect

The influence of plasma membrane lipid components on the activity of the H{sup +}-ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.

Sandstrom, R.P.; Cleland, R. (Portland State Univ., OR (USA) Univ. of Washington, Seattle (USA))



Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles[W][OPEN  

PubMed Central

To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein–tagged MyoB1/2 localize to vesicles that traffic in a myosin XI–dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A–sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment. PMID:23995081

Peremyslov, Valera V.; Morgun, Eva A.; Kurth, Elizabeth G.; Makarova, Kira S.; Koonin, Eugene V.; Dolja, Valerian V.



An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight  

PubMed Central

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers – 1% muscle length (ML) amplitude and 150 Hz oscillation frequency – were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance. PMID:24115062

Wang, Qian; Newhard, Christopher S.; Ramanath, Seemanti; Sheppard, Debra; Swank, Douglas M.



Neural crest specification by inhibition of the ROCK/Myosin II pathway.  


Neural crest is a population of multipotent progenitor cells that form at the border of neural and non-neural ectoderm in vertebrate embryos, and undergo epithelial-mesenchymal transition and migration. According to the traditional view, the neural crest is specified in early embryos by signaling molecules including BMP, FGF and Wnt proteins. Here we identify a novel signaling pathway leading to neural crest specification, which involves Rho-associated kinase (ROCK) and its downstream target non-muscle Myosin II. We show that ROCK inhibitors promote differentiation of human embryonic stem cells into neural crest-like progenitors (NCPs) that are characterized by specific molecular markers and ability to differentiate into multiple cell types, including neurons, chondrocytes, osteocytes and smooth muscle cells. Moreover, inhibition of Myosin II was sufficient for generating NCPs at high efficiency. Whereas Myosin II has been previously implicated in the self-renewal and survival of human pluripotent ES cells, we demonstrate its role in neural crest development during ES cell differentiation. Inhibition of this pathway in Xenopus embryos expanded neural crest in vivo, further indicating that neural crest specification is controlled by ROCK-dependent Myosin II activity. We propose that changes in cell morphology in response to ROCK and Myosin II inhibition initiate mechanical signaling leading to neural crest fates. Stem Cells 2014. PMID:25346532

Kim, Kyeongmi; Ossipova, Olga; Sokol, Sergei Y



Capping of surface receptors and concomitant cortical tension are generated by conventional myosin.  


We have investigated the role of cytoskeletal contraction in the capping of surface proteins crosslinked by concanavalin A on mutant Dictyostelium cells lacking conventional myosin. Measurements of cellular deformability to indicate the development of cortical tension show that cells of the wild-type parental strain, AX4, stiffen early during capping and relax back towards the softer resting state as the process is completed. Mutant cells lacking myosin (mhcA-) have a lower resting-state stiffness, and fail to stiffen and to cap crosslinked proteins on binding concanavalin A. Hence conventional myosin is essential both for capping and for the concomitant increase in cell stiffness. Furthermore, depletion of cellular ATP by azide causes a 'rigor' contraction in AX4 cells which makes them stiffen and become spherical. By contrast, the mhcA- cells fail to respond in these ways. These measurements of cortical tension in non-muscle cells can thus be directly correlated with the presence of conventional myosin, demonstrating that contractile tension generated by myosin can drive both a change of cell shape and the capping of crosslinked surface receptors. PMID:2797182

Pasternak, C; Spudich, J A; Elson, E L



Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.  


The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats. PMID:1534798

Kandarian, S C; Schulte, L M; Esser, K A



Dynamic coupling of regulated binding sites and cycling myosin heads in striated muscle.  


In an activated muscle, binding sites on the thin filament and myosin heads switch frequently between different states. Because the status of the binding sites influences the status of the heads, and vice versa, the binding sites and myosin heads are dynamically coupled. The functional consequences of this coupling were investigated using MyoSim, a new computer model of muscle. MyoSim extends existing models based on Huxley-type distribution techniques by incorporating Ca(2+) activation and cooperative effects. It can also simulate arbitrary cross-bridge schemes set by the researcher. Initial calculations investigated the effects of altering the relative speeds of binding-site and cross-bridge kinetics, and of manipulating cooperative processes. Subsequent tests fitted simulated force records to experimental data recorded using permeabilized myocardial preparations. These calculations suggest that the rate of force development at maximum activation is limited by myosin cycling kinetics, whereas the rate at lower levels of activation is limited by how quickly binding sites become available. Additional tests investigated the behavior of transiently activated cells by driving simulations with experimentally recorded Ca(2+) signals. The unloaded shortening profile of a twitching myocyte could be reproduced using a model with two myosin states, cooperative activation, and strain-dependent kinetics. Collectively, these results demonstrate that dynamic coupling of binding sites and myosin heads is important for contractile function. PMID:24516189

Campbell, Kenneth S



The role of myosin-II in force generation of DRG filopodia and lamellipodia.  


Differentiating neurons process the mechanical stimulus by exerting the protrusive forces through lamellipodia and filopodia. We used optical tweezers, video imaging and immunocytochemistry to analyze the role of non-muscle myosin-II on the protrusive force exerted by lamellipodia and filopodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. When the activity of myosin-II was inhibited by 30??M Blebbistatin protrusion/retraction cycles of lamellipodia slowed down and during retraction lamellipodia could not lift up axially as in control condition. Inhibition of actin polymerization with 25?nM Cytochalasin-D and of microtubule polymerization with 500?nM Nocodazole slowed down the protrusion/retraction cycles, but only Cytochalasin-D decreased lamellipodia axial motion. The force exerted by lamellipodia treated with Blebbistatin decreased by 50%, but, surprisingly, the force exerted by filopodia increased by 20-50%. The concomitant disruption of microtubules caused by Nocodazole abolished the increase of the force exerted by filopodia treated with Blebbistatin. These results suggest that; i- Myosin-II controls the force exerted by lamellipodia and filopodia; ii- contractions of the actomyosin complex formed by filaments of actin and myosin have an active role in ruffle formation; iii- myosin-II is an essential component of the structural stability of GCs architecture. PMID:25598228

Sayyad, Wasim A; Amin, Ladan; Fabris, Paolo; Ercolini, Erika; Torre, Vincent



Myosin 1 controls membrane shape by coupling F-Actin to membrane  

PubMed Central

Cellular functions are intimately associated with rapid changes in membrane shape. Different mechanisms interfering with the lipid bilayer, such as the insertion of proteins with amphipatic helices or the association of a protein scaffold, trigger membrane bending. By exerting force on membranes, molecular motors can also contribute to membrane remodeling. Previous studies have shown that actin and myosin 1 participate in the invagination of the plasma membrane during endocytosis while kinesins and dynein with microtubules provide the force to elongate membrane buds at recycling endosomes and at the trans-Golgi network (TGN). Using live cell imaging we have recently shown that a myosin 1 (myosin 1b) regulates the actin dependent post-Golgi traffic of cargo and generates force that controls the assembly of F-actin foci and promotes with the actin cytoskeleton the formation of tubules at the TGN. Our data provide evidence that actin and myosin 1 can regulate membrane remodeling of organelles as well as having an unexpected role in the spatial organization of the actin cytoskeleton. Here, we discuss our results together with the role of actin and other myosins that have been implicated in the traffic of cargo. PMID:22754614

Coudrier, Evelyne; Almeida, Claudia G.



Probing myosin structural conformation in vivo by second-harmonic generation microscopy  

PubMed Central

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo. PMID:20385845

Pavone, F. S.



Characterization of Na+K+-ATPase in bovine sperm.  


Existing as a ubiquitous transmembrane protein, Na(+)K(+)-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na(+)K(+)-ATPase is a dimer of ? and ? subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na(+)K(+)-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against ?1 and 3, and all ? isoforms. Relative quantity of Na(+)K(+)-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that ?3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of ?1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of ?1 revealed three distinct spots. Based on the unique quantity, location and structure Na(+)K(+)-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm. PMID:22284223

Hickey, Katie D; Buhr, Mary M



Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte  

PubMed Central

The plasma membrane H+-ATPase provides the driving force for solute transport via an electrochemical gradient of H+ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H+-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H+-ATPase (pT H+-ATPase) and non-pT H+-ATPase as in the green algae, and that pT H+-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H+-ATPase genes, designated PpHA (Physcomitrella patens H+-ATPase). Six isoforms are the pT H+-ATPase; a remaining isoform is non-pT H+-ATPase. An apparent 95-kD protein was recognized by anti-H+-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H+-ATPase. Furthermore, we could not detect the pT H+-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H+-ATPase most likely appeared for the first time in bryophyte. PMID:22836495

Okumura, Masaki; Takahashi, Koji; Inoue, Shin-ichiro; Kinoshita, Toshinori



The mitochondrial ATPase6 gene is more susceptible to mutation than the ATPase8 gene in breast cancer patients  

PubMed Central

Background Breast cancer is the most common malignancy in women throughout the world. Mitochondria play important roles in cellular energy production, free radical generation and apoptosis. Identification of mitochondrial DNA mutations and/or polymorphisms as cancer biomarkers is rapidly developing in molecular oncology research. Methods In this study, the DNA alterations of the mitochondrial ATPase 6 and 8 genes were investigated in 49 breast cancer patients using PCR amplification and direct DNA sequencing on mtDNA. A possible association between these variants and tumorigenesis was assessed. Furthermore, the impact of non-synonymous substitutions on the amino acid sequence was evaluated using the PolyPhen-2 software. Results Twenty eight distinct somatic mitochondrial DNA variants were detected in tumor tissues but not in the corresponding adjacent non-tumor tissues. Among these variants, 9 were observed for the first time in breast cancer patients. The mtDNA variants of A8384 (T7A), T8567C (I14T), G8572A (G16S), A9041G (H172R) and G9055A (A177T) showed the most significant effects probably due to damaging changes to the resulting protein. Furthermore, non-synonymous amino acid changing variants were more frequent in the ATPase6 gene compared to the ATPase8 gene. Conclusion Our results showed that the ATPase6 gene is more susceptible to variations in breast cancer and may play an important role in tumorigenesis by changing the energy metabolism level in cancer cells. PMID:24588805



NMR assignment and secondary structure of coiled coil domain of C-terminal myosin binding subunit of myosin phosphatase.  


Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGI?, PKG-I?(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil ?-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations. PMID:24693955

Sharma, Alok K; Rigby, Alan C



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

E-print Network

cycle. An especially usefulStanford University approach has been to apply a load to an active motor in the chemomechanical cycle (Block et al.,Philadelphia, Pennsylvania 19104 2003; Wang et al., 1998).3 Department Myosin VI, also a processive motor (Rock et al., 2001),optical trapping, we observed myosin VI stepping

Spudich, James A.


Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells  

Microsoft Academic Search

PurposePhosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells.

M. Satpathy; P. Gallagher; M. Lizotte-Waniewski; S. P. Srinivas



Myosin phosphorylation and cyclic adenosine 3',5'-monophosphate in relaxation of arterial smooth muscle by vasodilators.  


Recent evidence indicates that contraction of vascular smooth muscle may be regulated by two calcium-dependent mechanisms: activation of myosin kinase, and calcium binding to a second, unknown regulatory site. This hypothesis implies that vasodilators could modify vascular tone by several mechanisms, including inactivation of myosin kinase. Since relaxation of the carotid artery following agonist removal may occur when myosin phosphorylation is at resting levels, we could determine whether dephosphorylation of myosin is necessarily involved in the molecular mechanisms mediating relaxation in response to vasodilators. The relaxant effects of adenosine, 3-isobutyl-1-methylxanthine, forskolin, sodium nitroprusside, and 8-bromo-cGMP were tested under conditions where myosin phosphorylation was at basal levels (0.08 +/- 0.02 mol Pi/mol light chain). All of these agents increased the rate of relaxation in nonsteady state experiments where relaxation was induced by stimulus washout. Steady state dose-response curves were obtained for forskolin and 8-bromo-cGMP in the presence of basal myosin phosphorylation. Forskolin caused a dose-dependent increase in cAMP levels at a rate consistent with a cause and effect relationship between relaxation and total tissue cAMP content. Both drugs relaxed the muscles, with no detectable change in myosin phosphorylation. Therefore, dephosphorylation of myosin is not a necessary event in the molecular mechanism of several vasodilators, including some which presumably act via cyclic nucleotides. PMID:6319039

Gerthoffer, W T; Trevethick, M A; Murphy, R A



Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain  

Microsoft Academic Search

We describe a quantitative analysis of Acanthamoeba castellanii myosin II rod domain images collected from atomic force microscope experiments. These images reveal that the rod domain forms a novel filament structure, most likely requiring unusual head-to-tail interactions. Similar filaments are seen also in negatively stained electron microscopy images. Truncated myosins from Acanthamoeba and other model organisms have been visualized before,

Daniel J. Rigotti; Bashkim Kokona; Theresa Horne; Eric K. Acton; Carl D. Lederman; Karl A. Johnson; Robert S. Manning; Suzanne Amador Kane; Walter F. Smith; Robert Fairman



Selective Expression of Myosin IC Isoform A in Mouse and Human Cell Lines and Mouse Prostate Cancer Tissues  

PubMed Central

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer. PMID:25259793

Ihnatovych, Ivanna; Sielski, Neil L.; Hofmann, Wilma A.



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

SciTech Connect

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

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



Long single ?-helical tail domains bridge the gap between structure and function of myosin VI  

PubMed Central

Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take ~36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single ?-helix of ~10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the ~36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization. PMID:18511944

Spink, Benjamin J; Sivaramakrishnan, Sivaraj; Lipfert, Jan; Doniach, Sebastian; Spudich, James A



Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.  


We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. PMID:18094930

Forer, A; Spurck, T; Pickett-Heaps, J D



An internal deletion mutant of a myosin heavy chain in Caenorhabditis elegans.  


Unc-54 I is the structural gene for a myosin heavy chain present in a major fraction of the total myosin of Caenorhabditis elegans. The allele e675, which possesses a normal amount of myosin but fails to assemble thick filaments, has been shown previously to contain a novel heavy chain of molecular weight 2 X 10(5), shorter by 10(4) than the wild-type (N2) unc-54 gene product. The structural alteration of the E675 heavy chain is an internal deletion of 10(4) molecular weight near the COOH terminus of the molecule. This has been determined by mapping the partial digestion products of heavy chain fragments labeled specifically at their NH2 termini. PMID:271956

MacLeod, A R; Waterston, R H; Brenner, S



Regulation of somatic myosin activity by Protein Phosphatase 1? controls Drosophila oocyte polarization  

PubMed Central

The Drosophila body axes are established in the oocyte during oogenesis. Oocyte polarization is initiated by Gurken, which signals from the germline through the epidermal growth factor receptor (Egfr) to the posterior follicle cells (PFCs). In response the PFCs generate an unidentified polarizing signal that regulates oocyte polarity. We have identified a loss-of-function mutation of flapwing, which encodes the catalytic subunit of Protein Phosphatase 1? (PP1?) that disrupts oocyte polarization. We show that PP1?, by regulating myosin activity, controls the generation of the polarizing signal. Excessive myosin activity in the PFCs causes oocyte mispolarization and defective Notch signaling and endocytosis in the PFCs. The integrated activation of JAK/STAT and Egfr signaling results in the sensitivity of PFCs to defective Notch. Interestingly, our results also demonstrate a role of PP1? in generating the polarizing signal independently of Notch, indicating a direct involvement of somatic myosin activity in axis formation. PMID:21490061

Sun, Yi; Yan, Yan; Denef, Natalie; Schüpbach, Trudi



Myosin Binding Protein-C: A Regulator of Actomyosin Interaction in Striated Muscle  

PubMed Central

Myosin-Binding protein-C (MyBP-C) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges, via direct interactions with both thick myosin and thin actin filaments. Three distinct MyBP-C isoforms have been characterized; cardiac, slow skeletal, and fast skeletal. Numerous mutations in the gene for cardiac MyBP-C (cMyBP-C) have been associated with familial hypertrophic cardiomyopathy (FHC) and have led to increased interest in the regulation and roles of the cardiac isoform. This review will summarize our current knowledge on MyBP-C and its role in modulating contractility, focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles. PMID:22028592

Ackermann, Maegen A.; Kontrogianni-Konstantopoulos, Aikaterini



Electron Microscopic Observation and Biochemical Properties of Carp Myosin B during Frozen Storage at -8°C with Cryoprotectants  

NASA Astrophysics Data System (ADS)

The cryoprotective effect of sorbitol (0.5M) and monosodium glutamate (0.3M) was examined on the freeze denaturation of carp myosin B filaments by observing the morphological changes in electron microscopy. Myosin B in the presence of 0.1 or 0.6M KCl was stored at -8°C that was higer temperature than the eutectic point of KCl and provided the concentrated KCl solution for causing the filamentous structure to deform. In the case of frozen storage in 0.1M KCl, the deformation of myosin B filaments was protected with both cryoprotectants. In the case of 0.6M KCl with monosodium glutamate, the deformation of the filaments was prevented. However, the granular matters deformed from myosin B were observed to some extent in 0.6M KCl with sorbitol. Morphological changes of the filaments in the electron microscopy agreed with the changes in biochemical properties of myosin B.

Inoue, Norio; Oguni, Moritoshi; Yamamoto, Mika; Shinano, Haruo


A bioinformatic and computational study of myosin phosphatase subunit diversity.  


Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. PMID:24898838

Dippold, Rachael P; Fisher, Steven A



Design principles governing the motility of myosin V.  


The molecular motor myosin V (MyoV) exhibits a wide repertoire of pathways during the stepping process, which is intimately connected to its biological function. The best understood of these is the hand-over-hand stepping by a swinging lever arm movement toward the plus end of actin filaments. Single-molecule experiments have also shown that the motor "foot stomps," with one hand detaching and rebinding to the same site, and back-steps under sufficient load. The complete taxonomy of MyoV's load-dependent stepping pathways, and the extent to which these are constrained by motor structure and mechanochemistry, are not understood. Using a polymer model, we develop an analytical theory to describe the minimal physical properties that govern motor dynamics. We solve the first-passage problem of the head reaching the target-binding site, investigating the competing effects of backward load, strain in the leading head biasing 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. We derive a stall force formula, determined by lever arm compliance and chemical cycle rates. By exploring the MyoV design space, we predict that it 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. PMID:24101499

Hinczewski, Michael; Tehver, Riina; Thirumalai, D



Functional dissection of myosin binding protein C phosphorylation  

PubMed Central

Cardiac myosin binding protein C (cMyBP-C) phosphorylation is differentially regulated in the normal heart and during disease development. Our objective was to examine in detail three phosphorylatable sites (Ser-273, Ser-282, and Ser-302) present in the protein’s cardiac-specific sequences, as these residues are differentially and reversibly phosphorylated during normal and abnormal cardiac function. Three transgenic lines were generated: DAA, which expressed cMyBP-C containing Asp-273, Ala-282, Ala302, in which a charged amino acid was placed at residue 273 and the remaining two sites rendered nonphosphorylatable by substituting alanines for the two serines; AAD containing Ala-273, Ala-282, Asp-302), in which aspartate was placed at residue 302 and the remaining two sites rendered nonphosphorylatable; and SDS containing Ser-273, Asp-282, Ser-302. These mice were compared to mice constructed previously along similar lines: wild type, in which normal cMyBP-C is transgenically expressed, AllP?, in which alanines were substituted and ADA mice as well. DAA and AAD mice showed pathology that was more severe than cMyBP-C nulls. DAA and AAD animals exhibited left ventricular chamber dilation, interstitial fibrosis, irregular cardiac rhythm and sudden cardiac death. Our results define the effects of the sites’ post-translational modifications on cMyBP-C functionality and together, give a comprehensive picture of the potential consequences of site-specific phosphorylation. Ser-282 is a key residue in controlling S2 interaction with the thick and thin filaments. The new DAA and AAD constructs show that phosphorylation at one site in the absence of the ability to phosphorylate the other sites, depending upon the particular residues involved, can lead to severe cardiac remodeling and dysfunction. PMID:24001940

Gupta, Manish K.; Gulick, James; James, Jeanne; Osinska, Hanna; Lorenz, John N.; Robbins, Jeffrey



Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin  

PubMed Central

Background Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. Methods To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Results Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). Conclusions This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. General Significance This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved. PMID:23747303

Roman, Horia Nicolae; Zitouni, Nedjma B.; Kachmar, Linda; Ijpma, Gijs; Hilbert, Lennart; Matusovskiy, Oleg; Benedetti, Andrea; Sobieszek, Apolinary; Lauzon, Anne-Marie



Structural changes in myosin motors and filaments during relaxation of skeletal muscle  

PubMed Central

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 ?m, 4°C were investigated by X-ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin-attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer-line reflections associated with the quasi-helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half-time of ca 60 ms. Recovery of the equatorial X-ray intensities was largely complete during chaotic relaxation, but the other X-ray signals recovered more slowly than force. PMID:19651765

Brunello, E; Fusi, L; Reconditi, M; Linari, M; Bianco, P; Panine, P; Narayanan, T; Piazzesi, G; Lombardi, V; Irving, M



Plasma membrane proton-ATPase of a turtle bladder epithelial cell line.  


Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme. PMID:2858486

Lubansky, H J; Arruda, J A



Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity.  


We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations. PMID:22459703

Kloster, Antonina; Olsen, Lars Folke



Circadian rhythmicity and photic plasticity of myosin gene transcription in fast skeletal muscle of Atlantic cod (Gadus morhua).  


The circadian rhythm is a fundamental adaptive mechanism to the daily environmental changes experienced by many organisms, including fish. Myosins constitute a large family of contractile proteins that are essential functional components of skeletal muscle. They are known to display thermal plasticity but the influence of light on myosin expression remains to be investigated in fish. In the present study, we have examined the circadian rhythmicity and photoperiodic plasticity of myosin gene transcription in Atlantic cod (Gadus morhua) fast skeletal muscle. In silico mining of the Atlantic cod genome resulted in the identification of 76 myosins representing different classes, many of which were hitherto uncharacterized. Among the 23 fast skeletal muscle myosin genes, myh_tc, myh_n1, myh_n4, myo18a_2, and myo18b_2 displayed circadian rhythmic expression and contained several circadian-related transcription factor binding sites (Creb, Mef2 and E-box motifs) within their putative promoter regions. Also, the circadian expression of these 5 myosins strongly correlated with the transcription pattern of clock genes in fast skeletal muscle. Under ex vivo conditions, myosin transcript levels lost their circadian rhythmicity. Nonetheless, different photoperiod regimes influenced the mRNA levels of myh_n4, myo18a_2 and myo18b_2 in fast skeletal muscle explants. Photoperiod manipulation in Atlantic cod juveniles revealed that continuous light significantly elevated mRNA levels of several myosins in fast skeletal muscle when compared to natural photoperiod. The daily rhythmicity observed in some fast skeletal muscle myosin genes suggests that they may be under circadian clock regulation. In addition, the influence of photoperiod on their expression implies that myosins may be involved in the photic plasticity of muscle growth observed in Atlantic cod. PMID:24856374

Lazado, Carlo C; Nagasawa, Kazue; Babiak, Igor; Kumaratunga, Hiruni P S; Fernandes, Jorge M O



V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells  

PubMed Central

The vacuolar (H+)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing. PMID:20702583

Jansen, Eric J. R.; Hafmans, Theo G. M.



Structural mechanism of the ATP-induced dissociation of rigor myosin from actin  

PubMed Central

Myosin is a true nanomachine, which produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle. The principle underlying each step is that structural changes in separate regions of the protein must be mechanically coupled. The step in which myosin dissociates from tightly bound actin (the rigor state) is triggered by the 30 ? distant binding of ATP. Large conformational differences between the crystal structures make it difficult to perceive the coupling mechanism. Energetically accessible transition pathways computed at atomic detail reveal a simple coupling mechanism for the reciprocal binding of ATP and actin. PMID:21518908

Kühner, Sebastian; Fischer, Stefan



And the dead shall rise: Actin and myosin return to the spindle  

PubMed Central

The spindle directs chromosome partitioning in eukaryotes and, for the last three decades, has been considered primarily a structure based on microtubules, microtubule motors, and other microtubule binding proteins. However, a surprisingly large body of both old and new studies suggests roles for actin filaments (F-actin) and myosins (F-actin-based motor proteins) in spindle assembly and function. Here we review these data, and conclude that in several cases the evidence for F-actin and myosins participation in spindle function is very strong, and in the situations where it is less strong, there is nevertheless enough evidence to warrant further investigation. PMID:21920311

Sandquist, Joshua C.; Kita, Angela M.; Bement, William M.



Comparison of the body wall myosin heavy chain sequences from Onchocerca volvulus and Brugia malayi.  


The complete coding sequence of Onchocerca volvulus myosin heavy chain has been determined from a series of overlapping cDNAs. The protein sequences from the 2 filarids, one responsible for subcutaneous filariasis, the other for lymphatic filariasis, show 92% identity, and are 1957 amino acids long. Each protein sequence is also equally related, with 75% identity, to MHC-B, the protein encoded by the unc-54 gene of the free-living nematode C.elegans. Such analysis is useful in phylogenetic studies among nematodes, as well as in structure-function relationships among myosin isolates. PMID:1741012

Werner, C; Rajan, T V



Novel Inhibitors of E. coli RecA ATPase Activity.  


The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents. PMID:20648224

Sexton, Jonathan Z; Wigle, Tim J; He, Qingping; Hughes, Mark A; Smith, Ginger R; Singleton, Scott F; Williams, Alfred L; Yeh, Li-An



AAA+ ATPases: achieving diversity of function with conserved machinery.  


AAA+ adenosine triphosphatases (ATPases) are molecular machines that perform a wide variety of cellular functions. For instance, they can act in vesicle transport, organelle assembly, membrane dynamics and protein unfolding. In most cases, the ATPase domains of these proteins assemble into active ring-shaped hexamers. As AAA+ proteins have a common structure, a central issue is determining how they use conserved mechanistic principles to accomplish specific biological actions. Here, we review the features and motifs that partially define AAA+ domains, describe the cellular activities mediated by selected AAA+ proteins and discuss the recent work, suggesting that various AAA+ machines with very different activities employ a common core mechanism. The importance of this mechanism to human health is demonstrated by the number of genetic diseases caused by mutant AAA+ proteins. PMID:17897320

White, Susan Roehl; Lauring, Brett



High-resolution 31P nuclear magnetic resonance study of Chlamydia trachomatis: induction of ATPase activity in elementary bodies.  

PubMed Central

ATPase activity of elementary bodies (EBs) of Chlamydia trachomatis was investigated by using high-resolution 31P nuclear magnetic resonance spectroscopy. ATPase activity was detected in EBs of C. trachomatis serovars A, B, and L2 after treatment with the reducing agents 2-mercaptoethanol and glutathione. ATPase activity was oligomycin sensitive and magnesium ion dependent. EBs heated at 60 degrees C for 10 min or pretreated with Triton X-100 before exposure to 2-mercaptoethanol did not exhibit ATPase activity. Monoclonal antibody to the major outer membrane protein abrogated ATPase activity of EBs, whereas monoclonal antibody to chlamydial lipopolysaccharide only marginally reduced the level of ATPase activity. These findings suggest that EBs possess intrinsic ATPase activity and that cysteine-rich outer membrane proteins of EBs are important in the regulation of ATPase activity. The major outer membrane protein may be the major route through which ATP accesses ATPase. Images PMID:2530175

Peeling, R W; Peeling, J; Brunham, R C



Molecular cloning of the plant plasma membrane H + ATPase  

Microsoft Academic Search

Summary  Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This\\u000a gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H+-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed\\u000a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

T. K. Surowy; M. R. Sussman



Mechanically driven ATP synthesis by F1ATPase  

Microsoft Academic Search

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed

Hiroyasu Itoh; Akira Takahashi; Kengo Adachi; Hiroyuki Noji; Ryohei Yasuda; Masasuke Yoshida; Kazuhiko Kinosita



The Role of the Plasma Membrane H+-ATPase in Plant–Microbe Interactions  

PubMed Central

Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant–pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility. PMID:21300757

Elmore, James Mitch; Coaker, Gitta



Influence of intramembrane electric charge on Na,K-ATPase.  


Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K(+)-, ADP-, and Na(+)-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na(+)-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB- had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl- by other anions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field. PMID:7876184

Klodos, I; Fedosova, N U; Plesner, L



V-ATPase as an effective therapeutic target for sarcomas  

SciTech Connect

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

Perut, Francesca, E-mail: [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)



The oligomeric state of the active Vps4 AAA ATPase.  


The cellular ESCRT (endosomal sorting complexes required for transport) pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including the human immunodeficiency virus. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type Saccharomyces cerevisiae and Sulfolobus solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size-exclusion chromatography and equilibrium analytical ultracentrifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p-Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double-ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits. PMID:24161953

Monroe, Nicole; Han, Han; Gonciarz, Malgorzata D; Eckert, Debra M; Karren, Mary Anne; Whitby, Frank G; Sundquist, Wesley I; Hill, Christopher P



GLUT1CBP(TIP2/GIPC1) Interactions with GLUT1 and Myosin VI: Evidence Supporting an Adapter Function for GLUT1CBPV?  

PubMed Central

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures. PMID:15975910

Reed, Brent C.; Cefalu, Christopher; Bellaire, Bryan H.; Cardelli, James A.; Louis, Thomas; Salamon, Joanna; Bloecher, Mari Anne; Bunn, Robert C.



Immunolocalization of the sarcolemmal Ca2+\\/Mg2+ ecto-ATPase (myoglein) in rat myocardium  

Microsoft Academic Search

Cardiac plasma membrane Ca2+\\/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+\\/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+\\/Mg2+ ecto-ATPase; no cross reaction was observed towards other

Subburaj Kannan; Vijayan Elimban; Robert R. Fandrich; Elissavet Kardami; Naranjan S. Dhalla



Further studies on the adaptation of fish myofibrillar ATPases to different cell temperatures  

Microsoft Academic Search

Summary  Previous studies on fish Mg2+Ca2+ activated myofibrillar ATPases have been extended to species inhabiting diverse thermal environments. Cold adapted ATPases\\u000a have considerably higher catalytic centred activities at low temperatures than warm adapted ATPases. Differences in cell temperature\\u000a have also lead to evolutionary modifications in thermodynamic activation parameters. The free energies (?G2+), enthalpies (?H2+) and entropies (?S2+) of activation of the

I. A. Johnston; N. J. Walesby; W. Davison; G. Goldspink



Abscisic acid suppresses hypocotyl elongation by dephosphorylating plasma membrane H(+)-ATPase in Arabidopsis thaliana.  


Plasma membrane H(+)-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of the penultimate threonine of H(+)-ATPase. However, regulation of the H(+)-ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by the H(+)-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H(+)-ATPase isoform in etiolated seedlings. Application of the phytohormone ABA to etiolated seedlings suppressed hypocotyl elongation within 30 min at the half-inhibitory concentration (4.2 µM), and induced dephosphorylation of the penultimate threonine of H(+)-ATPase without affecting the amount of H(+)-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H(+)-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H(+)-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate threonine of H(+)-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H(+)-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through, at least in part, dephosphorylation of H(+)-ATPase in etiolated seedlings. PMID:24492258

Hayashi, Yuki; Takahashi, Koji; Inoue, Shin-Ichiro; Kinoshita, Toshinori



X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns.  


Fruit fly (Drosophila melanogaster) is one of the most useful animal models to study the causes and effects of hereditary diseases because of its rich genetic resources. It is especially suitable for studying myopathies caused by myosin mutations, because specific mutations can be induced to the flight muscle-specific myosin isoform, while leaving other isoforms intact. Here we describe an X-ray-diffraction-based method to evaluate the structural effects of mutations in contractile proteins in Drosophila indirect flight muscle. Specifically, we describe the effect of the headless myosin mutation, Mhc (10) -Y97, in which the motor domain of the myosin head is deleted, on the X-ray diffraction pattern. The loss of general integrity of the filament lattice is evident from the pattern. A striking observation, however, is the prominent meridional reflection at d = 14.5 nm, a hallmark for the regularity of the myosin-containing thick filament. This reflection has long been considered to arise mainly from the myosin head, but taking the 6th actin layer line reflection as an internal control, the 14.5-nm reflection is even stronger than that of wild-type muscle. We confirmed these results via electron microscopy, wherein image analysis revealed structures with a similar periodicity. These observations have major implications on the interpretation of myosin-based reflections. PMID:25400584

Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto; Suggs, Jennifer A; Bernstein, Sanford I



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

PubMed Central

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

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



X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns  

PubMed Central

Fruit fly (Drosophila melanogaster) is one of the most useful animal models to study the causes and effects of hereditary diseases because of its rich genetic resources. It is especially suitable for studying myopathies caused by myosin mutations, because specific mutations can be induced to the flight muscle-specific myosin isoform, while leaving other isoforms intact. Here we describe an X-ray-diffraction-based method to evaluate the structural effects of mutations in contractile proteins in Drosophila indirect flight muscle. Specifically, we describe the effect of the headless myosin mutation, Mhc10-Y97, in which the motor domain of the myosin head is deleted, on the X-ray diffraction pattern. The loss of general integrity of the filament lattice is evident from the pattern. A striking observation, however, is the prominent meridional reflection at d = 14.5 nm, a hallmark for the regularity of the myosin-containing thick filament. This reflection has long been considered to arise mainly from the myosin head, but taking the 6th actin layer line reflection as an internal control, the 14.5-nm reflection is even stronger than that of wild-type muscle. We confirmed these results via electron microscopy, wherein image analysis revealed structures with a similar periodicity. These observations have major implications on the interpretation of myosin-based reflections. PMID:25400584

Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto; Suggs, Jennifer A.; Bernstein, Sanford I.



Motor-motor interactions in ensembles of muscle myosin: using theory to connect single molecule to ensemble measurements  

NASA Astrophysics Data System (ADS)

Interactions between the proteins actin and myosin drive muscle contraction. Properties of a single myosin interacting with an actin filament are largely known, but a trillion myosins work together in muscle. We are interested in how single-molecule properties relate to ensemble function. Myosin's reaction rates depend on force, so ensemble models keep track of both molecular state and force on each molecule. These models make subtle predictions, e.g. that myosin, when part of an ensemble, moves actin faster than when isolated. This acceleration arises because forces between molecules speed reaction kinetics. Experiments support this prediction and allow parameter estimates. A model based on this analysis describes experiments from single molecule to ensemble. In vivo, actin is regulated by proteins that, when present, cause the binding of one myosin to speed the binding of its neighbors; binding becomes cooperative. Although such interactions preclude the mean field approximation, a set of linear ODEs describes these ensembles under simplified experimental conditions. In these experiments cooperativity is strong, with the binding of one molecule affecting ten neighbors on either side. We progress toward a description of myosin ensembles under physiological conditions.

Walcott, Sam



Structure and Interactions of Myosin-binding Protein C Domain C0  

PubMed Central

Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction. PMID:21297165

Ratti, Joyce; Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark




E-print Network

) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting

Thomas, David D.


Predicting allosteric communication in myosin via a pathway of conserved residues.  


We present a computational method that predicts a pathway of residues that mediate protein allosteric communication. The pathway is predicted using only a combination of distance constraints between contiguous residues and evolutionary data. We applied this analysis to find pathways of conserved residues connecting the myosin ATP binding site to the lever arm. These pathway residues may mediate the allosteric communication that couples ATP hydrolysis to the lever arm recovery stroke. Having examined pre-stroke conformations of Dictyostelium, scallop, and chicken myosin II as well as Dictyostelium myosin I, we observed a conserved pathway traversing switch II and the relay helix, which is consistent with the understood need for allosteric communication in this conformation. We also examined post-rigor and rigor conformations across several myosin species. Although initial residues of these paths are more heterogeneous, all but one of these paths traverse a consistent set of relay helix residues to reach the beginning of the lever arm. We discuss our results in the context of structural elements and reported mutational experiments, which substantiate the significance of the pre-stroke pathways. Our method provides a simple, computationally efficient means of predicting a set of residues that mediate allosteric communication. We provide a refined, downloadable application and source code (on to share this tool with the wider community ( PMID:17900617

Tang, Susan; Liao, Jung-Chi; Dunn, Alexander R; Altman, Russ B; Spudich, James A; Schmidt, Jeanette P



Structural dynamics of the myosin relay helix by time-resolved EPR and FRET.  


We have used two complementary time-resolved spectroscopic techniques, dipolar electron-electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF(x)) or ADP.P(i) (ADP.AlF(4)) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF(4) was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition. PMID:19966224

Agafonov, Roman V; Negrashov, Igor V; Tkachev, Yaroslav V; Blakely, Sarah E; Titus, Margaret A; Thomas, David D; Nesmelov, Yuri E



Muscle activity and aging affect myosin structural distribution and force generation in rat fibers  

E-print Network

on the tibial branch of the ischiatic nerve. The protocol consisted of 5 sets of 6­10 maximal isometric-binding myosin (P 0.01). These data support the hypothesis that changes in muscle activity affect muscle strength mass, or muscle quality, is diminished in arm and leg muscles of elderly men and women compared

Thomas, David D.


Kinesin and Myosin-driven Steps of Vesicle Recruitment for Ca 2 1 -regulated Exocytosis  

Microsoft Academic Search

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exo- cytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca 2 1 -regulated exocytosis

Guo-Qiang Bi; Robert L. Morris; Guochun Liao; Janet M. Alderton; Jonathan M. Scholey; Richard A. Steinhardt


Contraction of myofibrils in the presence of antibodies to myosin subfragment 2.  

PubMed Central

In a muscle-based version of in vitro motility assays, the unloaded shortening velocity of rabbit skeletal myofibrils has been determined in the presence and absence of affinity-column-purified polyclonal antibodies directed against the subfragment-2 region of myosin. Contraction was initiated by photohydrolysis of caged ATP and the time dependence of shortening was monitored by an inverted microscope equipped with a video camera. Antibody-treated myofibrils undergo unloaded shortening in a fast phase with initial rates and half-times comparable to control (untreated) myofibrils, despite a marked reduction in the isometric force of skinned muscle fibers in the presence of the antibodies. In antibody-treated myofibrils, this process is followed by a much slower phase of contraction, terminating in elongated structures with well-defined sarcomere spacings (approximately 1 micron) in contrast to the supercontracted globular state of control myofibrils. These results suggest that although the unloaded shortening of myofibrils (and in vitro motility of actin filaments over immobilized myosin heads) can be powered by myosin heads, the subfragment-2 region as well as the myosin head contributes to force production in actively contracting muscle. Images PMID:2217176

Harrington, W F; Karr, T; Busa, W B; Lovell, S J



Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.  


Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase. PMID:25131196

Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo



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

ERIC Educational Resources Information Center

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

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



Opposite changes in myosin heavy chain composition of human masseter and biceps brachii muscles during aging  

Microsoft Academic Search

The myosin heavy chain (MyHC) content in functionally different parts of the human masseter muscle of six elderly and five young adult subjects (mean age 74 and 22 years, respectively) was determined, using gel electrophoresis. The MyHC composition of the old masseter was also studied by enzyme- and immunohistochemical methods and compared with previous data for young adults. For comparison,

M. Monemi; P.-O. Eriksson; F. Kadi; G. S. Butler-Browne; L.-E. Thornell



Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers  

E-print Network

. Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers (force/cross-sectional area) in skeletal muscle from aged an- imals results from structural changes-related decline in skeletal muscle force generation. spectroscopy; specific tension; force; weakness; aging

Thomas, David D.


Effect of Ca 2+ -independent myosin light chain kinase on different skinned smooth muscle fibers  

Microsoft Academic Search

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca2+-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca2+. While the Ca2+ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus

M. Gagelmann; U. Mrwa; S. Bostrom; J. C. Rüegg; D. Hartshorne