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1

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.  

PubMed Central

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+. Images

Chacko, S; Conti, M A; Adelstein, R S

1977-01-01

2

ATPase Activity of Myosin Correlated with Speed of Muscle Shortening  

PubMed Central

Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca++-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca++-activated myosin ATPase was the same as that of the speed of shortening, Q10 about 2. Higher Q10 values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q10 values close to 2 could be obtained for the Mg++-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.

Barany, Michael

1967-01-01

3

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

PubMed

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production. PMID:23589853

Muretta, Joseph M; Petersen, Karl J; Thomas, David D

2013-04-30

4

Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational changeq  

Microsoft Academic Search

We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin- activated ATPase activity of M5Full was significantly activated by Ca2þ, whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca2þ and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca2þ-induced conformational transition from 14S

Xiang-dong Li; Katsuhide Mabuchi; Reiko Ikebe; Mitsuo Ikebea

5

ATPase activity of myosin in hair bundles of the bullfrog's sacculus.  

PubMed Central

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor. Images FIGURE 1 FIGURE 3

Burlacu, S; Tap, W D; Lumpkin, E A; Hudspeth, A J

1997-01-01

6

The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase  

Microsoft Academic Search

The microvillus ll0-kD protein-calmodulin complex (designated ll0K-CM) shares several proper- ties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, ll0K- CM is an ATPase with diagnostically myosin-like diva- lent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K + and EDTA (0.24 ~tmol Pi\\/mg per min) or in the presence of Ca

Karen A. Conzelman; Mark S. Mooseker

1987-01-01

7

[Role of light chains of myosin in the regulation of contraction of vertebrate striated muscles].  

PubMed

The data of the study on Ca2+ sensitivity of ATPase activity of myosin from vertebrate striated muscles in the presence of actin and the conditions of its manifestation and disappearance are presented. The role of Ca2+ sensitivity of actin-activated myosin ATPase in the regulation of contraction of vertebrate striated muscles is discussed. PMID:21033344

Malyshev, S L; Fre?dina, N A; Vikhliantsev, I M; Bledzhiants, D A; Karaduleva, E V; Shumilina, Iu V; Udal'tsov, S N; Marsagishvili, L G; Bobylev, A G; Podlubnaia, Z A

2010-01-01

8

Functional Analysis of Myosin Mutations That Cause Familial Hypertrophic Cardiomyopathy  

Microsoft Academic Search

We have studied the actin-activated ATPase activities of three mutations in the motor domain of the myosin heavy chain that cause familial hypertrophic cardiomyopathy. We placed these mutations in rodent ?-cardiac myosin to establish the relevance of using rodent systems for studying the biochemical mechanisms of the human disease. We also wished to determine whether the biochemical defects in these

Osha Roopnarine; Leslie A. Leinwand

1998-01-01

9

Polymorphic myosin as the common determinant of myofibrillar ATPase in different haemodynamic and thyroid states  

Microsoft Academic Search

Summary Chronic alterations in haemodynamic load or thyroid state affect the ATPase activity of myofibrils from the rat heart. In order to determine the limits of such an adaptational reaction, the Ca2+-dependent activation of myofibrils containing only myosin V1 or V3 was investigated. These limiting states were achieved by changes in the thyroid state, i.e., myosin V1 myofibrils were obtained

H. Rupp

1982-01-01

10

Human myosin Vc is a low duty ratio nonprocessive motor.  

PubMed

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. K(ATPase) of the actin-activated ATPase was 62 microm, which is much higher than that of myosin Va ( approximately 1 mum). The rate of ADP release from actomyosin Vc was 12.7 s(-1), which was 2 times greater than the entire ATPase cycle rate, 6.5 s(-1). P(i) burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms. PMID:18079121

Watanabe, Shinya; Watanabe, Tomonobu M; Sato, Osamu; Awata, Junya; Homma, Kazuaki; Umeki, Nobuhisa; Higuchi, Hideo; Ikebe, Reiko; Ikebe, Mitsuo

2008-04-18

11

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells  

PubMed Central

Summary Stimulation of Na+/K+-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na+/K+-ATPase recruitment to the plasma membrane by increasing the average speed of Na+/K+-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na+/K+-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na+/K+-ATPase-containing vesicles, as well as the Na+/K+-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na+/K+-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na+/K+-ATPase to the plasma membrane and thus increased activity.

Lecuona, Emilia; Minin, Alexander; Trejo, Humberto E.; Chen, Jiwang; Comellas, Alejandro P.; Sun, Haiying; Grillo, Doris; Nekrasova, Oxana E.; Welch, Lynn C.; Szleifer, Igal; Gelfand, Vladimir I.; Sznajder, Jacob I.

2009-01-01

12

Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor*  

PubMed Central

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. KATPase of the actin-activated ATPase was 62 ?m, which is much higher than that of myosin Va (?1 ?m). The rate of ADP release from actomyosin Vc was 12.7 s-1, which was 2 times greater than the entire ATPase cycle rate, 6.5 s-1. Pi burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.

Watanabe, Shinya; Watanabe, Tomonobu M.; Sato, Osamu; Awata, Junya; Homma, Kazuaki; Umeki, Nobuhisa; Higuchi, Hideo; Ikebe, Reiko; Ikebe, Mitsuo

2008-01-01

13

Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.  

PubMed

Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube. PMID:15122921

Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

2004-05-11

14

Orthovanadate and Orthophosphate Inhibit Muscle Force via Two Different Pathways of the Myosin ATPase Cycle  

PubMed Central

Measurements of the half-sarcomere stiffness during activation of skinned fibers from rabbit psoas (sarcomere length 2.5 ?m, temperature 12°C) indicate that addition of 0.1 mM orthovanadate (Vi) to the solution produces a drop to ?1/2 in number of force-generating myosin motors, proportional to the drop in steady isometric force (T0), an effect similar to that produced by the addition of 10 mM phosphate (Pi). However, in contrast to Pi, Vi does not change the rate of isometric force development. The depression of T0 in a series of activations in presence of Vi is consistent with an apparent second-order rate constant of ?1 × 103 M?1 s?1. The rate constant of T0 recovery in a series of activations after removal of Vi is 3.5 × 10?2 s?1. These results, together with the finding in the literature that the ATPase rate is reduced by Vi in proportion to isometric force, are reproduced with a kinetic model of the acto-myosin cross-bridge cycle where binding of Vi to the force-generating actomyosin-ADP state induces detachment from actin to form a stable myosin-ADP-Vi complex that is not able to complete the hydrolysis cycle and reenters the cycle only via reattachment to actin upon activation in Vi-free solution.

Caremani, Marco; Lehman, Steve; Lombardi, Vincenzo; Linari, Marco

2011-01-01

15

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).

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

2013-01-01

16

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

PubMed Central

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

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

2014-01-01

17

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

PubMed

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 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 MV 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 (J Gen Physiol 50:197-218, 1967). PMID:22930330

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

2012-12-01

18

Inhibiting Myosin-ATPase Reveals Dynamic Range of Mitochondrial Respiratory Control in Skeletal Muscle  

PubMed Central

Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (?20-300 ?M) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >?250 and ?80 ?M in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ?2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30°C but not 37°C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 ?M) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ?1560 ?M, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Perry, Christopher G.R.; Kane, Daniel A.; Lin, Chien-Te; Kozy, Rachel; Cathey, Brook L.; Lark, Daniel S.; Kane, Constance L.; Brophy, Patricia M.; Gavin, Timothy P; Anderson, Ethan J.; Neufer, P. Darrell

2013-01-01

19

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.

KAKEI, Toshihito; SUMIYOSHI, Hiroki; HIGASHI-FUJIME, Sugie

2012-01-01

20

Mechanism for coupling free energy in ATPase to the myosin active site.  

PubMed

Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBeFx-S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPA1F4-S1 and MgADPScFx-S1) falling in between in the order MgADPBeFx > MgADPA1F4 > MgADPScFx > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the gamma-phosphate analog metal from the oxygen connecting it to the beta-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X-ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the beta- and the gamma-phosphate, stresses the active site of S1 through protein-nucleotide contacts at the gamma-phosphate and nucleotide base. The stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure. PMID:9116016

Park, S; Ajtai, K; Burghardt, T P

1997-03-18

21

Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light.  

PubMed

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in vitro motility of class II myosins. In cells, it has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage. PMID:15641783

Sakamoto, Takeshi; Limouze, John; Combs, Christian A; Straight, Aaron F; Sellers, James R

2005-01-18

22

Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1.  

PubMed

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 ?m). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 ?m/s, respectively, from which the value for full-length ATM1 is calculated to be ?0.2 ?m/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ?90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

2014-05-01

23

Conformationally trapping the actin-binding cleft of myosin with a bifunctional spin label.  

PubMed

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

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

2013-02-01

24

Erythrocyte Protein 4.1 Binds and Regulates Myosin  

NASA Astrophysics Data System (ADS)

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

Pasternack, Gary R.; Racusen, Richard H.

1989-12-01

25

Human myosin Vc is a low duty ratio, nonprocessive molecular motor.  

PubMed

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration. PMID:18201966

Takagi, Yasuharu; Yang, Yi; Fujiwara, Ikuko; Jacobs, Damon; Cheney, Richard E; Sellers, James R; Kovács, Mihály

2008-03-28

26

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension.  

PubMed

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells. PMID:16186105

Henn, Arnon; De La Cruz, Enrique M

2005-11-25

27

Evidence that myosin does not contribute to force production in chromosome movement  

PubMed Central

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.

1982-01-01

28

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle  

Microsoft Academic Search

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response\\u000a to exposure to cold or to overfeeding. The identification of the “furnace” that is responsible for increased heat generation\\u000a in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX),\\u000a with a

Roger Cooke

2011-01-01

29

Opening of the myosin nucleotide triphosphate binding domain during the ATPase cycle.  

PubMed

A series of ATP analogs, in which moieties of various sizes have been added to the gamma-phosphorus of ATP, bind to the active site of myosin and to the actomyosin complex in myofibrils and in chemically skinned fibers. The affinity of the analogs for the active site shows only a slight dependence on the size of the added moiety. Addition of even our smallest group (CH3) reduced the binding affinity of ATPgamma-CH3 for S1 to 40 microM, a factor of 10(5) less than observed for ATP. Computer molecular docking of ATP-gammaCH3 into the myosin-ADP.BeF3 crystal structure of Dictyostelium discoideum indicates no steric interference to prevent binding. This suggests that the maintenance of charge at the gamma-phosphate is crucial for tight nucleotide binding. Addition of larger groups, (1) an EPR probe (ATP-gammaSL) or (2) ADP (i.e., P1, P5-diadenosine pentaphosphate, AP5A), reduced the affinity by only approximately a factor of 10 over that of ATP-gammaCH3. In the crystal structure of S1 complexed with nucleotides, the phosphates are buried within a protein structure called "the phosphate tube". Both the bulk of the modifying groups and the lack of dependence on the size of the group are incompatible with threading of the phosphates down the Pi-tube, showing that the tube must open. Similar domain movements have been found in other proteins including members of the G-protein superfamily, a family that has structural homologies to myosin. PMID:9315852

Pate, E; Naber, N; Matuska, M; Franks-Skiba, K; Cooke, R

1997-10-01

30

Action of peptide-B from bovine fibrinogen on ATPase activity and superprecipitation of myosin B.  

PubMed

Bovine peptide-B from fibrinogen was capable of accelerating the structural and enzymatic effects associated with superprecipitation of myosin B. The rate of superprecipitation coupled with the hydrolysis of ATP are increased during the structural transformation. In the concentration range from 10-8 to 10-4 M peptide-B, the rate of superprecipitation is increased 12-fold while the hydrolysis of ATP doubles and the time to reach the final extent of superprecipitation is decreased 68%. Under these same conditions, the hydrolysis of ATP by myosin A was unaffected. The concentrations of magnesium and calcium were between 10 and 20 muM, and no additional divalent metal ions were added to the system. Superprecipitation was treated as a model for muscle contraction to explain the in vivo studies of bovine peptide-B in which the peptide behaves as a vasopressor substance producing vascular vasoconstriction. A possible mechanism for the participation of bovine peptide-B in the model for muscle contraction, based on the polarizing interaction of the highly charged density of negativity of the peptide with the actomyosin complex, is presented. Furthermore, bovine peptide-B is speculated as participating in vasoconstriction via attachment to some smooth muscle receptor. PMID:123418

Osbahr, A J; Custodio, R

1975-02-01

31

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

PubMed

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

2014-01-01

32

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

PubMed Central

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

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

2014-01-01

33

[Seasonal changes in the isoform composition of the myosin heavy chains in skeletal muscles of hibernating ground squirrels Spermophilus undulatus].  

PubMed

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca(2+)-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isorfom decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca(2+)-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions. PMID:23272578

Lazareva, M V; Trapeznikova, K O; Vikhliantsev, I M; Bobylev, A G; Klimov, A A; Podlubnaia, Z A

2012-01-01

34

Calmodulin dissociation regulates brush border myosin I (110-kD- calmodulin) mechanochemical activity in vitro  

PubMed Central

110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin- dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium- inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium- sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.

1990-01-01

35

The kinase domain alters the kinetic properties of the myosin IIIA motor.  

PubMed

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells. PMID:18229949

Dosé, Andréa C; Ananthanarayanan, Shobana; Moore, Judy E; Corsa, Amoreena C; Burnside, Beth; Yengo, Christopher M

2008-02-26

36

Cardiac and skeletal muscle expression of mutant ?-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy.  

PubMed

Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the ?-myosin heavy chain (?-MHC) gene. In this study, the expression of the mutant ?-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of ?-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant ?-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant ?-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein. PMID:22213221

Di Domenico, Marina; Casadonte, Rita; Ricci, Pietroantonio; Santini, Mario; Frati, Giacomo; Rizzo, Antonietta; Carratelli, Caterina Romano; Lamberti, Monica; Parrotta, Elvira; Quaresima, Barbara; Faniello, Concetta M; Costanzo, Francesco; Cuda, Giovanni

2012-10-01

37

Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle.  

PubMed Central

We studied papillary muscle mechanics and energetics, myosin phenotype, and ATPase activities in left ventricles from rats bearing a growth hormone (GH)--secreting tumor. 18 wk after tumor induction, animals exhibited a dramatic increase in body weight (+101% vs. controls) but no change in the ventricular weight/body weight ratio. The maximum isometric force of papillary muscles normalized per cross-sectional area rose markedly (+42%, P less than 0.05 vs. controls), whereas the maximum unloaded shortening velocity did not change. This was observed despite a marked isomyosin shift towards V3 (32 +/- 5% vs. 8 +/- 2% in controls, P less than 0.001). Increased curvature of the force-velocity relationship (+64%, P less than 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium- and actin-activated myosin ATPase activities assayed on quickly frozen left ventricular sections were similar in tumor-bearing rats and in controls. After alkaline preincubation, these activities only decreased in tumor-bearing rats, demonstrating that V3 enzymatic sites were involved in total ATPase activity. These data demonstrate that chronic GH hypersecretion in the rat leads to a unique pattern of myocardial adaptation which allows the muscle to improve its contractile performance and economy simultaneously, thanks to myosin phenoconversion and an increase in the number of active enzymatic sites. Images

Timsit, J; Riou, B; Bertherat, J; Wisnewsky, C; Kato, N S; Weisberg, A S; Lubetzki, J; Lecarpentier, Y; Winegrad, S; Mercadier, J J

1990-01-01

38

Cardiac contractile dysfunction in Lep \\/ Lep obesity is accompanied by NADPH oxidase activation, oxidative modification of sarco(endo)plasmic reticulum Ca 2+ ATPase and myosin heavy chain isozyme switch  

Microsoft Academic Search

Aims\\/hypothesis  Obesity is an independent risk factor for heart diseases but the underlying mechanism is not clear. This study examined cardiac contraction, oxidative stress, oxidative modification of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and the myosin heavy chain (MHC) isoform switch in obese mice.Methods  Mechanical properties were evaluated in ventricular myocytes from C57BL\\/6J lean and Lep\\/Lep obese mice (formerly known as ob\\/ob mice), including

S.-Y. Li; X. Yang; A. F. Ceylan-Isik; M. Du; N. Sreejayan; J. Ren

2006-01-01

39

Myosin types in human skeletal muscle fibers  

Microsoft Academic Search

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

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

1980-01-01

40

Calcium-Dependent Myosin from Insect Flight Muscles  

Microsoft Academic Search

SUMMARY Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin mole- cule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent.

WILLIAM LEHMAN; BELINDA BULLARD; KATHLEEN HAMMOND

1974-01-01

41

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

SciTech Connect

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

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

2013-11-29

42

Improvement of the yields of recombinant actin and myosin V-HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture.  

PubMed

Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V-HMM at late exponential phase (11.3 × 10(6) cells/ml) to that at early exponential phase (1.0 × 10(6) cells/ml). The relative yields of actin and myosin V-HMM were approximately equal at both phases (typically 200 mg of actin and 5 mg of myosin V-HMM per 10(10) cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V-HMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay. PMID:22990978

Ohki, Takashi; Mikhailenko, Sergey V; Arai, Tomomi; Ishii, Shuya; Ishiwata, Shin'ichi

2012-10-01

43

Sucrose increases the activation energy barrier for actin-myosin strong binding.  

PubMed

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin-myosin (A-M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37±3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucrose's effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V. PMID:24370736

Jackson, Del R; Webb, Milad; Stewart, Travis J; Phillips, Travis; Carter, Michael; Cremo, Christine R; Baker, Josh E

2014-06-15

44

Myosin chaperones?  

PubMed Central

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

Hellerschmied, Doris; Clausen, Tim

2014-01-01

45

Myosin V: regulation by calcium, calmodulin, and the tail domain  

Microsoft Academic Search

alcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due

Dimitry N. Krementsov; Elena B. Krementsova; Kathleen M. Trybus

2004-01-01

46

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis.  

PubMed

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function. PMID:9755051

DiSanto, M E; Wang, Z; Menon, C; Zheng, Y; Chacko, T; Hypolite, J; Broderick, G; Wein, A J; Chacko, S

1998-10-01

47

Influence of the Cardiac Myosin Hinge Region on Contractile Activity  

Microsoft Academic Search

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the

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

1991-01-01

48

A hearing loss-associated myo1c mutation (R156W) decreases the myosin duty ratio and force sensitivity.  

PubMed

myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch 1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c(3IQ)) and mutant myo1c (R156W-myo1c(3IQ)) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited >4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but the rate of actin gliding is substantially reduced at low surface densities of R156W-myo1c(3IQ). We used a frictional loading assay to measure the affect of resisting forces on the rate of actin gliding and found that R156W-myo1c(3IQ) is less force-sensitive than myo1c(3IQ). Taken together, these results indicate that myo1c with the R156W mutation has a lower duty ratio than the wild-type protein and motile properties that are less sensitive to resisting forces. PMID:21265502

Lin, Tianming; Greenberg, Michael J; Moore, Jeffrey R; Ostap, E Michael

2011-03-22

49

pH sensitivity of myosin adenosine triphosphatase and subtypes of myofibres in porcine muscle  

Microsoft Academic Search

Summary  Myofibres from pig were classified generally into two types based on their reaction for myosin ATPase. Type I had a strong reaction for acid-stable myosin ATPase and a weak or negative reaction for alkali-stable ATPase; type II had the reciprocal properties. Types SM, SS and MS were defined and considered to represent types intermediate between I and II; they possibly

A. Suzuki; R. G. Cassens

1980-01-01

50

Ventricular myosin modifies in vitro step-size when phosphorylated.  

PubMed

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8nm, and a rare 3nm displacement for ? cardiac myosin (?Mys). S15 phosphorylation in ?Mys is now shown to change step-size distribution by advancing the 8nm step frequency. After phosphorylation, the 8nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament. PMID:24726887

Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P

2014-07-01

51

Preparation and Characterization of Myosin Proteins.  

ERIC Educational Resources Information Center

Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

Caldwell, Elizabeth; Eftink, Maurice R.

1985-01-01

52

Thiol groups of gizzard myosin heavy chains  

SciTech Connect

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

Bailin, G.

1986-05-01

53

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

PubMed

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

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

2014-02-18

54

Modification of Interface between Regulatory and Essential Light Chains Hampers Phosphorylation-dependent Activation of Smooth Muscle Myosin*  

PubMed Central

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, Vmax, and KATPase of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca2+ binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218–8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.

Ni, Shaowei; Hong, Feng; Haldeman, Brian D.; Baker, Josh E.; Facemyer, Kevin C.; Cremo, Christine R.

2012-01-01

55

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.

Shaffer, Justin F.; Kier, William M.

2012-01-01

56

Degradation of myocardiac myosin and creatine kinase in rats given alkaline ionized water.  

PubMed

Recently, the authors have shown that marked necrosis and fibrosis of myocardium were observed in rats given alkaline ionized water (AKW). To clarify the cause of myocardial lesions, the activities of myosin ATPase, actomyosin ATPase and creatine kinase (CK) in myocardium of rats given AKW at 15 weeks-old were compared with those in myocardium of rats given tap water (TPW). Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardiac myosin and isoelectric focusing (IEF) of myocardiac CK were performed which revealed a distinct difference between AKW and TPW groups. The activities of myosin ATPase and actomyosin ATPase in the AKW group were higher than those in the TPW group, and these elevated activities were caused by the degradation of myosin in the AKW group judging from the SDS-PAGE pattern of myosin. On the other hand, the activity of CK in the AKW group was lower than that in the TPW group, and the IEF pattern of CK showed leakage of myocardiac CK. These results indicate that increases in actomyosin ATPase activity and myosin ATPase activity, plus the decrease in CK activity caused the disorder of coupled reaction in male rats given AKW at 15 weeks-old. It is concluded that this disorder of coupled reaction may cause marked myocardiac necrosis and fibrosis in rats given AKW. PMID:9524951

Watanabe, T; Kishikawa, Y

1998-02-01

57

Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts  

PubMed Central

Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: (i) binds to rabbit muscle actin and is dissociated from it by ATP, (ii) has an ATPase activity that is suppressed by Mg2+ in 0.6 M KCl and is activated by rabbit muscle actin in the presence of Mg2+ in 14 mM KCl, (iii) forms thin bipolar aggregates in 0.1 M KCl when viewed in the electron microscope, (iv) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function. Images

Adelstein, Robert S.; Conti, Mary Anne; Johnson, George S.; Pastan, Ira; Pollard, Thomas D.

1972-01-01

58

Physical and enzymatic properties of myosin from porcine brain.  

PubMed Central

Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58-60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum.

Hobbs, D S; Frederiksen, D W

1980-01-01

59

ELECTRON MICROSCOPE OBSERVATIONS ON MYOSIN FROM PHYSARUM POLYCEPHALUM  

PubMed Central

Myosin has been separated from Physarum polycephalum actomyosin in confirmation of the results of Hatano and Tazawa. In an intermediate step, myosin-enriched actomyosin has also been obtained. The mean yield of free myosin was 4.4 mg from 100 g of mold. It was obtained as water-clear solutions at µ = 0.055 with calcium ATPase activity of up to 0.5 µM Pi/min per mg. Negatively stained preparations were examined by electron microscopy. Physarum myosin in 0.5 M KCl interacted with actin from rabbit skeletal muscle to form polarized arrowhead complexes similar to but less regular than those of natural actomyosin from muscle or myosin-enriched Physarum actomyosin. The Physarum myosin-enriched actomyosin at low ionic strength displayed evidence of head-to-tail and tail-to-tail aggregation attributable to the myosin component. Yet Physarum myosin alone did not produce detectable filaments at µ = 0.055 at pH 7, 6.5, or 5.8, nor when dialyzed against 0.01 M ammonium acetate, nor when the dielectric constant of the medium was reduced. However, aggregation approaching the extent of ‘thick filaments’ up to 0.3 µ long was found in some preparations of myosin-enriched actomyosin put into solutions containing adenosine triphosphate. Myosin alone in such solutions did not form filaments. The results are compatible with the idea that head-to-tail aggregations are favored by actin-myosin interactions in Physarum, possibly due to alignment of the extended or tail portions of this myosin molecule.

Nachmias, Vivianne T.

1972-01-01

60

Mutations in the ?-myosin rod cause myosin storage myopathy via multiple mechanisms  

PubMed Central

Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, ?-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the ?-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule.

Armel, Thomas Z.; Leinwand, Leslie A.

2009-01-01

61

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

PubMed

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

Liu, Jiao; Puolanne, Eero; Ertbjerg, Per

2014-10-01

62

Amino acid composition of dynein and comparison with myosin.  

PubMed

A comparison is made between dynein [flagellar ATPase; EC 3.6.1.3], purified from sea urchin sperm flagella, and muscle myosin. The amino acid composition of dynein was found to be statistically different from that of myosin. The same was true of their tryptic fragments retaining ATPase activity, i.e., Fragment A of dynein and heavy meromyosin. At low ionic strength, no superprecipitation took place when ATP was added to a mixture of dynein and actin, and stimulation of the Mg2+-ATPase activity of dynein remained below 50% even when a one-hundred-fold excess of actin was present. No viscosity drop was caused by adding ATP to a solution containing dynein and actin. Anti-myosin antiserum did not react with dynein, while anti-Fragment A antiserum formed no precipit-n line against myosin. Furthermore, the amount of dynein that combined with F-actin was less than one-fifth of the amount of dynein that fully combined with microtubules. These results are consistent with the dissimilarity in enzymatic and other physiocochemical properties of these two proteins. PMID:129468

Ogawa, K; Okuno, M; Mohri, H

1975-10-01

63

Characterization and localization of myosin in the brush border of intestinal epithelial cells  

PubMed Central

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 ?mol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 ?mol P(i)/mg-min), and low with Mg++ (0.01 ?mol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.

Mooseker, MS; Pollard, TD

1978-01-01

64

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.

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

2013-01-01

65

Hybrids of Physarum myosin light chains and desensitized scallop myofibrils  

PubMed Central

The two light chains of Physarum myosin have been purified in a 1:1 ratio with a yield of 0.5-1 mg/100 g of plasmodium and a purity of 40- 70%; the major contaminant is a 42,000-dalton protein. The 17,700 Mr Physarum myosin light chain (PhLC1) binds to scallop myofibrils, providing the regulatory light chains (ScRLC) have been removed. The 16,500 Mr light (PhLC2) does not bind to scallop myofibrils. The calcium control of scallop myosin ATPase is lost by the removal of one of the two ScRLC's and restored equally well by the binding of either PhLC1 or rabbit skeletal myosin light chains. When both ScRLC's are removed, replacement by two plasmodial light chains does not restore calcium control as platelet or scallop light chains do. Purified plasmodial actomyosin does not bind calcium in 10(-6) M free calcium, 1 mM MgCl2. No tropomyosin was isolated from Physarum by standard methods. Because the Physarum myosin light chains can substitute only partially for light chains from myosin linked systems, because calcium does not bind to the actomyosin, and because tropomyosin is apparently absent, the regulation of plasmodial actomyosin by micromolar Ca++ may involve other mechanisms, possibly phosphorylation.

1981-01-01

66

Myosin-I moves actin filaments on a phospholipid substrate: implications for membrane targeting  

PubMed Central

Acanthamoeba myosin-I bound to substrates of nitrocellulose or planar lipid membranes on glass moved actin filaments at an average velocity of 0.2 micron/s. This movement required ATP and phosphorylation of the myosin-I heavy chain. We prepared planar lipid membranes on a glass support by passive fusion of lipid vesicles (Brian, A. A., and H. M. McConnell. 1984. Proc. Natl. Acad. Sci. USA. 81:6159-6163) composed of phosphatidylcholine and containing 0-40% phosphatidylserine. The mass of lipid that bound to the glass was the same for membranes of 2 and 20% phosphatidylserine in phosphatidylcholine and was sufficient to form a single bilayer. Myosin-I moved actin filaments on planar membranes of 5-40% but not 0-2% phosphatidylserine. At the low concentrations of phosphatidylserine, actin filaments tended to detach suggesting that less myosin-I was bound. We used the cooperative activation of Acanthamoeba myosin-I ATPase by low concentrations of actin to assess the association of phospholipids with myosin-I. Under conditions where activity depends on the binding of actin to the tail of myosin-I (Albanesi, J. P., H. Fujisaki, and E. D. Korn. 1985. J. Biol. Chem. 260:11174-11179), phospholipid vesicles with 5-40% phosphatidylserine inhibited ATPase activity. The motility and ATPase results demonstrate a specific interaction of the tail of myosin-I with physiological concentrations of phosphatidylserine. This interaction is sufficient to support motility and may provide a mechanism to target myosin-I to biological membranes.

1992-01-01

67

Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site.  

PubMed Central

We have designed an "antipeptide" capable of firmly and specifically interacting with a preselected stretch of myosin S-1 heavy chain. Covalent attachment of this antipeptide to its target stretch, residues 633-642, does not affect the intrinsic ATPase activities of the protein but significantly reduces the actin-binding capabilities of the myosin head. Images

Chaussepied, P; Morales, M F

1988-01-01

68

Unregulated smooth-muscle myosin in human intestinal neoplasia  

Microsoft Academic Search

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc\\/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP.

Pia Alhopuro; Denis Phichith; Sari Tuupanen; Heli Sammalkorpi; Miranda Nybondas; Juha Saharinen; James P. Robinson; Zhaohui Yang; Li-Qiong Chen; Torben Orntoft; Jukka-Pekka Mecklin; Heikki Järvinen; Charis Eng; Gabriela Moeslein; Darryl Shibata; Richard S. Houlston; Anneke Lucassen; Ian P. M. Tomlinson; Virpi Launonen; Ari Ristimäki; Diego Arango; Auli Karhu; H. Lee Sweeney; Lauri A. Aaltonen

2008-01-01

69

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

Microsoft Academic Search

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

K. Kawasaki; Y. Saeki; Y. Ohnuki

1997-01-01

70

Conventional myosins – unconventional functions  

Microsoft Academic Search

While the discovery of unconventional myosins raised expectations that their actions were responsible for most aspects of\\u000a actin-based cell motility, few anticipated the wide range of cellular functions that would remain the purview of conventional\\u000a two-headed myosins. The three nonsarcomeric, cellular myosins—M2A, M2B and M2C—participate in diverse roles including, but\\u000a not limited to: neuronal dynamics, axon guidance and synaptic transmission;

Peter D. Chantler; Steven R. Wylie; Caroline P. Wheeler-Jones; Imelda M. McGonnell

2010-01-01

71

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

PubMed Central

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

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

2011-01-01

72

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain  

PubMed Central

We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting “SH1” thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.

Agafonov, Roman V.; Nesmelov, Yuri E.; Titus, Margaret A.; Thomas, David D.

2008-01-01

73

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain.  

PubMed

We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting "SH1" thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account. PMID:18765799

Agafonov, Roman V; Nesmelov, Yuri E; Titus, Margaret A; Thomas, David D

2008-09-01

74

Coevolution of head, neck, and tail domains of myosin heavy chains  

PubMed Central

Myosins, a large family of actin-based motors, have one or two heavy chains with one or more light chains associated with each heavy chain. The heavy chains have a (generally) N-terminal head domain with an ATPase and actin-binding site, followed by a neck domain to which the light chains bind, and a C-terminal tail domain through which the heavy chains self-associate and/or bind the myosin to its cargo. Approximately 140 members of the myosin superfamily have been grouped into 17 classes based on the sequences of their head domains. I now show that a phylogenetic tree based on the sequences of the combined neck and tail domains groups 144 myosins, with a few exceptions, into the same 17 classes. For the nine myosin classes that have multiple members, phylogenetic trees based on the head domain or the combined neck/tail domains are either identical or very similar. For class II myosins, very similar phylogenetic trees are obtained for the head, neck, and tail domains of 47 heavy chains and for 29 essential light chains and 19 regulatory light chains. These data strongly suggest that the head, neck, and tail domains of all myosin heavy chains, and light chains at least of class II myosins, have coevolved and are likely to be functionally interdependent, consistent with biochemical evidence showing that regulated actin-dependent MgATPase activity of Dictyostelium myosin II requires isoform specific interactions between the heavy chain head and tail and light chains.

Korn, Edward D.

2000-01-01

75

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

PubMed Central

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

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

2013-01-01

76

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

PubMed

Two functions are proposed for the conserved family of UCS proteins: helping to fold myosin motor proteins and stimulating the motor function of folded myosins. We examined both functions in yeast. The fission yeast UCS protein (Rng3p) concentrates in nodes containing myosin-II (Myo2) and other proteins that condense into the cytokinetic contractile ring. Both the N-terminal (central) and C-terminal (UCS) domains of Rng3p can concentrate independently in contractile rings, but only full-length Rng3p supports contractile ring function in vivo. The presence of Rng3p in ATPase assays doubles the apparent affinity (K(ATPase)) of both native Myo2 and recombinant heads of Myo2 for actin filaments. Rng3p promotes gliding of actin filaments by full-length Myo2 molecules, but not Myo2 heads alone. Myo2 isolated from mutant strains defective for Rng3p function is soluble and supports actin filament gliding. In budding yeast the single UCS protein (She4p) acts on both myosin-I isoforms (Myo3p and Myo5p) and one of two myosin-V isoforms (Myo4p). Myo5p turns over approximately 10 times faster in she4Delta cells than wild-type cells, reducing the level of Myo5p in cells 10-fold and in cortical actin patches approximately 4-fold. Nevertheless, Myo5p isolated from she4Delta cells has wild-type ATPase and motility activities. Thus, a fraction of this yeast myosin can fold de novo in the absence of UCS proteins, but UCS proteins promote myosin stability and interactions with actin. PMID:18523008

Lord, Matthew; Sladewski, Thomas E; Pollard, Thomas D

2008-06-10

77

The kinetics of bivalent metal ion dissociation from myosin subfragments.  

PubMed Central

Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.

Bennett, A J; Bagshaw, C R

1986-01-01

78

Catalytic strategy used by the myosin motor to hydrolyze ATP.  

PubMed

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?O4 (2-) 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

2014-07-22

79

Potent inhibition of arterial smooth muscle tonic contractions by the selective myosin II inhibitor, blebbistatin.  

PubMed

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC(50) approximately 5 microM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited beta-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC(50) approximately 20 microM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II. PMID:17132816

Eddinger, Thomas J; Meer, Daniel P; Miner, Amy S; Meehl, Joel; Rovner, Arthur S; Ratz, Paul H

2007-02-01

80

Potent stimulation of myofilament force and ATPase activity of skeletal muscle by eudistomin M, a novel Ca(++)-sensitizing agent from a Caribbean tunicate.  

PubMed

In the course of our survey of biologically active compounds from natural sources, eudistomins were isolated from a Caribbean tunicate Eudistoma olivaceum. In the present experiments, eudistomin M (Eud-M, > 10(-5) M) caused a concentration-dependent increase in the contractile response of skinned fibers from guinea pig skeletal psoas muscles to Ca++. The superprecipitation and ATPase activity of myosin B from fast skeletal muscles of rabbit back and leg were potentiated by this compound (> 10(-5) M) in a concentration-dependent manner. In skinned fibers, superprecipitation and the ATPase activity of myosin B, Eud-M shifted the concentration-response curve for Ca++ to the upper direction. Ca(++)-, K(+)-EDTA- or Mg(++)-ATPase was not affected by Eud-M. This compound had no effect on the ATPase activity of actomyosin reconstituted from actin and myosin in the presence or absence of troponin. However, the ATPase activity of actin-myosin-troponin-tropomyosin reconstituted system was increased significantly by Eud-M. These results suggest that Eud-M increases the Ca++ sensitivity of the contractile apparatus in skeletal muscles at least partially mediated through troponin-tropomyosin system and thus enhances the ATPase activity of myosin B and the contractile force of myofilament. PMID:9580615

Ohizumi, Y; Matsunaga, K; Nakatani, K; Kobayashi, J

1998-05-01

81

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

PubMed Central

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

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

2004-01-01

82

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

PubMed

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

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

2011-06-01

83

Acto-myosin based response to stiffness and rigidity sensing  

PubMed Central

Cells sense the rigidity of their environment and respond to it. Most studies have been focused on the role of adhesion complexes in rigidity sensing. In particular, it has been clearly shown that proteins of the adhesion complexes were stretch-sensitive and could thus trigger mechano-chemical signaling in response to applied forces. In order to understand how this local mechano-sensitivity could be coordinated at the cell scale, we have recently carried out single cell traction force measurements on springs of varying stiffness. We found that contractility at the cell scale (force, speed of contraction, mechanical power) was indeed adapted to external stiffness and reflected ATPase activity of non-muscle myosin II and acto-myosin response to load. Here we suggest a scenario of rigidity sensing where local adhesions sensitivity to force could be coordinated by adaptation of the acto-myosin dependent cortical tension at the global cell scale. Such a scenario could explain how spreading and migration are oriented by the rigidity of the cell environment.

Fouchard, Jonathan; Mitrossilis, Demosthene

2011-01-01

84

Phosphorylation of Human Platelet Myosin  

PubMed Central

A preparation extracted from human blood platelets, which incorporates 32P from ?-labeled AT32P into one of the two light chains of platelet myosin and platelet myosin head is described. This phosphorylation, which appears to be due to an endogenous kinase, is specific for the myosin light chain in that no other protein extracted in 0.6 M KCl-15 mM Tris·HCl (pH 7.5) is phosphorylated. The phosphorylated light chain, which has been purified by gel filtration, releases the covalently bound phosphate after incubation in alkali and not after incubation in acid. Images

Adelstein, Robert S.; Conti, Mary Anne; Anderson, William

1973-01-01

85

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

NASA Technical Reports Server (NTRS)

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

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

1981-01-01

86

A STUDY OF THE ADENOSINE TRIPHOSPHATASE ACTIVITY OF MYOSIN AND ACTOMYOSIN  

PubMed Central

1. An experimental study was made on the adenosine triphosphatase action of crystalline myosin and actomyosin preparations under different conditions. 2. No enzymatic activity was found in the absence of salts. Activation was given by KCl and CaCl2, whereas MgCl2 in the presence of other ions inhibited. 3. The effect of pH is complex. In stabilizing buffers or at low temperature, there are two optima (pH 6.2 to 6.5 and pH 9.2) provided Ca is present. Without Ca only the acid optimum is found. The highest activities are reached in glycine buffer at pH 9.2 in the presence of Ca. 4. The study of the Mg-Ca antagonism revealed that the inhibition due to Mg is fully developed with Mg:Ca ratios less than 1, the inhibition usually exceeding 90 per cent. 5. It is shown that in the muscle the myosin-ATPase is most probably also subjected to the inhibitory action of the Mg ions. 6. From data in the literature it is calculated that the liberation of inorganic phosphate during muscular activity takes place at a rate of at least 0.200 mg. P per mg. myosin per minute. 7. From the results of the present study it is found that the myosin in the muscle can liberate inorganic phosphate from ATP at a rate of at most 0.003 mg. P per mg. myosin per minute. 8. It is concluded therefore that myosin-ATPase cannot be responsible for the liberation of the main part of the phosphate in contracting muscle, and therefore cannot have the rôle in muscular metabolism ascribed to it in recent hypotheses and discussions.

Mommaerts, W. F. H. M.; Seraidarian, Krikor

1947-01-01

87

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.

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

2013-01-01

88

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish.  

PubMed

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site. PMID:11281274

Gauvry, L; Ennion, S; Ettelaie, C; Goldspink, G

2000-12-01

89

Smooth muscle myosin: regulation and properties.  

PubMed Central

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.

Somlyo, Avril V; Khromov, Alexander S; Webb, Martin R; Ferenczi, Michael A; Trentham, David R; He, Zhen-He; Sheng, Sitong; Shao, Zhifeng; Somlyo, Andrew P

2004-01-01

90

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function.  

PubMed

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

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

2014-05-01

91

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

PubMed Central

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

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

2012-01-01

92

[ATPase activity and the contractile capacity of the muscle tissue in chick embryos during development].  

PubMed

The value of ATPase activity of the myofibril preparations and the value and duration of actomyosin superprecipitation were estimated for different muscles during the chick embryonic development. The ATPase level increases during embryogenesis 4.5-fold, in the leg muscle this change takes place distinctly earlier than in the leg muscle. The value and rate of actomyosin superprecipitation also markedly increase, to a lesser extent for m. soleus than for m. pectoralis. It is suggested that these changes and differences are mainly due to the delay in synthesis of certain types of the embryonic myosin light chains. PMID:2966919

Kalamkarova, M B; Kofman, E B; Ale?nikova, K S; Szoor, A; Kalapos, I

1988-01-01

93

Conformational changes of myosin by gamma irradiation  

NASA Astrophysics Data System (ADS)

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

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

2000-05-01

94

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

PubMed

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

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

2014-05-01

95

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation.  

PubMed

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin. PMID:24361582

Taylor, Kenneth A; Feig, Michael; Brooks, Charles L; Fagnant, Patricia M; Lowey, Susan; Trybus, Kathleen M

2014-03-01

96

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.  

PubMed

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum. PMID:1378447

Espindola, F S; Espreafico, E M; Coelho, M V; Martins, A R; Costa, F R; Mooseker, M S; Larson, R E

1992-07-01

97

A New Role for Myosin II in Vesicle Fission  

PubMed Central

An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ? 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.

Cabeza, Jose M.; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

2014-01-01

98

Structural kinetics of myosin by transient time-resolved FRET.  

PubMed

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

2011-02-01

99

Structural kinetics of myosin by transient time-resolved FRET  

PubMed Central

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)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET 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)2FRET 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.

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

2011-01-01

100

Unregulated smooth-muscle myosin in human intestinal neoplasia.  

PubMed

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia. PMID:18391202

Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Järvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S; Lucassen, Anneke; Tomlinson, Ian P M; Launonen, Virpi; Ristimäki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H Lee; Aaltonen, Lauri A

2008-04-01

101

Unregulated smooth-muscle myosin in human intestinal neoplasia  

PubMed Central

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz–Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P.; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Jarvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S.; Lucassen, Anneke; Tomlinson, Ian P. M.; Launonen, Virpi; Ristimaki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H. Lee; Aaltonen, Lauri A.

2008-01-01

102

Mn2+-Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance  

PubMed Central

Pulsed electron paramagnetic resonance at the microwave Ka band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn2+ at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron–nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn·nucleotide complexes. Therefore, to directly detect binding of the Mn·nucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron–electron double resonance to detect spin probe–manganese dipolar interaction. We found that under substoichiometric conditions, both Mn·AMPPNP and Mn·ADP·AlF4 form a complex with myosin, and Mn·ADP does not form such a complex. This correlates well with the biological dissociation of Mg·ADP from myosin after the hydrolysis of ATP. The analysis of 31P ENDOR spectra reveals that in Mn·AMPPNP, Mn·ATP, and Mn·ADP at myosin or in solution, the nucleotide is coordinated to Mn2+ by two phosphate groups, whereas in Mn·ADP·AlF4, only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn·ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal–protein binding when such binding does not provide reliable spectroscopic signatures.

Astashkin, Andrei V.; Nesmelov, Yuri E.

2013-01-01

103

Genomic structure of the human unconventional myosin VI gene  

Microsoft Academic Search

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin

Nadav Ahituv; Tama Sobe; Nahid G. Robertson; Cynthia C. Morton; R. Thomas Taggart; Karen B. Avraham

2000-01-01

104

Porcine myosin-VI: characterization of a new mammalian unconventional myosin  

PubMed Central

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.

1994-01-01

105

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.

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

2013-01-01

106

Carbonylation of myosin heavy chains in rat heart during diabetes.  

PubMed

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague-Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+dP/dt) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca(2+)- and Mg(2+)-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-alpha to MHC-beta ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-alpha and MHC-beta. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca(2+)-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +dP/dt induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes. PMID:20359464

Shao, Chun-Hong; Rozanski, George J; Nagai, Ryoji; Stockdale, Frank E; Patel, Kaushik P; Wang, Mu; Singh, Jaipaul; Mayhan, William G; Bidasee, Keshore R

2010-07-15

107

Reversible inactivation of myosin subfragment 1 activity by mechanical immobilization.  

PubMed Central

The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, epsilonADP binds aggregated S1 with reduced affinity, and the bound epsilonADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1 x MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.

Highsmith, S; Duignan, K; Franks-Skiba, K; Polosukhina, K; Cooke, R

1998-01-01

108

Myosin lever arm directs collective motion on cellular actin network.  

PubMed

The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions. PMID:24591646

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

2014-03-18

109

Switches in fish myosin genes induced by environment temperature in muscle of the carp.  

PubMed

Fish are cold blooded animals and their muscle function is expected to be greatly affected by environmental temperature. Species that live in the Antarctic ocean have evolved a different contractile system to fish that live in the tropical waters. In the case of Antarctic fish they have a higher specific myofibrillar ATPase activity but 'the trade off' seems to be a lower thermal stability. They are thus capable of a greater muscle power output at low temperatures but the lower thermal stability means they are restricted to living at temperatures below +4 degrees C. Some species, however, experience a wide range of seasonal variations in temperature. We found that these species adapt by changing their myofibrillar apparatus so that they have a higher specific ATPase which physiological studies indicate is due to a different type of myosin crossbridge for low temperature swimming. This is reversible and they develop a contractile system with a greater thermal stability and a commensurate loss of ATPase activity when their environment warms up again. There were several possibilities by which this may be achieved including expression of different isoform genes or the post- translational processing of existing proteins. To elucidate the mechanism we made a carp genomic library and screened this for myosin heavy chain gene using mammalian cDNA sequences under moderate stringency conditions. The clones were restriction mapped which resulted in 28 non overlapping sequences. This indicated that the carp had a reasonably large family of myosin heavy chain genes that is about twice the size of that in mammals. Rather fortuitously the first sequence to be identified was from the gene that is predominantly expressed in white muscle at warm temperatures. This was done by extracting the RNA from red and white muscle of fish acclimated to different 25 degrees C, 18 degrees C or 8 degrees C and carrying out Northern analysis using the gene fragment as the probe. The time course for the expression of this gene when carp maintained at a low temperature were acclimated to a warm temperature was slightly in advance of the change in myofibrillar ATPase which suggested that this strategy for adaptation is regulated at the transcriptional level. Hence these species of fish can adapt to seasonal changes in temperature by expressing different myosin heavy chain isoform genes and rebuilding their myofibrils for either warm or cold temperature swimming. At the present time we are characterising the 5' regulatory (promoter) sequence of this gene to see how a temperature switch may operate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1341032

Goldspink, G; Turay, L; Hansen, E; Ennion, S; Gerlach, G

1992-01-01

110

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

PubMed Central

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

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

2014-01-01

111

New insights into myosin evolution and classification.  

PubMed

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites. PMID:16505385

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

2006-03-01

112

Myosin II Dynamics during Embryo Morphogenesis  

NASA Astrophysics Data System (ADS)

During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

Kasza, Karen

2013-03-01

113

Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain  

PubMed Central

We have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, in order to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final anisotropy cooperatively and to a similar extent, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of saturating ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin.

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

2013-01-01

114

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

PubMed

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

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

2014-01-01

115

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

PubMed Central

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

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

2014-01-01

116

Homology model of nonmuscle myosin heavy chain IIA and binding mode analysis with its inhibitor blebbistatin.  

PubMed

Nonmuscle myosin heavy chain IIA (NMMHC IIA, gene code: MYH9) plays a critical role in physiological and pathological functions. A homology model of NMMHC IIA was constructed based on the crystal structure of smooth muscle myosin II. Blebbistatin, a myosin II ATPase inhibitor, had been found to bind to NMMHC IIA with Leu228 as the important amino acid residue and van der Waals contacts as the main force of the interaction. The final complex demonstrated that the destruction of the salt bridge occurred between the Arg204 and Glu427 residues when blebbistatin was present. Molecular dynamic simulation of the complex showed that the binding affinity of blebbistatin to NMMHC IIA was strongly sensitive to the nucleotide binding region and actin binding region. The disturbance of the two regions increased the enhancement of the binding cavity with blebbistatin and resulted in a slightly more expanded conformation in the nucleotide binding region and actin binding region. A combined pharmacophore- and docking-based virtual screening was performed to identify several saponins as potential inhibitors for NMMHC IIA. These findings introduce new insights on the binding mode of blebbistatin and NMMHC IIA and novel leading compounds from natural products for NMMHC IIA-related diseases. PMID:23315199

Lv, Yanni; Lu, Shuai; Lu, Tao; Kou, Junping; Yu, Boyang

2013-04-01

117

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

PubMed Central

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

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

2010-01-01

118

Thermal denaturation and aggregation of myosin subfragment 1 isoforms with different essential light chains.  

PubMed

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

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

2010-01-01

119

Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics  

PubMed Central

Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.

Wylie, Steven R.

2008-01-01

120

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance.  

PubMed

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin. PMID:18339764

Nesmelov, Yuri E; Agafonov, Roman V; Burr, Adam R; Weber, Ralph T; Thomas, David D

2008-07-01

121

Structure and Dynamics of the Force-Generating Domain of Myosin Probed by Multifrequency Electron Paramagnetic Resonance  

PubMed Central

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.Vi-bound biochemical state of myosin, which presumably mimics the S1.ADP.Pi state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.Vi biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

Nesmelov, Yuri E.; Agafonov, Roman V.; Burr, Adam R.; Weber, Ralph T.; Thomas, David D.

2008-01-01

122

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

PubMed Central

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

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

2012-01-01

123

Expression of myosin VIIA during mouse embryogenesis  

Microsoft Academic Search

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

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

1997-01-01

124

Motoring around the plant cell: insights from plant myosins.  

PubMed

Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negative myosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins. PMID:20491672

Sparkes, Imogen A

2010-06-01

125

Myosin-VIIb, a novel unconventional myosin, is a constituent of microvilli in transporting epithelia.  

PubMed

Mouse myosin-VIIb, a novel unconventional myosin, was cloned from the inner ear and kidney. The human myosin-VIIb (HGMW-approved symbol MYO7B) sequence and exon structure were then deduced from a human BAC clone. The mouse gene was mapped to chromosome 18, approximately 0.5 cM proximal to D18Mit12. The human gene location at 2q21.1 was deduced from the map location of the BAC and confirmed by fluorescence in situ hybridization. Myosin-VIIb has a conserved myosin head domain, five IQ domains, two MyTH4 domains coupled to two FERM domains, and an SH3 domain. A phylogenetic analysis based on the MyTH4 domains suggests that the coupled MyTH and FERM domains were duplicated in myosin evolution before separation into different classes. Myosin-VIIb is expressed primarily in kidney and intestine, as shown by Northern and immunoblot analyses. An antibody to myosin-VIIb labeled proximal tubule cells of the kidney and enterocytes of the intestine, specifically the distal tips of apical microvilli on these transporting epithelial cells. PMID:11401444

Chen, Z Y; Hasson, T; Zhang, D S; Schwender, B J; Derfler, B H; Mooseker, M S; Corey, D P

2001-03-15

126

Myosin Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells  

Microsoft Academic Search

Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin

James H. Sabry; Sheri L. Moores; Shannon Ryan; Ji-Hong Zang; James A. Spudich

1997-01-01

127

Human myosin-Vc is a novel class V myosin expressed in epithelial cells.  

PubMed

Class V myosins are one of the most ancient and widely distributed groups of the myosin superfamily and are hypothesized to function as motors for actin-dependent organelle transport. We report the discovery and initial characterization of a novel member of this family, human myosin-Vc (Myo5c). The Myo5c protein sequence shares approximately 50% overall identity with the two other class V myosins in vertebrates, myosin-Va (Myo5a) and myosin-Vb (Myo5b). Systematic analysis of the mRNA and protein distribution of these myosins indicates that Myo5a is most abundant in brain, whereas Myo5b and Myo5c are expressed chiefly in non-neuronal tissues. Myo5c is particularly abundant in epithelial and glandular tissues including pancreas, prostate, mammary, stomach, colon and lung. Immunolocalization in colon and exocrine pancreas indicates that Myo5c is expressed chiefly in epithelial cells. A dominant negative approach using a GFP-Myo5c tail construct in HeLa cells reveals that the Myo5c tail selectively colocalizes with and perturbs a membrane compartment containing the transferrin receptor and rab8. Transferrin also accumulates in this compartment, suggesting that Myo5c is involved in transferrin trafficking. As a class V myosin of epithelial cells, Myo5c is likely to power actin-based membrane trafficking in many physiologically crucial tissues of the human body. PMID:11870218

Rodriguez, Olga C; Cheney, Richard E

2002-03-01

128

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

NASA Technical Reports Server (NTRS)

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

Sharma, Prasanta; Cheung, Herbert C.

1989-01-01

129

Dictyostelium myosin II G680V suppressors exhibit overlapping spectra of biochemical phenotypes including facilitated phosphate release.  

PubMed Central

We have biochemically characterized 13 intragenic suppressors of the G680V mutation of Dictyostelium myosin II. In the absence of the G680V mutation, the suppressors result in a number of deviant behaviors, most commonly an increase in the basal (actin-independent) ATPase of the motor. This phenotype is complementary to that of the G680V mutant and supports our proposal that the latter impairs phosphate release. Different subsets of the mutants also suffer from poor ATPase enhancement by 1 mg/ml actin, failure to release from actin in the presence of ATPgammaS (or ADP and salt), and excessive release from actin in the presence of ADP. The patterns of suppressor behaviors suggest that, in general, they are facilitating P(i)-releasing state(s) of the motor, but that different individual suppressors may secondarily perturb other states or actions of the motor.

Wu, Y; Nejad, M; Patterson, B

1999-01-01

130

Duplex RNA activated ATPases (DRAs)  

PubMed Central

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

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

2013-01-01

131

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

PubMed

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

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

2014-06-15

132

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis  

PubMed Central

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3–activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

2013-01-01

133

Prolonged Ca2+ and force transients in myosin RLC transgenic mouse fibers expressing malignant and benign FHC mutations.  

PubMed

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force. PMID:16837010

Wang, Ying; Xu, Yuanyuan; Kerrick, W Glenn L; Wang, Yingcai; Guzman, Georgianna; Diaz-Perez, Zoraida; Szczesna-Cordary, Danuta

2006-08-11

134

Unconventional Myosins in Inner-Ear Sensory Epithelia  

PubMed Central

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-I? is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-I? is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-I?, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-I? probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

Hasson, Tama; Gillespie, Peter G.; Garcia, Jesus A.; MacDonald, Richard B.; Zhao, Yi-dong; Yee, Ann G.; Mooseker, Mark S.; Corey, David P.

1997-01-01

135

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

PubMed

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

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

2014-09-01

136

Biological motors: Conventional and Unconventional Myosins  

NASA Astrophysics Data System (ADS)

Molecular motors are smart, soft machines that regulate their dynamics and energy consumption for efficient tuning to their cell-biological role and mechanics of their cargo. The efficiency is derived partly from harnessing the chaotic thermal fluctuations nano-scale machines experience, rather than struggle against them. Reciprocal coupling between the enzymatic chemistry, structural changes, and mechanical steps is expected from the thermodynamics of an energy-transducing nano-machine. Strong evidence for this bidirectional coupling exists for muscle (conventional) myosin and unconventional myosins. The structural dynamics of myosin leading to translocation along actin are detectable by Optical Trap Mechanical Nanometry (OTNM), Single-Molecule Fluorescence Polarization Microscopy (SMFPM), Fluorescence Imaging at One Nanometer Accuracy (FIONA) and various combinations of these methods. We are in an Acronym Rich Environment (ARE). Progress and puzzles make this a lively research area.

Goldman, Yale E.

2006-03-01

137

On archaebacterial ATPase from Halobacterium saccharovorum  

NASA Technical Reports Server (NTRS)

The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

1984-01-01

138

Class VI Unconventional Myosin is Required for Spermatogenesis in Drosophila  

PubMed Central

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

Hicks, Jennifer L.; Deng, Wu-Min; Rogat, Aaron D.; Miller, Kathryn G.; Bownes, Mary

1999-01-01

139

Rho Kinase, Myosin-II, and p42/44 MAPK Control Extracellular Matrix-mediated Apical Bile Canalicular Lumen Morphogenesis in HepG2 Cells  

PubMed Central

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.

Herrema, Hilde; Czajkowska, Dominika; Theard, Delphine; van der Wouden, Johanna M.; Kalicharan, Dharamdajal; Zolghadr, Behnam; Hoekstra, Dick

2006-01-01

140

Genetics Home Reference: Myosin storage myopathy  

MedlinePLUS

... heavy chain. This protein is found in heart (cardiac) muscle and in type I skeletal muscle fibers, one ... the muscles that the body uses for movement. Cardiac ?-myosin heavy chain is the major component of the thick filament in muscle cell structures called sarcomeres. Sarcomeres, which are made ...

141

Myosin Vb Is Associated with Plasma Membrane Recycling Systems  

PubMed Central

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Lapierre, Lynne A.; Kumar, Ravindra; Hales, Chadwick M.; Navarre, Jennifer; Bhartur, Sheela G.; Burnette, Jason O.; Provance, D. William; Mercer, John A.; Bahler, Martin; Goldenring, James R.

2001-01-01

142

Relationship of the membrane ATPase from Halobacterium saccharovorum to vacuolar ATPases  

NASA Technical Reports Server (NTRS)

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

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

1991-01-01

143

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases  

NASA Technical Reports Server (NTRS)

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

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

1991-01-01

144

Single-Molecule Adhesion Forces and Attachment Lifetimes of Myosin-I Phosphoinositide Interactions  

PubMed Central

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3–2.5 pN), with rates exceeding 360 s?1 at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.

Pyrpassopoulos, Serapion; Shuman, Henry; Ostap, E. Michael

2010-01-01

145

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.

1992-01-01

146

Cell Contact-dependent Regulation of Epithelial-Myofibroblast Transition via the Rho-Rho Kinase-Phospho-Myosin Pathway  

PubMed Central

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell–cell junctions is a prerequisite for transforming growth factor-?1 (TGF-?1)-induced transdifferentiation of kidney tubular cells into ?-smooth muscle actin (SMA)–expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca2+ removal–induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-?1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-?1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca2+-removal– ± TGF-?1–induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC–dependent nuclear accumulation of MRTF.

Fan, Lingzhi; Sebe, Attila; Peterfi, Zalan; Masszi, Andras; Thirone, Ana C.P.; Rotstein, Ori D.; Nakano, Hiroyasu; McCulloch, Christopher A.; Szaszi, Katalin; Mucsi, Istvan

2007-01-01

147

Phosphate release coupled to rotary motion of F1-ATPase  

PubMed Central

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

Okazaki, Kei-ichi; Hummer, Gerhard

2013-01-01

148

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

PubMed Central

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

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

2013-01-01

149

Dissociation of force from myofibrillar MgATPase and stiffness at short sarcomere lengths in rat and toad skeletal muscle.  

PubMed Central

1. Single fast-twitch fibres from the extensor digitorum longus muscle of the rat, Rattus norvegicus, and single twitch fibres from the iliofibularis muscle of the cane toad, Bufo marinus, were mechanically skinned and then used to measure maximally Ca2+-activated [( Ca2+] greater than 0.03 mmol l-1) isometric force production, myofibrillar MgATPase activity and fibre stiffness at different sarcomere lengths. MgATP hydrolysis was linked by an enzyme cascade to the oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) and was monitored by a microfluorimetric system. Fibre stiffness was measured from the amplitude of force oscillations generated by small sinusoidal length changes. 2. At sarcomere lengths which were optimal for isometric force production (around 2.7 microns for rat and 2.2 microns for toad fibres) the myofibrillar MgATPase activity (mean +/- S.E.M.) at 21-22 degrees C was found to be 3.80 +/- 0.53 molecules MgATP hydrolysed s-1 per myosin head for eight rat fibres and 6.35 +/- 0.77 s-1 per myosin head for four toad fibres. 3. At sarcomere lengths shorter than 2.7 microns in rat fibres and 2.2 microns in toad fibres, MgATPase and stiffness remained elevated and close to their respective values at 2.7 microns in rat fibres and 2.2 microns in toad fibres even when the isometric force decreased to near zero levels. 4. The dissociation at short sarcomere lengths of myofibrillar MgATPase activity and fibre stiffness from isometric force suggests that the cross-bridge cycle is not greatly affected by double actin filament overlap with the myosin filaments at short sarcomere lengths. Moreover, the results suggest that cross-bridges can be formed by myosin with actin filaments projecting from the nearest Z-line and from the Z-line in the other half of the sarcomere. 5. These results help to reconcile energetic and mechanical data obtained by others at short sarcomere lengths and can be explained within the framework of the sliding filament theory.

Stephenson, D G; Stewart, A W; Wilson, G J

1989-01-01

150

Roles for myosin Va in RNA transport and turnover.  

PubMed

Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA. PMID:23176491

McCaffrey, Mary W; Lindsay, Andrew J

2012-12-01

151

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.

Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bartoli, Fulvia; Salazar, Leiria; Zhao, Fa-Qing; Craig, Roger; Padron, Raul

2008-01-01

152

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

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

1993-01-01

153

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

PubMed

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

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

2012-08-15

154

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

NASA Astrophysics Data System (ADS)

The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

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

2013-03-01

155

Shared gene structures and clusters of mutually exclusive spliced exons within the metazoan muscle myosin heavy chain genes.  

PubMed

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

Kollmar, Martin; Hatje, Klas

2014-01-01

156

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.

Kollmar, Martin; Hatje, Klas

2014-01-01

157

Relation between myocardial function and expression of sarcoplasmic reticulum Ca(2+)-ATPase in failing and nonfailing human myocardium.  

PubMed

Expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase was shown to be reduced in failing human myocardium. The functional relevance of this finding, however, is not known. We investigated the relation between myocardial function and protein levels of SR Ca(2+)-ATPase in nonfailing human myocardium (8 muscle strips from 4 hearts) and in myocardium from end-stage failing hearts with dilated (10 muscle strips from 9 hearts) or ischemic (7 muscle strips from 5 hearts) cardiomyopathy. Myocardial function was evaluated by the force-frequency relation in isometrically contracting muscle strip preparations (37 degrees C, 30 to 180 min-1). In nonfailing myocardium, twitch tension rose with increasing rates of stimulation and was 76% higher at 120 min-1 compared with 30 min-1 (P < .02). In failing myocardium, there was no significant increase in average tension at stimulation rates above 30 min-1. At 120 min-1, twitch tension was decreased by 59% (P < .05) in dilated cardiomyopathy and 76% (P < .05) in ischemic cardiomyopathy compared with nonfailing myocardium. Protein levels of SR Ca(2+)-ATPase, normalized per total protein or per myosin, were reduced by 36% (P < .02) or 32% (P < .05), respectively, in failing compared with nonfailing myocardium. SR Ca(2+)-ATPase protein levels were closely related to SR Ca2+ uptake, measured in homogenates from the same hearts (r = .70, n = 16, and P < .005).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8062417

Hasenfuss, G; Reinecke, H; Studer, R; Meyer, M; Pieske, B; Holtz, J; Holubarsch, C; Posival, H; Just, H; Drexler, H

1994-09-01

158

The biosynthesis of plasmodial myosin during starvation of Physarum polycephalum  

PubMed Central

The actomyosin protein complex of Physarum polycephalum was prepared from vegetative and starved plasmodia. The yield of actomyosin per unit wet wt. was the same from both types of plasmodia. Myosin was resolved from the complex by gel filtration and purified by ion-exchange chromatography. The Ca2+-stimulated adenosine triphosphatase activities of myosin preparations from vegetative and starved plasmodia were not appreciably different. Synthesis of myosin de novo was shown to occur during the starvation phase of the life-cycle by the isolation of labelled myosin preparations from plasmodia starved in the presence of [2-14C]glycine. Fractionation of polyacrylamide gels after gel filtration of labelled myosin confirmed the presence of label in the adenosine triphosphatase-active myosin band. It is concluded that during starvation myosin synthesis continues although there is a net loss of approx. 50% of the total protein. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of Physarum myosin showed the presence of low-molecular-weight components of the molecule, similar to those of muscle myosins. The content and composition of the free amino acid pool of Physarum was measured at various time-intervals during the vegetative and starvation phases of the life-cycle.

White, F. Harry; Lascelles, June

1973-01-01

159

Myosin I contributes to the generation of resting cortical tension.  

PubMed Central

The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

Dai, J; Ting-Beall, H P; Hochmuth, R M; Sheetz, M P; Titus, M A

1999-01-01

160

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana  

PubMed Central

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

Krzeszowiec, Weronika

2007-01-01

161

Myosin light chain kinases and phosphatase in mitosis and cytokinesis  

PubMed Central

Summary At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.

Matsumura, Fumio; Yamakita, Yoshihiko; Yamashiro, Shigeko

2011-01-01

162

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle  

PubMed Central

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.

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

2011-01-01

163

Evolution of plant p-type ATPases.  

PubMed

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

2012-01-01

164

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

PubMed

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

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

2013-10-29

165

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

NASA Technical Reports Server (NTRS)

BACKGROUND: Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. RESULTS: Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. CONCLUSIONS: Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

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

2001-01-01

166

Actin-myosin viscoelastic ?ow in the keratocyte lamellipod  

Microsoft Academic Search

ABSTRACT The lamellipod, the locomotory region of migratory cells, is shaped by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow of the viscoelastic actin network. Recently, quantitative flow data was obtained, yet there is no detailed theory explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin

Boris Rubinstein; Maxime F. Fourniery

167

Role of a Novel Myosin Isoform in Prostate Cancer Metastasis.  

National Technical Information Service (NTIS)

The objective of the project for the reporting period was to generate a number prostate cancer cell lines that that are either myosin IC isoform A deficient or that constitutively express GFP-myosin IC isoform A. We used shRNA to generate isoform Anegativ...

W. A. Hofmann

2013-01-01

168

Molecular Characterization of Myosin Phosphatase in Endothelium  

PubMed Central

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 paper, our new data demonstrates 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.

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

2011-01-01

169

Neuromuscular Development and Regulation of Myosin Expression  

NASA Technical Reports Server (NTRS)

The proposed experiments were designed to determine whether the absence of gravity during embryogenesis influences the postnatal development of the neuromuscular system. Further, we examined the effects of reduced gravity on hindlimb muscles of the pregnant rats. Microgravity may have short and long-term effects on the development of muscle fiber type differentiation and force producing capabilities. Microgravity will reduce muscle fiber size and cause a shift in myosin heavy chain expression from slow to fast in hindlimb muscles of the adult pregnant rats.

Bodine, Sue

1997-01-01

170

Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation  

PubMed Central

Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin–myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin–myosin crossbridges. Actin–myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin–myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

Matsui, Tsubasa S.; Kaunas, Roland; Kanzaki, Makoto; Sato, Masaaki; Deguchi, Shinji

2011-01-01

171

Sodium, potassium-atpases in algae and oomycetes.  

PubMed

We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells. PMID:16167182

Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña

2005-08-01

172

Sarcoplasmic Reticulum Ca2+-ATPase Activity and Glycogen Content in Various Fiber Types after Intensive Exercise in Thoroughbred Horses  

PubMed Central

To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca2+-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO2max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. SR Ca2+-ATPase activity of the muscle and glycogen content of each muscle fiber type were determined with biochemical analysis and quantitative histochemical staining, respectively. As compared to the pre value, the glycogen content of each muscle fiber type was reduced by 15–27% at 1 min, 20 min, and 60 min after the exercise and recovered to the pre value at 24 hr after exercise test. These results indicate that 24 hr is enough time to recover glycogen content after short-term intensive exercise. The mean value of the SR Ca2+-ATPase activity showed a slight decrease (not significant) immediately after exercise, and complete recovery at 60 min after exercise. There were no significant relationship between the changes in glycogen content of each muscle fiber type and SR Ca2+-ATPase. Although further studies are needed, SR Ca2+-ATPase is not a useful parameter to detect muscle fitness, at least in Thoroughbred horses.

MINAMI, Yoshio; YAMANO, Seiko; KAWAI, Minako; HIRAGA, Atsushi; MIYATA, Hirofumi

2009-01-01

173

Localization of various ATPases in fresh and cryopreserved bovine spermatozoa  

Microsoft Academic Search

Different ATPases may control the various functional changes that spermatozoa undergo just prior to fertilization, with the enzyme's specific location within the cell reflecting its function. The activities of Mg2+-ATPase, Ca2+?Mg2+-ATPase, Na+?K+-ATPase and Ca2+-ATPase were determined for head plasma membranes (HPM) and sperm body membrane (SBM) from both fresh (n = 4) and cryopreserved bovine spermatozoa (n = 4) and

Yuyuan Zhao; Mary M. Buhr

1996-01-01

174

Roles for kinesin and myosin during cytokinesis.  

PubMed Central

Cytokinesis in higher plants involves the phragmoplast, a complex cytoplasmic structure that consists of microtubules (MTs), microfilaments (MFs) and membrane elements. Both MTs and MFs are essential for cell plate formation, although it is not clear which motor proteins are involved. Some candidate processes for motor proteins include transport of Golgi vesicles to the plane of the cell plate and the spatiotemporal organization of the cytoskeletal elements in order to achieve proper deposition and alignment of the cell plate. We have focused on the kinesin-like calmodulin binding protein (KCBP) and, more broadly, on myosins. Using an antibody that constitutively activates KCBP, we find that this MT motor, which is minus-end directed, contributes to the organization of the spindle and phragmoplast MTs. It does not participate in vesicle transport; rather, because of the orientation of the phragmoplast MTs, it is supposed that plus-end kinesins fill this role. Myosins, on the other hand, based on their inhibition with 2,3-butanedione monoxime and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), are associated with the process of post-mitotic spindle/phragmoplast alignment and with late lateral expansion of the cell plate. They are also not the principal motors involved in vesicle transport.

Hepler, Peter K; Valster, Aline; Molchan, Tasha; Vos, Jan W

2002-01-01

175

Myosin V from head to tail  

PubMed Central

Myosin V (myoV), a processive cargo transporter, has arguably been the most well-studied unconventional myosin of the past decade. Considerable structural information is available for the motor domain, the IQ motifs with bound calmodulin or light chains, and the cargo-binding globular tail, all of which have been crystallized. The repertoire of adapter proteins that link myoV to a particular cargo is becoming better understood, enabling cellular transport processes to be dissected. MyoV is processive, meaning that it takes many steps on actin filaments without dissociating. Its extended lever arm results in long 36 nm steps, making it ideal for single molecule studies of processive movement. In addition, electron microscopy revealed the structure of the inactive, folded conformation of myoV when it is not transporting cargo. This review provides a background on myoV, and highlights recent discoveries that show why myoV will continue to be an active focus of investigation.

Trybus, Kathleen M.

2008-01-01

176

Tripolyphosphate hydrolysis by bovine fast and slow myosin subfragment 1 isoforms  

PubMed Central

Polyphosphates are used in the meat industry to increase the water holding capacity of meat products. Tripolyphosphate (TPP) is a commonly used polyphosphate and it is metabolized into pyrophosphate and monophosphate in meat. The enzymes responsible for its metabolism have not been fully characterized. The motor domain of myosin (subfragment 1 or S1) is a likely candidate. The objectives of this study were to determine if bovine S1 hydrolyzes TPP, to characterize the TPPase activity of the fast (cutaneous trunci) and slow (masseter) isoforms, and to determine the influence of pH on S1 TPPase activity. S1 hydrolyzed TPP and in comparison with ATP as substrate, it hydrolyzed TPP 16 – 32% more slowly. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The Vmax was 0.94 and 5.0 nmole Pi/mg S1 protein/min while the Km was 0.38 and 0.90 mM TPP for slow and fast S1, respectively. Pyrophosphate was a strong inhibitor of TPPase activity with a Ki of 88 and 8.3 ?M PPi for fast and slow S1 isoforms, respectively. Both ATPase and TPPase activities were influenced by pH with the activity being higher at low pH for both fast and slow S1 isoforms. The activity at pH 5.4 was 1.5 to 4 fold higher than that at pH 7.6 for the different isoforms and substrates. These data show that myosin S1 readily hydrolyzes TPP and suggest that it is a major TPPase in meat.

Yamazaki, Marie; Shen, Qingwu W.; Swartz, Darl R.

2010-01-01

177

Auditory mechanotransduction in the absence of functional myosin-XVa  

PubMed Central

In hair cells of all vertebrates, a mechanosensory bundle is formed by stereocilia with precisely graded heights. Unconventional myosin-XVa is critical for formation of this bundle because it transports whirlin and perhaps other molecular components responsible for programmed elongation of stereocilia to the stereocilia tips. A tip of a stereocilium is the site of stereocilia growth and one of the proposed sites of mechano-electrical transduction. In adult shaker 2 mice, a mutation that disables the motor function of myosin-XVa results in profound deafness and abnormally short stereocilia that lack stereocilia links, an indispensable component of mechanotransduction machinery. Therefore, it was assumed that myosin-XVa is required for proper formation of the mechanotransduction apparatus. Here we show that in young postnatal shaker 2 mice, abnormally short stereocilia bundles of auditory hair cells have numerous stereocilia links and ‘wild type’ mechano-electrical transduction. We compared the mechanotransduction current in auditory hair cells of young normal-hearing littermates, myosin-XVa-deficient shaker 2 mice, and whirler mice that have similarly short stereocilia but intact myosin-XVa at the stereocilia tips. This comparison revealed that the absence of functional myosin-XVa does not disrupt adaptation of the mechanotransduction current during sustained bundle deflection. Thus, the hair cell mechanotransduction complex forms and functions independently from myosin-XVa-based hair bundle morphogenesis.

Stepanyan, Ruben; Belyantseva, Inna A; Griffith, Andrew J; Friedman, Thomas B; Frolenkov, Gregory I

2006-01-01

178

Auditory mechanotransduction in the absence of functional myosin-XVa.  

PubMed

In hair cells of all vertebrates, a mechanosensory bundle is formed by stereocilia with precisely graded heights. Unconventional myosin-XVa is critical for formation of this bundle because it transports whirlin and perhaps other molecular components responsible for programmed elongation of stereocilia to the stereocilia tips. A tip of a stereocilium is the site of stereocilia growth and one of the proposed sites of mechano-electrical transduction. In adult shaker 2 mice, a mutation that disables the motor function of myosin-XVa results in profound deafness and abnormally short stereocilia that lack stereocilia links, an indispensable component of mechanotransduction machinery. Therefore, it was assumed that myosin-XVa is required for proper formation of the mechanotransduction apparatus. Here we show that in young postnatal shaker 2 mice, abnormally short stereocilia bundles of auditory hair cells have numerous stereocilia links and 'wild type' mechano-electrical transduction. We compared the mechanotransduction current in auditory hair cells of young normal-hearing littermates, myosin-XVa-deficient shaker 2 mice, and whirler mice that have similarly short stereocilia but intact myosin-XVa at the stereocilia tips. This comparison revealed that the absence of functional myosin-XVa does not disrupt adaptation of the mechanotransduction current during sustained bundle deflection. Thus, the hair cell mechanotransduction complex forms and functions independently from myosin-XVa-based hair bundle morphogenesis. PMID:16973713

Stepanyan, Ruben; Belyantseva, Inna A; Griffith, Andrew J; Friedman, Thomas B; Frolenkov, Gregory I

2006-11-01

179

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.

Padilla-Benavides, Teresita; McCann, Courtney J.; Arguello, Jose M.

2013-01-01

180

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics  

PubMed Central

The ventricles of small mammals express mostly ?-myosin heavy chain (?-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ?10% ?-MHC on a predominately ?-MHC (slow isoform) background. In failing human ventricles, the amount of ?-MHC is dramatically reduced, leading to the hypothesis that even small amounts of ?-MHC on a predominately ?-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (fapp) and detachment (gapp) for myosins composed of 100% ?-MHC or ?-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% ?-MHC or 100% ?-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca2+ concentrations. The rate of ATP utilization was ?2.5-fold higher in preparations expressing 100% ?-MHC compared with those expressing only ?-MHC, whereas ktr was 2-fold faster in the ?-MHC myocardium. From these variables, we calculated fapp to be approximately threefold higher for ?-MHC than ?-MHC and gapp to be twofold higher in ?-MHC. Mathematical modeling of isometric twitches predicted that small increases in ?-MHC significantly increased the rate of force development. These results suggest that low-level expression of ?-MHC has significant effects on contraction kinetics.

Locher, Matthew R.; Razumova, Maria V.; Stelzer, Julian E.; Norman, Holly S.; Patel, Jitandrakumar R.; Moss, Richard L.

2009-01-01

181

Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides  

Microsoft Academic Search

BACKGROUND: Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. RESULTS: In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3

Peter G Gillespie; Janet L Cyr

2002-01-01

182

Androcam is a tissue-specific light chain for myosin VI in the Drosophila testis.  

PubMed

Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue. PMID:16790438

Frank, Deborah J; Martin, Stephen R; Gruender, Bridget N T; Lee, Yung-Sheng R; Simonette, Rebecca A; Bayley, Peter M; Miller, Kathryn G; Beckingham, Kathleen M

2006-08-25

183

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

SciTech Connect

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

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

2011-02-25

184

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

PubMed Central

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 endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F- actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.

1995-01-01

185

Actin filaments on myosin beds: The velocity distribution  

NASA Astrophysics Data System (ADS)

In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

1995-12-01

186

Cell adhesion: Ushering in a new understanding of myosin VII  

Microsoft Academic Search

The myosin VII motor protein has recently been found to have a role in cell adhesion. This new function is conserved from amoebae to man and provides an explanation for deafness in Usher syndrome patients.

Markus Maniak

2001-01-01

187

Cell adhesion: ushering in a new understanding of myosin VII.  

PubMed

The myosin VII motor protein has recently been found to have a role in cell adhesion. This new function is conserved from amoebae to man and provides an explanation for deafness in Usher syndrome patients. PMID:11369222

Maniak, M

2001-04-17

188

Cooperativity of myosin molecules through strain-dependent chemistry.  

PubMed Central

There is mounting evidence that the myosin head domain contains a lever arm which amplifies small structural changes that occur at the nucleotide-binding site. The mechanical work associated with movement of the lever affects the rates at which the products of ATP hydrolysis are released. During muscle contraction, this strain-dependent chemistry leads to cooperativity of the myosin molecules within a thick filament. Two aspects of cooperative action are discussed, in the context of a simple stochastic model. (i) A modest motion of the lever arm on ADP release can serve to regulate the fraction of myosin bound to the thin filament, in order to recruit more heads at higher loads. (ii) If the lever swings through a large angle when phosphate is released, the chemical cycles of the myosin molecules can be synchronized at high loads. This leads to stepwise sliding of the filaments and suggests that the isometric condition is not a steady state.

Duke, T

2000-01-01

189

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?  

NASA Technical Reports Server (NTRS)

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

Hochstein, L. I.

1996-01-01

190

FXYD Proteins Stabilize Na,K-ATPase  

PubMed Central

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

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

2011-01-01

191

Identification of an organelle receptor for myosin-Va  

Microsoft Academic Search

Little is known about how molecular motors bind to their vesicular cargo. Here we show that myosin-Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor–protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin-Va. Melanophilin creates

Xufeng S. Wu; Kang Rao; Hong Zhang; Fei Wang; James R. Sellers; Lydia E. Matesic; Neal G. Copeland; Nancy A. Jenkins; John A. Hammer

2002-01-01

192

Smooth-muscle contraction without smooth-muscle myosin  

Microsoft Academic Search

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal

Gui-Xuan Chai; Leonidas G. Baltas; Valeria Lamounier-Zepter; Gudrun Lutsch; Monika Kott; Hannelore Haase; Ingo Morano; Michael Bader

2000-01-01

193

Nonlinear elasticity and an 8-nm working stroke of single myosin molecules in myofilaments.  

PubMed

Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rod cofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin's subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin's internal structural changes to physiological force generation and filament sliding. PMID:20689017

Kaya, Motoshi; Higuchi, Hideo

2010-08-01

194

A mechanosensory system governs myosin II accumulation in dividing cells  

PubMed Central

The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress–responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane–cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.

Kee, Yee-Seir; Ren, Yixin; Dorfman, Danielle; Iijima, Miho; Firtel, Richard; Iglesias, Pablo A.; Robinson, Douglas N.

2012-01-01

195

Structure of androcam supports specialized interactions with myosin VI.  

PubMed

Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca(2+) and is locked in a "closed" conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca(2+)-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca(2+) signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins. PMID:22851764

Joshi, Mehul K; Moran, Sean; Beckingham, Kathleen M; MacKenzie, Kevin R

2012-08-14

196

Structure of androcam supports specialized interactions with myosin VI  

PubMed Central

Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca2+ and is locked in a “closed” conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca2+-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca2+ signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins.

Joshi, Mehul K.; Moran, Sean; Beckingham, Kathleen M.; MacKenzie, Kevin R.

2012-01-01

197

A role for myosin II in mammalian mitochondrial fission.  

PubMed

Mitochondria are dynamic organelles, undergoing both fission and fusion regularly in interphase cells. Mitochondrial fission is thought to be part of a quality-control mechanism whereby damaged mitochondrial components are segregated from healthy components in an individual mitochondrion, followed by mitochondrial fission and degradation of the damaged daughter mitochondrion. Fission also plays a role in apoptosis. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer's disease. Mitochondrial fission requires the dynamin GTPase Drp1, which assembles in a ring around the mitochondrion and appears to constrict both outer and inner mitochondrial membranes. However, mechanisms controlling Drp1 assembly on mammalian mitochondria are unclear. Recent results show that actin polymerization, driven by the endoplasmic reticulum-bound formin protein INF2, stimulates Drp1 assembly at fission sites. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or small interfering RNA (siRNA)-mediated suppression of myosin IIA or myosin IIB causes an increase in mitochondrial length in both control cells and cells expressing constitutively active INF2. Active myosin II accumulates in puncta on mitochondria in an actin- and INF2-dependent manner. In addition, myosin II inhibition decreases Drp1 association with mitochondria. Based on these results, we propose a mechanistic model in which INF2-mediated actin polymerization leads to myosin II recruitment and constriction at the fission site, enhancing subsequent Drp1 accumulation and fission. PMID:24485837

Korobova, Farida; Gauvin, Timothy J; Higgs, Henry N

2014-02-17

198

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

PubMed

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

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

2014-02-15

199

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast  

PubMed Central

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

Stark, Benjamin C.; Sladewski, Thomas E.; Pollard, Luther W.

2010-01-01

200

Tropomyosin and myosin-II cellular levels promote actomyosin ring assembly in fission yeast.  

PubMed

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system. PMID:20110347

Stark, Benjamin C; Sladewski, Thomas E; Pollard, Luther W; Lord, Matthew

2010-03-15

201

Complete sequence of the Drosophila nonmuscle myosin heavy-chain transcript: conserved sequences in the myosin tail and differential splicing in the 5' untranslated sequence.  

PubMed Central

We have sequenced a cDNA that encodes the nonmuscle myosin heavy chain from Drosophila melanogaster. An alternatively spliced exon at the 5' end generates two distinct heavy-chain transcripts: the longer transcripts inserts an additional start codon upstream of the primary translation start site and encodes a myosin heavy chain with a 45-residue extension at its amino terminus. The remainder of the coding sequence reveals extensive homology with other conventional myosins, especially metazoan nonmuscle and smooth muscle myosin isoforms. Comparisons among available myosin heavy-chain sequences establish that characteristic differences in sequence throughout the length of both the globular myosin head and extended rod-like tail readily distinguish nonmuscle and smooth muscle myosins from striated muscle isoforms and predict a basis for their functional diversity. Images

Ketchum, A S; Stewart, C T; Stewart, M; Kiehart, D P

1990-01-01

202

Na+/K+-ATPase: Activity and inhibition  

NASA Astrophysics Data System (ADS)

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

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

2009-09-01

203

Enhancement of phagocytosis by Compound 48/80 and its influence upon Ca++-dependent ATP-ase.  

PubMed

A new activity of p-methoxy-phenylethyl-methyl-amine condensed by formaldehyde (Cpd 48/80) has been established: enhancement of phagocytosis. This property seems to be correlated to its ability to activate lysosomes. Enhancement of phagocytosis is discussed in relation to the virus penetration process. Furthermore it was found that Cpd 48/80 and to a smaller degree the dimer inhibit the Ca++-dependent ATP-ase (E. C. 3.6.1.3.) from human red blood cells. The enzyme from bovine myosin is not inhibited by the dimer, but somewhat more by poly-THIQ (7-methoxy-tetrahydroisoquinoline condensed by formaldehyde) than by Cpd 48/80. These properties are discussed in relation to a general hypothesis of virus-induced giant cell formation. Finally it has been shown that the drug does not influence the PHA-stimulation (phytohemagglutinin) of lymphocytes, indicating selectivity of its activity. PMID:170946

Falke, D; Lemmel, E M; Richardson, L S; Wolf, H U

1975-08-01

204

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.

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

2013-01-01

205

Neonatal myosin in bovine and pig tensor tympani muscle fibres.  

PubMed Central

In previous studies of middle ear muscles, the classification of fibre types by histochemical methods was particularly difficult in the bovine and porcine tensor tympani muscle, suggesting the presence of immature fibres. We therefore reexamined the tensor tympani from pigs and cattle of various ages immunohistochemically, using a panel of antimyosin antibodies, including one (anti-NE) specific for neonatal and embryonic myosins. Fibres positive to anti-NE were found in tensor tympani in both species in all ages examined; only a few of these fibres reacted exclusively with this antibody; some also contained slow myosin and the majority also contained adult fast (type IIA) myosin. Furthermore, although the remaining fibres included some of the classical types I and IIA, the majority of them showed a mismatch between their histochemical and immunohistochemical profiles. The morphological appearance of the muscle, the widespread presence of neonatal myosin (often together with another myosin in the same fibre) and the persistence of this composition from birth to adulthood, could be explained by an incomplete development of the muscle fibres, resulting in a 'muscle' much better suited to the role of a ligament. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Scapolo, P A; Rowlerson, A; Mascarello, F; Veggetti, A

1991-01-01

206

Actin and myosin contribute to mammalian mitochondrial DNA maintenance  

PubMed Central

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ?-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ?-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ?-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

2011-01-01

207

Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum  

NASA Technical Reports Server (NTRS)

Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg(2+) or Mn(2+) and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 micro-M. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90 percent.

Kristjansson, H.; Hochstein, L. I.

1985-01-01

208

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.

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

2013-01-01

209

A family of unconventional myosins from the nematode Caenorhabditis elegans 1 1 Edited by J. Karn  

Microsoft Academic Search

The unconventional myosins are a superfamily of actin-based motor proteins that are expressed in a wide range of cell types and organisms. Thirteen classes of unconventional myosin have been defined, and current efforts are focused on elucidating their individual functions in vivo. Here, we report the identification of a family of unconventional myosin genes in Caenorhabditis elegans. The hum-1, hum-2,

Jeffrey P. Baker; Margaret A. Titus

1997-01-01

210

Expression, splicing, and evolution of the myosin gene family in plants.  

PubMed

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

2011-03-01

211

Hydrodynamic Models of Viscous Coupling between Myosin and Endoplasm in Characean Algae Motile  

Microsoft Academic Search

Cytoplasmic streaming in characean algae is thought to be driven by interaction between stationary subcortical actin bundles and motile endoplasmic myosin. Implicit in this mechanism is a requirement for some form of coupling to transfer motive force from the moving myosin to the endoplasm. Three models of viscous coupling between myosin and endoplasm are presented here, and the hydrodynamic feasibility

EUGENE A. NOTHNAGEL; W. W. WEBB

1982-01-01

212

A single class II myosin modulates T cell motility and stopping, but not synapse formation  

Microsoft Academic Search

Upon encountering an antigen, motile T cells stop crawling, change morphology and ultimately form an 'immunological synapse'. Although myosin motors are thought to mediate various aspects of this process, the molecules involved and their exact roles are not defined. Here we show that nonmuscle myosin heavy chain IIA, or MyH9, is the only class II myosin expressed in T cells

Jordan Jacobelli; Stephen A Chmura; Denis B Buxton; Mark M Davis; Matthew F Krummel

2004-01-01

213

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.

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

2011-01-01

214

Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking  

PubMed Central

Background Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments. Results In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1). Conclusion All results favored the peripheral dynamic tethering hypothesis.

Provance, D William; Addison, Erin J; Wood, Patrick R; Chen, David Z; Silan, Colleen M; Mercer, John A

2008-01-01

215

Actin Network Architecture Can Determine Myosin Motor Activity  

PubMed Central

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture is maintained in a dynamic steady-state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization myosins selectively contracted and disassembled anti-parallel actin structures while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

Reymann, Anne-Cecile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guerin, Christophe; Cao, Wenxiang; Chin, Harvey F.; De La Cruz, Enrique M.; Thery, Manuel; Blanchoin, Laurent

2013-01-01

216

Organization of the human skeletal myosin heavy chain gene cluster.  

PubMed Central

Myosin is an important structural and enzymatic component of skeletal muscle. Multiple myosin isoforms are encoded by a multigene family and are expressed in different developmental stages and fiber types. In humans and mice, skeletal myosin heavy chain (MYH) genes are clustered on a single chromosome (17p and 11, respectively). Since the structural organization of the gene cluster may affect its expression as well as shed light on MYH genetic alterations, a physical map of the human MYH gene cluster was constructed. Nine yeast artificial chromosomes containing MYH genes were isolated and used to construct a contiguous set (contig) of overlapping yeast artificial chromosomes. This contig encompasses a genetic marker mapped to 17p13.1. Six MYH genes were located within a 500-kilobase segment of human DNA. The order of the genes within this cluster does not correspond to the developmental pattern of expression of individual members. Images

Yoon, S J; Seiler, S H; Kucherlapati, R; Leinwand, L

1992-01-01

217

Myosin-dependent targeting of transmembrane proteins to neuronal dendrites  

PubMed Central

The distinct electrical properties of axonal and dendritic membranes are largely a result of specific transport of vesicle-bound membrane proteins to each compartment. How this specificity arises is unclear because kinesin motors that transport vesicles cannot autonomously distinguish dendritically projecting microtubules from those projecting axonally. We hypothesized that interaction with a second motor might enable vesicles containing dendritic proteins to preferentially associate with dendritically projecting microtubules and avoid those that project to the axon. Here we show that in rat cortical neurons, localization of several distinct transmembrane proteins to dendrites is dependent on specific myosin motors and an intact actin network. Moreover, fusion with a myosin-binding domain from Melanophilin targeted Channelrhodopsin-2 specifically to the somatodendritic compartment of neurons in mice in vivo. Together, our results suggest that dendritic transmembrane proteins direct the vesicles in which they are transported to avoid the axonal compartment through interaction with myosin motors.

Lewis, Tommy L; Mao, Tianyi; Svoboda, Karel; Arnold, Don B

2010-01-01

218

Bafilomycins: A Class of Inhibitors of Membrane ATPases from Microorganisms, Animal Cells, and Plant Cells  

Microsoft Academic Search

Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles,

Emma Jean Bowman; Annette Siebers; Karlheinz Altendorf

1988-01-01

219

Electrostatic origin of the unidirectionality of walking myosin V motors  

PubMed Central

Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ?2–3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors.

Mukherjee, Shayantani; Warshel, Arieh

2013-01-01

220

Electrostatic origin of the unidirectionality of walking myosin V motors.  

PubMed

Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ?2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors. PMID:24106304

Mukherjee, Shayantani; Warshel, Arieh

2013-10-22

221

CHARACTERIZATION OF TIGHTLY-ASSOCIATED SMOOTH MUSCLE MYOSIN-MYOSIN LIGHT CHAIN KINASE-CALMODULIN COMPLEXES*  

PubMed Central

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ~1 nM free [Ca2+]. There were two MLCK pools that bound unphosphorylated SMM with Kd ~10 ?M and 0.2 ?M and phosphorylated SMM with a Kd ~ 20 ?M and 0.2 ?M. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca2+ and phosphorylation of SMM occurred at a pCa50 of 6.1 and Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip ELISA assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.

Hong, Feng; Haldeman, Brian D.; John, Olivia A.; Brewer, Paul D.; Wu, Yi-Ying; Ni, Shaowei; Wilson, David P.; Walsh, Michael P.; Baker, Jonathan E.; Cremo, Christine R.

2009-01-01

222

Kinetic properties and small-molecule inhibition of human myosin-6  

PubMed Central

Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion.

Heissler, Sarah M.; Selvadurai, Jayashankar; Bond, Lisa M.; Fedorov, Roman; Kendrick-Jones, John; Buss, Folma; Manstein, Dietmar J.

2012-01-01

223

The smooth muscle myosin seven amino acid heavy chain insert's kinetic role in the crossbridge cycle for mouse bladder.  

PubMed

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle. PMID:12562924

Karagiannis, Peter; Babu, Gopal J; Periasamy, Muthu; Brozovich, Frank V

2003-03-01

224

Model of Rho-Mediated Myosin Recruitment to the Cleavage Furrow during Cytokinesis  

NASA Astrophysics Data System (ADS)

The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During cytokinesis, the myosin attached to the cell's cortex progressively disassembles at the flanking regions and concentrates in the equator [1]. This recruitment depends on myosin motor activity and activation by Rho proteins. Central spindle and astral microtubules establish a spatial pattern of differential Rho activity [2]. We propose a reaction-diffusion model for the dynamics of myosin and Rho proteins during cytokinesis. In the model, the mitotic spindle activates Rho at the equator. Active Rho promotes, in a switch-like manner, myosin assembly into cortical minifilaments. Mechanical stress by cortical myosin causes disassembly of myosin minifilaments and deactivates Rho. Our results explain both the recruitment of myosin to the cleavage furrow and the observed damped myosin oscillations in the cell's flanking regions [1]. Spatial extent, period and decay rate of myosin oscillations are calculated. Various regimes of myosin recruitment are predicted. [1] Zhou & Wang, Mol. Biol. Cell 19:318 (2008) [2] Murthy & Wadsworth, J. Cell Sci. 121:2350 (2008)

Veksler, Alexander; Vavylonis, Dimitrios

2010-03-01

225

The in vitro motility activity of ?-cardiac myosin depends on the nature of the ?-myosin heavy chain gene mutation in hypertrophic cardiomyopathy  

Microsoft Academic Search

Several mutations in the ?-myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro\\u000a velocities of translocation of fluorescently-labelled actin by ?-myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy\\u000a patients with seven distinct ?-myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His,\\u000a and Leu908Val mutations; and (2) motility activity of ?-myosin purified from

GIOVANNI CUDA; LAMEH FANANAPAZIR; NEAL D. EPSTEIN; JAMES R. SELLERS

1997-01-01

226

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

PubMed Central

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

Raposo, Graca; Cordonnier, Marie-Neige; Tenza, Daniele; Menichi, Bernadette; Durrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

1999-01-01

227

Adiabatic Pumping Mechanism for Ion Motive ATPases  

NASA Astrophysics Data System (ADS)

An ion motive ATPase is a membrane protein that pumps ions across the membrane at the expense of the chemical energy of adenosine triphosphate (ATP) hydrolysis. Here we describe how an external electric field, by inducing transitions between several protein configurations, can also power this pump. The underlying mechanism may be very similar to that of a recently constructed adiabatic electron pump [

M. Switkes et al., Science 283, 1905 (1999)
].

Astumian, R. Dean

2003-09-01

228

The plasma membrane calcium ATPase and disease  

Microsoft Academic Search

The plasma membrane calcium ATPase (PMCA) uses energy to pump calcium (Ca2+) ions out of the cytosol into the extracellular milieu, usually against a strong chemical gradient. This energy expenditure\\u000a is necessary to maintain a relatively low intracellular net Ca2+ load. Mammals have four genes (ATP2B1-ATP2B4), encoding the proteins PMCA1 through PMCA4. Transcripts from each of these genes are alternatively

B. L Tempel; D. J. Shilling

229

Optical trapping studies of acto-myosin motor proteins  

NASA Astrophysics Data System (ADS)

Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical work. This paper focuses on studies of the acto-myosin motor system that is responsible for muscle contraction and a host of other cellular motilities. Myosin works by binding to filamentous actin, pulling and then releasing. Each cycle of interaction produces a few nanometres movement and a few piconewtons force. Individual interactions can be observed directly by holding an individual actin filament between two optically trapped microspheres and positioning it in the immediate vicinity of a single myosin motor. When the chemical fuel (adenosine triphosphate or ATP) is present the myosin undergoes repeated cycles of interaction with the actin filament producing square-wave like displacements and forces. Analysis of optical trapping data sets enables the size and timing of the molecular motions to be deduced.

Farrow, Rachel E.; Rosenthal, Peter B.; Mashanov, Gregory I.; Holder, Anthony A.; Molloy, Justin E.

2007-09-01

230

Engineering controllable bidirectional molecular motors based on myosin.  

PubMed

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. PMID:22343382

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

2012-04-01

231

Tryptic digestion as a probe of myosin S-1 conformation.  

PubMed Central

One of the products of the limited tryptic hydrolysis of chymotryptic myosin subfragment 1 is the 27,000-dalton NH2-terminal fragment. This fragment is generated by two parallel routes from either the 75,000- or 95,000-dalton peptide of the heavy chain: (i) through a 20,500-dalton precursor or (ii) directly without participation of a precursor. Lowering of pH and temperature and increasing of ionic strength inhibited route i digestion in comparison to route ii. MgATP and its derivatives in millimolar concentration substantially suppressed route i digestion. Suppression of route i digestion depended on the concentration of MgATP. It occurred after a lag phase when the ratio of MgATP to subfragment 1 concentrations was greater than 0.5. In contrast, the MgATP-induced increase in tryptophan fluorescence of myosin subfragment 1 appeared without a lag phase. The generation of the 27,000-dalton fragment by either route was not affected by F-actin however, the suppression of route i digestion induced by MgADP was abolished when myosin subfragment 1 was in ternary complex with actin and MgADP. We conclude that the 27,000/50,000-dalton hinge region is a flexible domain of the myosin head and that conformation of this region is sensitive to the presence of nucleotides and actin and to variations in ambient factors. Images

Muhlrad, A; Hozumi, T

1982-01-01

232

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

2012-04-01

233

Cooperative actions between myosin heads bring effective functions  

Microsoft Academic Search

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1,

Seiji Esaki; Yoshiharu Ishii; Masatoshi Nishikawa; Toshio Yanagida

2007-01-01

234

Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium.  

PubMed Central

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

Lowes, B D; Minobe, W; Abraham, W T; Rizeq, M N; Bohlmeyer, T J; Quaife, R A; Roden, R L; Dutcher, D L; Robertson, A D; Voelkel, N F; Badesch, D B; Groves, B M; Gilbert, E M; Bristow, M R

1997-01-01

235

The different roles of myosin IIA and myosin IIB in contraction of 3D collagen matrices by human fibroblasts.  

PubMed

Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model - all conventionally referred to as "contraction" - involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation. PMID:24768700

Liu, Zhenan; Ho, Chin-Han; Grinnell, Frederick

2014-08-15

236

Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate  

SciTech Connect

The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz/sub 2/ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz/sub 2/ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF/sub 1/) with a binding constant of 3.2 x 10/sup 5/ M/sup -1/. Bz/sub 2/ATP is also a substrate for the ATPase activity of SF/sub 1/ in the presence of different cations. The irradiation of SF/sub 1/ with (/sup 3/H)Bz/sub 2/ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF/sub 1/ after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling (/sup 3/H)Bz/sub 2/ATP is trapped on SF/sub 1/ by cross-linking the two reactive thiols, SH/sub 1/ and SH/sub 2/, by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified (/sup 14/C)Bz/sub 2/ATP-SF/sub 1/, after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography.

Mahmood, R.

1985-01-01

237

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.

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

2013-01-01

238

Myosin-1c interacts with hair-cell receptors through its calmodulin-binding IQ domains.  

PubMed

Myosin-1c plays an essential role in adaptation of hair-cell mechanoelectrical transduction. To mediate adaptation, myosin-1c must interact directly or indirectly with other components of the transduction apparatus, including the mechanically gated transduction channel. As a first step toward identifying myosin-1c receptors, we used recombinant myosin-1c fragments to identify specific binding sites in hair cells and to biochemically characterize their interaction with myosin-1c. Myosin-1c fragments bound to tips of hair-cell stereocilia, the location of transduction and adaptation. Surprisingly, this interaction did not depend on the C-terminal tail of myosin-1c, proposed previously to be the receptor-binding site of the molecule. Instead, the interaction of myosin-1c with stereociliary receptors depended on its calmodulin-binding IQ domains. This interaction was blocked by calmodulin, which probably bound to a previously unoccupied IQ domain of myosin-1c. The calcium-sensitive binding of calmodulin to myosin-1c may therefore modulate the interaction of the adaptation motor with other components of the transduction apparatus. PMID:11923413

Cyr, Janet L; Dumont, Rachel A; Gillespie, Peter G

2002-04-01

239

Gelsolin and Non-muscle Myosin IIA Interact to Mediate Calcium-regulated Collagen Phagocytosis*  

PubMed Central

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca2+ and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca2+]i, which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca2+]i were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca2+]i, also required gelsolin. Ionomycin-induced increases of [Ca2+]i overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca2+-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.

Arora, Pamma D.; Wang, Yongqiang; Janmey, Paul A.; Bresnick, Anne; Yin, Helen L.; McCulloch, Christopher A.

2011-01-01

240

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

PubMed Central

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

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

2013-01-01

241

Myosin Va Is Required for P Body but Not Stress Granule Formation*  

PubMed Central

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5?–3? mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.

Lindsay, Andrew J.; McCaffrey, Mary W.

2011-01-01

242

Gene expression pattern of myosin Va during spermatogenesis of Chinese mitten crab, Eriocheir sinensis.  

PubMed

Myosin Va is an F-actin dependent molecular motor with multiple functions that are essential for acrosome formation in mouse spermiogenesis. The spermatozoon of the crab has a complicated acrosome surrounded by a cup-shaped nucleus. In the present study, the myosin Va cDNA was cloned from the testis of the Chinese mitten crab Eriocheir sinensis using degenerate PCR and rapid amplification of cDNA ends (RACE). The myosin Va cDNA consists of a 125 bp 5'-untranslated region (5' UTR), a 5331 bp open reading frame (ORF) and a 590 bp 3' UTR. The putative myosin Va protein contains the head domain, neck domain and tail domain. Multiple alignment and phylogenetic tree showed that E. sinensis myosin Va is more closely related to the vertebrate myosin Va than to the invertebrate myosin V. E. sinensis myosin Va was expressed in various tissues. In situ hybridization demonstrated that myosin Va mRNA is located in the entire process of spermatogenesis. Quantitative real-time PCR indicated that the expression level at the mitotic and meiotic phases is higher than the spermiogenesis phase. Taken together, our work suggests that myosin Va may function in E. sinensis spermatogenesis. PMID:22846366

Sun, Xiao; Mao, Hai-Tao; Yang, Wan-Xi

2012-10-15

243

Nonmuscle myosin IIA is associated with poor prognosis of esophageal squamous cancer.  

PubMed

Nonmuscle myosin IIA (myosin IIA) is a force-producing protein involved in the process of cell migration. Its expression has been considered as a bad prognostic indicator in stage I lung adenocarcinoma. However, the expression and clinical significance of myosin IIA in esophageal cancer has not been explored. In this study, we investigate the expression level of myosin IIA in 50 esophageal squamous cancer and 30 adjacent normal esophageal tissues by immunohistochemical staining and correlated its expression with clinicopathological features. Myosin IIA was expressed in all esophageal squamous cancer tissues (100%) and 8 of 30 adjacent normal tissues (26.7%, P = 0.000). In cancer tissues, elevated myosin IIA expression level was significantly correlated with increasing metastatic lymph nodes, poorer cancer differentiation, and advanced tumor stage. Further univariate analysis suggested that strong myosin IIA expression was associated with a significantly shorter overall survival (P = 0.021). In addition, MYH9 SiRNA was transfected into esophageal squamous cancer cell line (KYSE-510) to study the role of myosin IIA in cell migration. SiRNA-mediated depletion of myosin IIA in KYSE-510 cells significantly increased cell-matrix adhesion and attenuated cell migration ability (P = 0.000). In conclusion, these findings indicate that overexpression of myosin IIA may contribute to the progression and poor prognosis of esophageal squamous cancer, and this effect may be associated with increased cancer cell migration. PMID:21951916

Xia, Z-K; Yuan, Y-C; Yin, N; Yin, B-L; Tan, Z-P; Hu, Y-R

2012-07-01

244

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

PubMed

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

Masuda, Tadashi

2013-09-01

245

ATP-synthesis in archaea: Structure-function relations of the halobacterial A-ATPase  

Microsoft Academic Search

The archaeal (A)-ATPase has been described as a chimeric energy converter with close relationship to both the vacuolar ATPase class in higher eukaryotes and the coupling factor (F)-ATPase class in eubacteria, mitochondria and chloroplasts. With respect to their structure and some inhibitor responses, A-ATPases are more closely related to the vacuolar ATPase type than to F-ATPase. Their function, ATP synthesis

Susanne Bickel-Sandkötter; Volker Wagner; Dietmar Schumann

1998-01-01

246

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

PubMed Central

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

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

2006-01-01

247

Cloning of the cDNA encoding the myosin heavy chain of a vertebrate cellular myosin.  

PubMed Central

The complete amino acid sequence of a vertebrate cellular myosin heavy chain (MHC; 1,959 amino acids, 226 kDa) has been deduced by using cDNA clones from a chicken intestinal epithelial cell library. RNA blot analysis of kidney, spleen, brain, liver, and intestinal epithelial cells as well as smooth muscle cells from the aorta and gizzard indicates the presence of a 7.3-kilobase (kb) message that is larger than the message for chicken smooth and striated muscle MHC. The chicken intestinal epithelial cell MHC shows overall similarity in primary structure to other MHCs in the areas of the reactive thiol residues and in areas contributing to the ATP binding site and actin binding site. The globular head domain is followed by an alpha-helical coiled-coil region, and as in smooth muscle MHC there is a short uncoiled sequence at the carboxyl terminus of the molecule. Comparison of amino acid sequences in the rod regions between human and chicken cellular MHCs shows a remarkable 92% identity. Images

Shohet, R V; Conti, M A; Kawamoto, S; Preston, Y A; Brill, D A; Adelstein, R S

1989-01-01

248

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

PubMed

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

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

2014-06-01

249

Regulation and Isoform Function of the V-ATPases  

PubMed Central

The vacuolar (H+)-ATPases are ATP-dependent proton pumps that function to acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in such normal physiological processes as receptor-mediated endocytosis, intracellular membrane traffic, pro-hormone processing, protein degradation and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, as well as in various disease processes, including renal tubular acidosis, osteopetrosis and tumor metastasis. V-ATPases are composed of a peripheral V1 domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V0 domain containing six different subunits that translocates protons. In mammalian cells most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V0 subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V1 and V0 domains, control of the tightness of coupling of proton transport and ATP hydrolysis and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both by control of coupling and by selective targeting. This review will begin with a brief introduction to the function, structure and mechanism of the V-ATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity.

Toei, Masashi; Saum, Regina; Forgac, Michael

2010-01-01

250

Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium  

PubMed Central

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca2+ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca2+ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines43/45 and threonine144 (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca2+ sensitivity required the presence of phosphorylatable cTnI. For comparison, the ?-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca2+ sensitivity. These ?-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca2+ and ATP, thereby promoting efficient ATP utilisation during contraction.

Pi, YeQing; Zhang, Dahua; Kemnitz, Kara R; Wang, Hao; Walker, Jeffery W

2003-01-01

251

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

PubMed

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

2013-02-01

252

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.

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

2013-01-01

253

A Genomic Screen for Yeast Vacuolar Membrane ATPase Mutants  

PubMed Central

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, ?4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl2. In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36? mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34?, vps15?, vps45?, and vps16?, caused both Vma? growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1? mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma? growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma? growth phenotype.

Sambade, Maria; Alba, Mercedes; Smardon, Anne M.; West, Robert W.; Kane, Patricia M.

2005-01-01

254

Control of cell membrane tension by myosin-I  

PubMed Central

All cell functions that involve membrane deformation or a change in cell shape (e.g., endocytosis, exocytosis, cell motility, and cytokinesis) are regulated by membrane tension. While molecular contacts between the plasma membrane and the underlying actin cytoskeleton are known to make significant contributions to membrane tension, little is known about the molecules that mediate these interactions. We used an optical trap to directly probe the molecular determinants of membrane tension in isolated organelles and in living cells. Here, we show that class I myosins, a family of membrane-binding, actin-based motor proteins, mediate membrane/cytoskeleton adhesion and thus, make major contributions to membrane tension. These studies show that class I myosins directly control the mechanical properties of the cell membrane; they also position these motor proteins as master regulators of cellular events involving membrane deformation.

Nambiar, Rajalakshmi; McConnell, Russell E.; Tyska, Matthew J.

2009-01-01

255

Purification and properties of an ATPase from Sulfolobus solfataricus  

NASA Technical Reports Server (NTRS)

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

Hochstein, Lawrence I.; Stan-Lotter, Helga

1992-01-01

256

P4 ATPases: Flippases in Health and Disease  

PubMed Central

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

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

2013-01-01

257

Rotational Model for Actin Filament Alignment by Myosin  

PubMed Central

Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allow us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors in a truly unique way.

Miller, Callie J; Ermentrout, G Bard; Davidson, Lance A

2012-01-01

258

Stepwise Sliding of Single Actin and Myosin Filaments  

Microsoft Academic Search

Dynamics of sliding were explored in isolated actin and myosin filaments. Sliding occurs in steps. The steps are integer multiples of 2.7nm, which is equal to the monomeric repeat along the actin filament. When filaments were forced to slide in the reverse direction, the size paradigm was the same. This size paradigm is parallel to that seen in the kinesin-microtubule

Xiumei Liu; Gerald H. Pollack

2004-01-01

259

Evolution and Classification of Myosins, a Paneukaryotic Whole-Genome Approach  

PubMed Central

Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animal multicellularity. Indeed, single-celled holozoans have the most complex myosin complement among eukaryotes, with paralogs of most myosins previously considered animal specific. We recover a dynamic evolutionary history, with several lineage-specific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family.

Sebe-Pedros, Arnau; Grau-Bove, Xavier; Richards, Thomas A.; Ruiz-Trillo, Inaki

2014-01-01

260

Evolution and classification of myosins, a paneukaryotic whole-genome approach.  

PubMed

Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animal multicellularity. Indeed, single-celled holozoans have the most complex myosin complement among eukaryotes, with paralogs of most myosins previously considered animal specific. We recover a dynamic evolutionary history, with several lineage-specific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family. PMID:24443438

Sebé-Pedrós, Arnau; Grau-Bové, Xavier; Richards, Thomas A; Ruiz-Trillo, Iñaki

2014-01-01

261

Identification and Characterization of Myosin from Rat Testicular Peritubular Myoid Cells1  

PubMed Central

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle—alpha-isoactin and F-actin—and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4°C, but did at 20°C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.

Fernandez, Dario; Bertoldi, Maria V.; Gomez, Laura; Morales, Alfonsina; Callegari, Eduardo; Lopez, Luis A.

2008-01-01

262

Insights into Human ?-Cardiac Myosin Function from Single Molecule and Single Cell Studies  

Microsoft Academic Search

?-Cardiac myosin is a mechanoenzyme that converts the energy from ATP hydrolysis into a mechanical force that drives contractility\\u000a in muscle. Thirty percent of the point mutations that result in hypertrophic cardiomyopathy are localized to MYH7, the gene encoding human ?-cardiac myosin heavy chain (?-MyHC). Force generation by myosins requires a tight and highly conserved\\u000a allosteric coupling between its different

Sivaraj Sivaramakrishnan; Euan Ashley; Leslie Leinwand; James A. Spudich

2009-01-01

263

Myosin light chain kinase in microvascular endothelial barrier function  

PubMed Central

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

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

2010-01-01

264

Myosin Vs organize actin cables in fission yeast  

PubMed Central

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

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

2012-01-01

265

Actin-Myosin Viscoelastic Flow in the Keratocyte Lamellipod  

PubMed Central

Abstract The lamellipod, the locomotory region of migratory cells, is shaped by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow of the viscoelastic actin network. Recently, quantitative flow data was obtained, yet there is no detailed theory explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin transport and solve the model equations numerically for the flat, fan-shaped lamellipodial domain of keratocytes. The solutions demonstrate that in the rapidly crawling cell, myosin concentrates at the rear boundary and pulls the actin network inward, so the centripetal actin flow is very slow at the front, and faster at the rear and at the sides. The computed flow and respective traction forces compare well with the experimental data. We also calculate the graded protrusion at the cell boundary necessary to maintain the cell shape and make a number of other testable predictions. We discuss model implications for the cell shape, speed, and bi-stability.

Rubinstein, Boris; Fournier, Maxime F.; Jacobson, Ken; Verkhovsky, Alexander B.; Mogilner, Alex

2009-01-01

266

Myosin I Is Required for Hypha Formation in Candida albicans†  

PubMed Central

The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5S366D were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.

Oberholzer, U.; Marcil, A.; Leberer, E.; Thomas, D. Y.; Whiteway, M.

2002-01-01

267

Light Chains of Myosins from White, Red, and Cardiac Muscles  

PubMed Central

Purified preparations of rabbit skeletal white, red, and cardiac muscle myosin (WM, RM, and CM) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences in both the molecular weights and number of light chains in these myosins were found. WM has three distinct light-chain components (LC1W, LC2W, LC3W) having molecular weights of 25,500, 17,400, and 15,100, respectively. No component with a molecular weight around 15,000 is present in RM or CM. RM and CM contain components of identical molecular weights close to 25,000 and 17,000 (LC1CR and LC2CR) which, however, clearly differ in molecular weight from the corresponding subunits in WM. RM has an additional component (LC1R) having a slightly higher molecular weight than LC1W and LC1CR. Thus differences and similarities in many biochemical properties between WM, RM, and CM, which have been described earlier, are also reflected in the light-chain components. The present results support the hypothesis that different sets of genes are active in producing components of myosin that make up different isozymic forms characteristic of each muscle type. Images

Sarkar, Satyapriya; Sreter, F. A.; Gergely, J.

1971-01-01

268

A Kinetic Model Describing the Processivity of Myosin-V  

PubMed Central

The precise details of how myosin-V coordinates the biochemical reactions and mechanical motions of its two head elements to engineer effective processive molecular motion along actin filaments remain unresolved. We compare a quantitative kinetic model of the myosin-V walk, consisting of five basic states augmented by two further states to allow for futile hydrolysis and detachments, with experimental results for run lengths, velocities, and dwell times and their dependence on bulk nucleotide concentrations and external loads in both directions. The model reveals how myosin-V can use the internal strain in the molecule to synchronize the motion of the head elements. Estimates for the rate constants in the reaction cycle and the internal strain energy are obtained by a computational comparison scheme involving an extensive exploration of the large parameter space. This scheme exploits the fact that we have obtained analytic results for our reaction network, e.g., for the velocity but also the run length, diffusion constant, and fraction of backward steps. The agreement with experiment is often reasonable but some open problems are highlighted, in particular the inability of such a general model to reproduce the reported dependence of run length on ADP concentration. The novel way that our approach explores parameter space means that any confirmed discrepancies should give new insights into the reaction network model.

Skau, Karl I.; Hoyle, Rebecca B.; Turner, Matthew S.

2006-01-01

269

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases  

Microsoft Academic Search

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump1. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a

Ramón Serrano; Morten C. Kielland-Brandt; Gerald R. Fink

1986-01-01

270

Dictyostelium myosin II mechanochemistry promotes active behavior of the cortex on long time scales  

PubMed Central

Cell cortices rearrange dynamically to complete cytokinesis, crawlin response to chemoattractant, build tissues, and make neuronal connections. Highly enriched in the cell cortex, actin, myosin II, and actin crosslinkers facilitate cortical movements. Because cortical behavior is the consequence of nanoscale biochemical events, it is essential to probe the cortex at this level. Here, we use high-resolution laser-based particle tracking to examine how myosin II mechanochemistry and dynacortin-mediated actin crosslinking control cortex dynamics in Dictyostelium. Consistent with its low duty ratio, myosin II does not directly drive active bead motility. Instead, myosin II and dynacortin antagonistically regulate other active processes in the living cortex.

Girard, Kristine D.; Kuo, Scot C.; Robinson, Douglas N.

2006-01-01

271

Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction  

PubMed Central

Background Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is a unique member of the myosin superfamily of actin-based motor proteins. Myosin VI is the only myosin known to move towards the minus or pointed ends of actin filaments. Although Myosin VI has been implicated in numerous cellular processes as both an anchor and a transporter, little is known about the role of Myosin VI in the nervous system. We previously recovered jar in a screen for genes that modify neuromuscular junction (NMJ) development and here we report on the genetic analysis of Myosin VI in synaptic development and function using loss of function jar alleles. Results Our experiments on Drosophila third instar larvae revealed decreased locomotor activity, a decrease in NMJ length, a reduction in synaptic bouton number, and altered synaptic vesicle localization in jar mutants. Furthermore, our studies of synaptic transmission revealed alterations in both basal synaptic transmission and short-term plasticity at the jar mutant neuromuscular synapse. Conclusions Altogether these findings indicate that Myosin VI is important for proper synaptic function and morphology. Myosin VI may be functioning as an anchor to tether vesicles to the bouton periphery and, thereby, participating in the regulation of synaptic vesicle mobilization during synaptic transmission.

2011-01-01

272

Myosin VI Regulates Actin Structure Specialization through Conserved Cargo-Binding Domain Sites  

PubMed Central

Actin structures are often stable, remaining unchanged in organization for the lifetime of a differentiated cell. Little is known about stable actin structure formation, organization, or maintenance. During Drosophila spermatid individualization, long-lived actin cones mediate cellular remodeling. Myosin VI is necessary for building the dense meshwork at the cones' fronts. We test several ideas for myosin VI's mechanism of action using domain deletions or site-specific mutations of myosin VI. The head (motor) and globular tail (cargo-binding) domains were both needed for localization at the cone front and dense meshwork formation. Several conserved partner-binding sites in the globular tail previously identified in vertebrate myosin VI were critical for function in cones. Localization and promotion of proper actin organization were separable properties of myosin VI. A vertebrate myosin VI was able to localize and function, indicating that functional properties are conserved. Our data eliminate several models for myosin VI's mechanism of action and suggest its role is controlling organization and action of actin assembly regulators through interactions at conserved sites. The Drosophila orthologues of interaction partners previously identified for vertebrate myosin VI are likely not required, indicating novel partners mediate this effect. These data demonstrate that generating an organized and functional actin structure in this cell requires multiple activities coordinated by myosin VI.

Isaji, Mamiko; Lenartowska, Marta; Noguchi, Tatsuhiko; Frank, Deborah J.; Miller, Kathryn G.

2011-01-01

273

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA.  

PubMed

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern. PMID:2208616

Zavodny, P J; Petro, M E; Lonial, H K; Dailey, S H; Narula, S K; Leibowitz, P J; Kumar, C C

1990-10-01

274

Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle  

PubMed Central

Length adaptation in airway smooth muscle (ASM) is attributed to reorganization of the cytoskeleton, and in particular the contractile elements. However, a constantly changing lung volume with tidal breathing (hence changing ASM length) is likely to restrict full adaptation of ASM for force generation. There is likely to be continuous length adaptation of ASM between states of incomplete or partial length adaption. We propose a new model that assimilates findings on myosin filament polymerization/depolymerization, partial length adaptation, isometric force, and shortening velocity to describe this continuous length adaptation process. In this model, the ASM adapts to an optimal force-generating capacity in a repeating cycle of events. Initially the myosin filament, shortened by prior length changes, associates with two longer actin filaments. The actin filaments are located adjacent to the myosin filaments, such that all myosin heads overlap with actin to permit maximal cross-bridge cycling. Since in this model the actin filaments are usually longer than myosin filaments, the excess length of the actin filament is located randomly with respect to the myosin filament. Once activated, the myosin filament elongates by polymerization along the actin filaments, with the growth limited by the overlap of the actin filaments. During relaxation, the myosin filaments dissociate from the actin filaments, and then the cycle repeats. This process causes a gradual adaptation of force and instantaneous adaptation of shortening velocity. Good agreement is found between model simulations and the experimental data depicting the relationship between force development, myosin filament density, or shortening velocity and length.

Ijpma, Gijs; Cairns, Simeon P.; Sieck, Gary C.

2011-01-01

275

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.

2009-02-01

276

Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments  

PubMed Central

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (?1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 ?M Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

Cao, Tracy T.; Chang, Wakam; Masters, Sarah E.; Mooseker, Mark S.

2004-01-01

277

Recent advances in understanding plant myosin function: life in the fast lane.  

PubMed

Plant myosins are required for organelle movement, and a role in actin organization has recently come to light. Myosin mutants display several gross morphological phenotypes, the most severe being dwarfism and reduced fecundity, and there is a correlation between reduced organelle movement and morphological defects. This review aims to discuss recent findings in plants relating to the role of myosins in actin dynamics, development, and organelle movement, more specifically the endoplasmic reticulum (ER). One overarching theme is that there still appear to be more questions than answers relating to plant myosin function and regulation. PMID:21772028

Sparkes, Imogen

2011-09-01

278

The basal Mg2(+)-dependent ATPase activity is not part of the (H(+)+K+)-transporting ATPase reaction cycle.  

PubMed Central

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the 'stretched-out' version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.

Van der Hijden, H T; Kramer-Schmitt, S; Grell, E; de Pont, J J

1990-01-01

279

Myosin individualized: single nucleotide polymorphisms in energy transduction  

PubMed Central

Background Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. Results An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. Conclusions Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function.

2010-01-01

280

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.

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

2012-01-01

281

Biochemical and bioinformatic analysis of the MYO19 motor domain  

PubMed Central

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ?40 MYO19 orthologs to ?2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-cosedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.

Adikes, Rebecca C.; Unrath, William C.; Yengo, Christopher M.; Quintero, Omar A.

2014-01-01

282

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail  

PubMed Central

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

283

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail.  

PubMed

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A; Korn, Edward D

2014-01-01

284

Potassium ion transport ATPase in insect epithelia.  

PubMed

K+ transport by the epithelia of midgut, salivary glands, Malpighian tubules, sensory sensilla, possibly rectum, and other organs of certain insects appears to use a unique K+ ATPase. Ouabain inhibition of transport-related events has not been demonstrated in these epithelia. The K+ pump is unlike the Na-K;ump but resembles the H;ump of phosphorylating membranes in its transport orientation, efficient thermodynamics, speculated two K+ per one MgATP2- stoichiometry, electrogenicity, and structure. Older electrochemical, tracer flux, and conductance evidence suggested that the K+ pump was on the apical plasma membrane of transporting cells in these epithelia. New X-ray microanalytical studies (XMA), reveal that the K+ concentration in all cells is more than 100 mM. Together with new microelectrode data these XMA results confirm the apical K+ pump location, resolve the K+ transport sport route, and suggest that the goblet cell cavity facilitates the generation of a large apical PD which may be used in nutrient absorption and pH regulation. K+ portasomes, which resemble F1-Fo ATPase particles, stud these K+ transporting apical membranes and are though to be the unit of active K+ transport. We have suggested a K+ transport mechanism in which two cations (2K+) are abandoned in an isolated domain of the portasomes during ATP2-hydrolysis and are repelled to the opposite membrane side via a K+ channel. Small peptides hydrolysed from the delta-endotoxin of Bacillus thuringiensis inhibit the K+ transport and may be useful as K+ pump inhibitors, apical membrane probes and insecticides. Goblet cell apical membrane fragments (GCAM) as well as fragments from columnar cell apical membrane (CCAM), lateral membrane (LM) and basal membranes (BM) were isolated as clean fractions using ultrasound, aspiration, and both differential and density gradient centrifugation; purification was monitored by electron microscopy. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) reveals that GCAM, CCAM, LM and BM have very different protein compositions. Preliminary enzymology is consistent with the K+ ATPase being on the apical plasma membrane of the goblet cells of midgut and enveloping cells of sensilla. PMID:6317792

Harvey, W R; Cioffi, M; Dow, J A; Wolfersberger, M G

1983-09-01

285

Myosin 1E localizes to actin polymerization sites in lamellipodia, affecting actin dynamics and adhesion formation  

PubMed Central

Summary Because the actin network in active lamellipodia is continuously assembling at the edge, moving inward and disassembling, there is a question as to how actin-binding proteins and other components are transported to the leading edge and how nascent adhesions are stabilized. Active transport could play a significant role in these functions but the components involved are unknown. We show here that Myosin 1E (a long tailed Myosin 1 isoform) rapidly moves to the tips of active lamellipodia and to actin-rich early adhesions, unlike Myosin 1G, 1B or 1C (short tailed isoforms). Myosin 1E co-localizes with CARMIL, FHOD1, Arp3 and ?3-integrin in those early adhesions. But these structures precede stable paxillin-rich adhesions. Myosin 1E movement depends upon actin-binding domains and the presence of an SH3 oligomerization domain. Overexpression of a Myosin 1E deletion mutant without the extreme C-terminal interacting (SH3) domain (Myosin 1E?SH3) increases edge fluctuations and decreases stable adhesion lifetimes. In contrast, overexpression of Myosin 1E full tail domain (TH1+TH2+TH3/SH3) decreases edge fluctuation. In Myosin 1E knockdown cells, and more prominently in cells treated with Myosin 1 inhibitor, cell–matrix adhesions are also short-lived and fail to mature. We suggest that, by moving to actin polymerization sites and early adhesion sites in active lamellipodia, Myosin 1E might play important roles in transporting not only important polymerizing proteins but also proteins involved in adhesion stabilization.

Gupta, Prabuddha; Gauthier, Nils C.; Cheng-Han, Yu; Zuanning, Yuan; Pontes, Bruno; Ohmstede, Malte; Martin, Rene; Knolker, Hans-Joachim; Dobereiner, Hans-Gunther; Krendel, Mira; Sheetz, Michael

2013-01-01

286

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.

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

2013-01-01

287

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

PubMed

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 98 th 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

2013-02-01

288

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.

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

2011-01-01

289

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

PubMed

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

2013-04-01

290

Specific Evolution of F1-Like ATPases in Mycoplasmas  

PubMed Central

F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the ?, ?, ? and ? subunits of F1 ATPases and could form an F1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F1-like structure is associated with a hypothetical X0 sector located in the membrane of mycoplasma cells.

Dautant, Alain; Bouyssou, Guillaume; Labroussaa, Fabien; Skollermo, Anna; Persson, Anja; Blanchard, Alain; Sirand-Pugnet, Pascal

2012-01-01

291

[Energetic applications: Na+/K+-ATPase and neuromuscular transmission].  

PubMed

Na/K-ATPase electrogenic activity and its indispensable role in maintaining gradients suggest that the modifications in isoform distribution and the functioning of the sodium pump have a major influence on both the neuronal functions, including excitability, and motor efficiency. This article proposes to clarify the involvement of Na/K-ATPase in the transmission of nerve influx within the peripheral nerve and then in the genesis, the maintenance, and the physiology of muscle contraction by comparing the data found in the literature with our work on neuron and muscle characterization of Na/K-ATPase activity and isoforms. PMID:19230940

Rigoard, P; Chaillou, M; Fares, M; Sottejeau, Y; Giot, J-P; Honfo-Ga, C; Rohan, J; Lapierre, F; Maixent, J-M

2009-03-01

292

NMR Structures of ?-Proteobacterial ATPase-Regulating ?-Subunits.  

PubMed

NMR structures of ?-subunits, which are recently discovered ?-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ?-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ?-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in ?-proteobacteria. PMID:24838125

Serrano, Pedro; Geralt, Michael; Mohanty, Biswaranjan; Wüthrich, Kurt

2014-07-15

293

Kinetic properties of mitochondrial ATPase during isoproterenol-induced cardiomyopathy.  

PubMed

1. The kinetic characteristics of the ATP hydrolysis by membrane-bound and Triton X-100 solubilized mitochondrial ATPase, during the isoproterenol-induced cardiomyopathy, were investigated. 2. An increase in the inhibitory action of the oligomycin, a decrease in the affinity of the ATP binding sites and an increase of both activation energy and rate of thermal inactivation were observed for mitochondrial ATPase. 3. The possibility that the changes described are related to the modifications of the active configuration of mitochondrial ATPase, during the isoproterenol-induced cardiomyopathy, is discussed. PMID:2143151

Curti, C; Uyemura, S A; Grecchi, M J; Leone, F A

1990-01-01

294

Electrogenic Na+ transport by Enterococcus hirae Na(+)-ATPase.  

PubMed

Energy-dependent generation of a membrane potential (delta psi) (-45 mV, interior negative) was observed in the F0F1, H(+)-ATPase-defective mutant of Enterococcus hirae. The generation of delta psi was found at high pH (but not at low pH), for which intracellular Na+ was required but not extracellular K+. The delta psi-generating activity was induced in cells cultured in media containing high concentrations of Na+, and was not observed in the Na(+)-ATPase mutants. These results suggest that E. hirae Na(+)-ATPase is responsible for the electrogenic sodium pump. PMID:7867809

Kakinuma, Y; Igarashi, K

1995-02-13

295

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

1981-01-01

296

Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy.  

PubMed

Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens. PMID:24478704

Hayek, Summer R; Lee, Samuel A; Parra, Karlett J

2014-01-01

297

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

Microsoft Academic Search

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

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

1993-01-01

298

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

Microsoft Academic Search

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

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

2000-01-01

299

Myosin, parvalbumin and myofibril expression in barbel ( Barbus barbus L.) lateral white muscle during development  

Microsoft Academic Search

Histo- and immunohistochemical techniques have recently been used to study the fibre type and myosin expression in fish muscle during development. In the present work, embryonic, larval and adult myosin isozymes (heavy and light chains) and parvalbumin isotypes were analyzed, from fertization to the adult stage, by polyacrylamide gel electrophoresis of barbel (Barbus barbus L.) trunk muscle extracts. The examined

Bruno Focant; Françoise Huriaux; Pierre Vandewalle; Manola Castelli; Guy Goessens

1992-01-01

300

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

PubMed

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

1997-04-01

301

Discovery of Omecamtiv Mecarbil the First, Selective, Small Molecule Activator of Cardiac Myosin  

PubMed Central

We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.

2010-01-01

302

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.

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

2013-01-01

303

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

PubMed Central

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

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

2012-01-01

304

Myosin-Va Transports the Endoplasmic Reticulum into the Dendritic Spines of Purkinje Neurons  

PubMed Central

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons (PNs) is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va1–3. Notably, the mechanism ensuring the ER's localization to spines has not been unraveled. While it has been proposed that animal class V myosins localize organelles by tethering them to the actin cytoskeleton4–7, we demonstrate here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into PN spines. Specifically, the myosin accumulates at the ER tip as the organelle moves into spines, and the myosin's ability to hydrolyze ATP is required for spine ER targeting. Moreover, myosin-Va is responsible for the vast majority of spine ER insertional events. Finally, attenuation of the myosin's ability to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we establish that an actin-based motor moves ER within animal cells, and we uncover the mechanism that mediates ER localization to PN spines, a prerequisite for synaptic plasticity.

Wagner, Wolfgang; Brenowitz, Stephan D.; Hammer, John A.

2012-01-01

305

The closed MTIP-MyosinA-tail complex from the malaria parasite invasion machinery  

PubMed Central

The Myosin A-tail Interacting Protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

Bosch, Jurgen; Turley, Stewart; Roach, Claudia M.; Daly, Thomas M.; Bergman, Lawrence W.; Hol, Wim G. J.

2009-01-01

306

Three-Dimensional Structure of Myosin Subfragment1: A Molecular Motor  

Microsoft Academic Search

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

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

1993-01-01

307

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

PubMed

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

2011-09-01

308

Diversity and variability of smooth muscle phenotypes of renal arterioles as revealed by myosin isoform expression  

Microsoft Academic Search

Diversity and variability of smooth muscle phenotypes of renal arterioles as revealed by myosin isoform expression. The contractility and extensibility of renal arterioles are important in the regulation of glomerular filtration. However, little is known regarding the characteristics of contractile proteins in these arterioles. Recently it was demonstrated that vascular smooth muscles contain two types of myosin heavy chain (MHC)

Kenjiro Kimura; Ryozo Nagai; Tatsuo Sakai; Masanori Aikawa; Makoto Kuro-o; Naoto Kobayashi; Isao Shirato; Tadashi Inagami; Masaya Oshi; Naoe Suzuki; Shigeyoshi Oba; Naobumi Mise; Akihiro Tojo; Yasunobu Hirata; Atsuo Goto; Yoshio Yazaki; Masao Omata

1995-01-01

309

Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish  

Microsoft Academic Search

Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the

Christoph Seiler; Orit Ben-David; Samuel Sidi; Oliver Hendrich; Alfons Rusch; Beth Burnside; Karen B. Avraham; Teresa Nicolsona

2004-01-01

310

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase  

PubMed Central

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force- producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.

1988-01-01

311

Substrate curvature sensing through Myosin IIa upregulates early osteogenesis.  

PubMed

Topographical cues mimicking the extracellular matrix (ECM) have demonstrated control over a diverse range of cellular behaviours including: initial adhesion, migration, cell growth, differentiation and death. How cells sense, and in turn translate, the topographical cues remains to be answered, but likely involves interactions through interfacial forces that influence cytoskeletal structure and integrin clustering, leading to the downstream activity of intracellular signalling cascades. Electrospun fibers have shown significant success as a biomimetic topography for bone tissue engineering applications, but mechanisms by which osteoprogenitor cells translate the fiber geometry into intracellular signalling activity is only recently being examined. We hypothesized that increased cellular differentiation observed on fibrous topography is due to acto-myosin contractility and cellular stiffness via the small GTPase RhoA. In order to evaluate this hypothesis, MC3T3-E1 osteoprogenitor cells were grown on poly(methyl methacrylate) (PMMA) fibers of 1.153 ± 0.310 ?m diameter. The elastic modulus of the cell surface was measured by atomic force microscopy (AFM) with a colloidal probe. Overall cellular stiffness was found to increase more than three-fold in osteoprogenitors adhered to a fiber, as opposed to those grown on a flat substrate. Pharmacological inhibition of RhoA signalling activity decreased cellular stiffness and cytoskeletal integrity of osteoprogenitors growing on fibrous substrates. Finally, we demonstrated not only RhoA activity through its effector Rho-associated coiled coil kinase II (ROCKII), but also Myosin IIa promotes early osteogenic differentiation, as shown by alkaline phosphatase (ALP) staining. Previous studies have demonstrated the importance of ROCKII on early differentiation. Our results shed light on mechanisms underlying geometry sensing by highlighting the role of Myosin IIa in addition to ROCKII and could ultimately contribute to scaffold design strategies. PMID:24104522

Ozdemir, Tugba; Xu, Li-Chong; Siedlecki, Christopher; Brown, Justin L

2013-11-01

312

[Clinical relevance of myosin isoforms in the diaphragm].  

PubMed

The diaphragm as a striated muscle is characterized by the repetition of a single element arranged in series: the sarcomere containing two kinds of myofilaments: a thick one constituted by the myosin, and a thin one primarily composed of actin. The myosin molecule consists of two heads where two myosin heavy chains (MHC) are fixed, a flexible hinge with two light (MLC) chains, and long rod-shaped tails. The diaphragm contains 4 MHC isoforms (MHC-slow, MHC-2A, MHC-2B, MHC-2X) and 6 MLC isoforms (MLC-1f, MLC-3f, MLC-1sa, MLC-1sb, MLC-2f, MLC-2s/v). In humans, the diaphragm contains mainly fibers expressing the isoforms MHC-slow, MHC-2A, and MLC-2f, MLC-2s et MLC-1f. For the mechanical properties of the different isoforms, there is a gradient from the MHC-slow to the MHC-2A, MHC-2B and MHC-2X/2B. According to the circumstances, the diaphragm will adapt towards a slow profile (COPD, cardiac failure and in animals: Duchenne muscular dystrophy, denervation-1 week, age-female, corticosteroids, chronic stimulation), or a fast profile (in animals: chronic hypoxia, denervation-2 weeks, age-males) or a more oxidative profile (in animals: cachexia, obesity). The reasons why the diaphragm adapts towards a slower or a faster muscle are not known. In fact, for a given pathological situation, several factors are able to influence the fiber composition of the diaphragm. Therefore, the net result of the influence of these different factors in terms of MHC and MLC diaphragm adaptation is difficult to predict. PMID:10939118

Gayan-Ramirez, G; Decramer, M

2000-06-01

313

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

NASA Astrophysics Data System (ADS)

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

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

2014-04-01

314

How are the cellular functions of myosin VI regulated within the cell?  

PubMed Central

This review, dedicated to the memory of Professor Setsuro Ebashi, focuses on our current work investigating the cellular functions and regulation of the unique unconventional motor, myosin VI. This myosin, unlike all the other myosins so far studied, moves towards the minus end of actin filaments and has been implicated in a wide range of cellular processes such as endocytosis, exocytosis, cell migration, cell division and cytokinesis. Myosin VI’s involvement in these cellular pathways is mediated by its interaction with specific adaptor proteins and is regulated by multiple regulatory signals and modifications such as calcium ions, PtdIns(4,5)P2 (PIP2) and phosphorylation. Understanding the functions of myosin VI within the cell and how it is regulated is now of utmost importance given the recent observations that it is associated with a number of human disorders such as deafness and cancers.

Buss, Folma; Kendrick-Jones, John

2008-01-01

315

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure  

PubMed Central

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5?-triphosphate (ATP) turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Malik, Fady I.; Hartman, James J.; Elias, Kathleen A.; Morgan, Bradley P.; Rodriguez, Hector; Brejc, Katjusa; Anderson, Robert L.; Sueoka, Sandra H.; Lee, Kenneth H.; Finer, Jeffrey T.; Sakowicz, Roman; Baliga, Ramesh; Cox, David R.; Garard, Marc; Godinez, Guillermo; Kawas, Raja; Kraynack, Erica; Lenzi, David; Lu, Pu Ping; Muci, Alexander; Niu, Congrong; Qian, Xiangping; Pierce, Daniel W.; Pokrovskii, Maria; Suehiro, Ion; Sylvester, Sheila; Tochimoto, Todd; Valdez, Corey; Wang, Wenyue; Katori, Tatsuo; Kass, David A.; Shen, You-Tang; Vatner, Stephen F.; Morgans, David J.

2014-01-01

316

Dynamics and regulation of contractile actin-myosin networks in morphogenesis  

PubMed Central

Contractile actin-myosin networks generate forces that drive cell shape changes and tissue remodeling during development. These forces can also actively regulate cell signaling and behavior. Novel features of actin-myosin network dynamics, such as pulsed contractile behaviors and the regulation of myosin localization by tension, have been uncovered in recent studies of Drosophila. In vitro studies of single molecules and reconstituted protein networks reveal intrinsic properties of motor proteins and actin-myosin networks, while in vivo studies have provided insight into the regulation of their dynamics and organization. Analysis of the complex behaviors of actin-myosin networks will be crucial for understanding force generation in actively remodeling cells and the coordination of cell shape and movement at the tissue level.

Kasza, Karen E.; Zallen, Jennifer A.

2012-01-01

317

Multimerization via Its Myosin Domain Facilitates Nuclear Localization and Inhibition of Core Binding Factor (CBF) Activities by the CBF Smooth Muscle Myosin Heavy Chain Myeloid Leukemia Oncoprotein  

Microsoft Academic Search

In CBF-SMMHC, core binding factor beta (CBF) is fused to the -helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba\\/F3 hematopoietic cells expressing a CBF-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBF-SMMHC(ACD) multimerized less effectively than either wild-type protein or a variant lacking

Tanawan Kummalue; Jianrong Lou; Alan D. Friedman

2002-01-01

318

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.

Zhao, Chenguang; Brown, Rebecca S. H.; Chase, Anna R.; Eisele, Markus R.; Schlieker, Christian

2013-01-01

319

The Na-K-ATPase and Calcium-Signaling Microdomains  

NSDL National Science Digital Library

The Na-K-ATPase is an energy-transducing ion pump that converts the free energy of ATP into transmembrane ion gradients. It also serves as a functional receptor for cardiotonic steroids such as ouabain and digoxin. Binding of ouabain to the Na-K-ATPase can activate calcium signaling in a cell-specific manner. The exquisite calcium modulation via the Na-K-ATPase is achieved by the ability of the pump to integrate signals from numerous protein and non-protein molecules, including ion transporters, channels, protein kinases/phosphatases, as well as cellular Na+. This review focuses on the unique properties of the Na-K-ATPase and its role in the formation of different calcium-signaling microdomains.

Jiang Tian (University of Toledo Health Science Campus Physiology and Pharmacology); Zi-jian Xie (University of Toledo Health Science Campus)

2008-08-01

320

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.

Betapudi, Venkaiah

2014-01-01

321

Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women.  

PubMed Central

Smooth muscle contraction is initiated primarily by an increase in intracellular Ca2+, Ca(2+)-dependent activation of myosin light chain kinase, and phosphorylation of myosin light chain. In this investigation, we identified pregnancy-associated alterations in myosin light chain phosphorylation, force of contraction, and content of contractile proteins in human myometrium. Steady-state levels of myosin light chain phosphorylation and contractile stress were correlated positively in both tissues, but the myometrial strips from pregnant women developed more stress at any given level of myosin light chain phosphorylation. During spontaneous contractions and during conditions that favor maximal generation of stress, the rate and extent of myosin light chain phosphorylation were attenuated in myometrial strips from pregnant women. The content of myosin and actin per milligram of protein and per tissue cross-sectional area was similar between myometrium of nonpregnant and pregnant women. Although cell size was significantly increased in tissues obtained from pregnant women, the amounts of contractile proteins per cellular cross-sectional area were similar. In addition, myosin light chain kinase and phosphatase activities were similar in the two tissues. The content of caldesmon was significantly increased in myometrium of pregnant women, whereas that of calponin (a smooth muscle-specific protein associated with the thin filaments) was not different. We conclude that adaptations of human myometrium during pregnancy include (a) cellular mechanisms that preclude the development of high levels of myosin light chain phosphorylation during contraction and (b) an increase in the stress generating capacity for any given level of myosin light chain phosphorylation. Images

Word, R A; Stull, J T; Casey, M L; Kamm, K E

1993-01-01

322

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.

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

2010-01-01

323

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.

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

2013-01-01

324

Regulation of endothelial signaling and migration by v-ATPase.  

PubMed

The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach. PMID:24254321

Rath, Sebastian; Liebl, Johanna; Fürst, Robert; Vollmar, Angelika M; Zahler, Stefan

2014-07-01

325

Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa  

Microsoft Academic Search

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

Tao Wang; Hua Li; Gang Lin; Chunyan Tang; Dongyang Li; Carl Nathan; K. Heran Darwin; Huilin Li

2009-01-01

326

Effect of Polygodial on the Mitochondrial ATPase of Saccharomyces cerevisiae  

Microsoft Academic Search

The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in Saccharomyces cerevisiae. In an acidification assay, polygodial completely suppressed the glucose-induced de- crease in external pH at 3.13 mg\\/ml, the same as the fungicidal concentration. Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p. Surprisingly, this ATPase was not directly inhibited by polygodial.

CHRISTOPHER S. LUNDE; ISAO KUBO

2000-01-01

327

V-ATPase expression in the mouse olfactory epithelium  

PubMed Central

The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa “A” subunit) and ATP6V1E1 (the ubiquitous 31-kDa “E” subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 (“B1,” typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 (“B2”) in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection.

Paunescu, Teodor G.; Jones, Abigail C.; Tyszkowski, Robert; Brown, Dennis

2008-01-01

328

Maternal–neonatal erythrocyte membrane Na + , K + ATPase and Mg 2+ ATPase activities in relation to the mode of delivery  

Microsoft Academic Search

Free radical production and high catecholamine levels are implicated in the modulation of Na+, K+-ATPase, and Mg2+-ATPase activities. The aim of this study was to investigate the effect of the mode of delivery on the above-mentioned enzyme\\u000a activities in maternal–neonatal erythrocyte membrane. Women with normal pregnancy (N = 30) were divided into two groups: Group A (N = 16) with normal labor and vaginal

Dimitrios G. Vlachos; Kleopatra H. Schulpis; Theodore Parthimos; Spyros Mesogitis; George D. Vlachos; Aris Antsaklis; Stylianos Tsakiris

2008-01-01

329

Vestibulo-ocular pathways modulate extraocular muscle myosin expression patterns.  

PubMed

The genetic and epigenetic influences that establish and maintain the unique phenotype of the extraocular muscles (EOMs) are poorly understood. The vestibulo-ocular reflex (VOR) represents an important input into the EOMs, as it stabilizes eye position relative to the environment and provides a platform for function of all other eye movement systems. A role for vestibular cues in shaping EOM maturation was assessed in these studies using the ototoxic nitrile compound 3',3'-iminodipropionitrile (IDPN) to eliminate the receptor hair cells that drive the vestibulo-ocular reflex. Intraperitoneal injections of IDPN were followed by a 2-week survival period, after which myosin heavy chain (MyHC) analysis of the EOMs was performed. When IDPN was administered to juvenile rats, the proportion of eye muscle fibers expressing developmental and fast myosins was increased, while EOM-specific MyHC mRNA levels were downregulated. By contrast, IDPN treatment in adult rats affected only the proportion of fibers expressing developmental MyHC isoforms, leaving the EOM-specific MyHC mRNA unaltered. These data provide evidence that the VOR modulates EOM-specific MyHC expression in development. The lack of significant changes in EOM-specific MyHC expression in adult EOM following IDPN administration suggests that there may be a critical period during development when alterations in vestibular activity have significant and permanent consequences for the eye muscles. PMID:10022967

Brueckner, J K; Ashby, L P; Prichard, J R; Porter, J D

1999-03-01

330

Dwell Time Distributions of the Molecular Motor Myosin V  

PubMed Central

The dwell times between two successive steps of the two-headed molecular motor myosin V are governed by non-exponential distributions. These distributions have been determined experimentally for various control parameters such as nucleotide concentrations and external load force. First, we use a simplified network representation to determine the dwell time distributions of myosin V, with the associated dynamics described by a Markov process on networks with absorbing boundaries. Our approach provides a direct relation between the motor’s chemical kinetics and its stepping properties. In the absence of an external load, the theoretical distributions quantitatively agree with experimental findings for various nucleotide concentrations. Second, using a more complex branched network, which includes ADP release from the leading head, we are able to elucidate the motor’s gating effect. This effect is caused by an asymmetry in the chemical properties of the leading and the trailing head of the motor molecule. In the case of an external load acting on the motor, the corresponding dwell time distributions reveal details about the motor’s backsteps.

Bierbaum, Veronika; Lipowsky, Reinhard

2013-01-01

331

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.

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

2012-01-01

332

Regulatory myosin light-chain genes of Caenorhabditis elegans.  

PubMed Central

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes. Images

Cummins, C; Anderson, P

1988-01-01

333

Distinct interactions between actin and essential myosin light chain isoforms.  

PubMed

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with ?-actin as well as ?-actin with alanin-mutated ELC binding site (?-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of ?-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to ?-actin (KD=575nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to ?-actin (KD=186nM). The reduced affinity of hALC-1 to ?-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1018M(-1)s(-1)) compared with kon of the hVLC-1/?-actin complex interaction (2908M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct ?-actin interactions. PMID:24857983

Petzhold, Daria; Simsek, Burcu; Meißner, Ralf; Mahmoodzadeh, Shokoufeh; Morano, Ingo

2014-07-01

334

Myosin binding protein C: implications for signal-transduction.  

PubMed

Myosin binding protein C (MYBPC) is a crucial component of the sarcomere and an important regulator of muscle function. While mutations in different myosin binding protein C (MYBPC) genes are well known causes of various human diseases, such as hypertrophic (HCM) and dilated (DCM) forms of cardiomyopathy as well as skeletal muscular disorders, the underlying molecular mechanisms remain not well understood. A variety of MYBPC3 (cardiac isoform) mutations have been studied in great detail and several corresponding genetically altered mouse models have been generated. Most MYBPC3 mutations may cause haploinsufficiency and with it they may cause a primary increase in calcium sensitivity which is potentially able to explain major features observed in HCM patients such as the hypercontractile phenotype and the well known secondary effects such as myofibrillar disarray, fibrosis, myocardial hypertrophy and remodelling including arrhythmogenesis. However the presence of poison peptides in some cases cannot be fully excluded and most probably other mechanisms are also at play. Here we shall discuss MYBPC interacting proteins and possible pathways linked to cardiomyopathy and heart failure. PMID:22173300

Knöll, Ralph

2012-05-01

335

ATPase kinetics for wild-type Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the beta-subunit Thr197-->Ser mutation.  

PubMed

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197-->Ser mutation in the beta subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thr197-->Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197-->Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197-->Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197-->Ser mutation as measured by determining the KiADP values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wild-type and mutant enzymes and the Thr197-->Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197-->Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197-->Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197-->Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197-->Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP. PMID:8026510

Mueller, D M; Indyk, V; McGill, L

1994-06-15

336

Non-muscle myosin IIB-like immunoreactivity is present at the drebrin-binding cytoskeleton in neurons.  

PubMed

Dendritic spines are extremely motile, providing a structural mechanism for synaptic plasticity. Actin-myosin interaction is thought to be responsible for the change in the shape of spine. We have already reported that drebrin, an actin-binding protein, inhibits actin-myosin interaction and is enriched in the dendritic spine of mature neurons. In this study, we prepared the actin cytoskeleton of dendritic spines as an immunoprecipitate with anti-drebrin antibody from adult guinea-pig brain, immunized mice with the cytoskeleton, and obtained a monoclonal antibody (MAb) called MAb G650. MAb G650 reacted with non-muscle myosin IIB, but it did not react with muscle myosin II or non-muscle myosin IIA. Immunoblotting with this antibody revealed that drebrin-binding cytoskeleton contains this myosin IIB-like immunreactivity. Immunohistochemistry using MAb G650 demonstrated that this myosin IIB-like immunreactivity can be detected in the neuronal cell bodies and their apical dendrites, where drebrin is hardly detected. These data demonstrate that a myosin subtype associated with drebrin-binding actin filaments in the dendritic spines is myosin IIB, although this myosin is widely distributed in somato-dendritic subdomains of neurons. Furthermore, it is indicated that the cytoskeletons in dendritic spine were uniquely characterized with actin-binding proteins such as drebrin, but not with myosins. PMID:10711814

Cheng, X T; Hayashi, K; Shirao, T

2000-02-01

337

In vitro motility assays and single molecule analyses reveal functional structural transitions in the molecular motor myosin  

NASA Astrophysics Data System (ADS)

The molecular basis of how myosin motors work has been significantly advanced by laser trap and other single molecule studies of myosins V and VI. Myosin V moves processively by stepping arm-over-arm, walking along the 36-nm pseudo-repeat of an actin filament by swinging its long lever arms through an angle of ˜70 ^o, and hydrolyzing one ATP per step. Compared to the laser trap, we have improved time resolution to submilliseconds by tracking single gold nanoparticle-myosin V conjugates using darkfield imaging, and have directly observed the behavior of the unbound head as the motor translocates. We have also developed a technique called single-molecule high resolution co-localization (SHREC), which allows simultaneous co-localization of two chromatically differing fluorophores only 10 nm apart. We used SHREC to directly observe myosin V molecules walking hand-over-hand. Myosin VI, a considerably different myosin family member, has been the biggest challenge to the lever arm hypothesis of myosin movement. It has a very short light chain binding domain (the conventional lever arm). Nevertheless, the molecule surprisingly steps processively 36 nm along an actin filament. Furthermore, myosin VI moves in the opposite direction to that of myosin II and myosin V. We now understand how this marvelous molecular motor achieves these feats.

Spudich, James

2010-03-01

338

Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI  

PubMed Central

SUMMARY Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C-terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30–36nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein we present new structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

Menetrey, Julie; Isabet, Tatiana; Ropars, Virginie; Mukherjea, Monalisa; Pylypenko, Olena; Liu, Xiaoyan; Perez, Javier; Vachette, Patrice; Sweeney, H. Lee; Houdusse, Anne M.

2012-01-01

339

Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex.  

PubMed

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia. PMID:11080149

Küssel-Andermann, P; El-Amraoui, A; Safieddine, S; Nouaille, S; Perfettini, I; Lecuit, M; Cossart, P; Wolfrum, U; Petit, C

2000-11-15

340

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

PubMed Central

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

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

1996-01-01

341

Involvement of myosin light-chain kinase in endothelial cell retraction  

SciTech Connect

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

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

1990-01-01

342

Differential Localization and Dynamics of Class I Myosins in the Enterocyte Microvillus  

PubMed Central

Epithelial cells lining the intestinal tract build an apical array of microvilli known as the brush border. Each microvillus is a cylindrical membrane protrusion that is linked to a supporting actin bundle by myosin-1a (Myo1a). Mice lacking Myo1a demonstrate no overt physiological symptoms, suggesting that other myosins may compensate for the loss of Myo1a in these animals. To investigate changes in the microvillar myosin population that may limit the Myo1a KO phenotype, we performed proteomic analysis on WT and Myo1a KO brush borders. These studies revealed that WT brush borders also contain the short-tailed class I myosin, myosin-1d (Myo1d). Myo1d localizes to the terminal web and striking puncta at the tips of microvilli. In the absence of Myo1a, Myo1d peptide counts increase twofold; this motor also redistributes along the length of microvilli, into compartments normally occupied by Myo1a. FRAP studies demonstrate that Myo1a is less dynamic than Myo1d, providing a mechanistic explanation for the observed differential localization. These data suggest that Myo1d may be the primary compensating class I myosin in the Myo1a KO model; they also suggest that dynamics govern the localization and function of different yet closely related myosins that target common actin structures.

Benesh, Andrew E.; Nambiar, Rajalakshmi; McConnell, Russell E.; Mao, Suli; Tabb, David L.

2010-01-01

343

Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis.  

PubMed

The human parasite Entamoeba histolytica is an ancient protozoan that expresses only one unconventional myosin, which has homology with myosin IB from other amoebae. Myosin IB is involved in phagocytosis of human cells by E. histolytica. In this work, we developed a microrheological technique, analysing magnetic phagosomes, which allowed us to probe the density of the F-actin network in living cells. Using this technique, we showed that overexpression of myosin IB led to an increase in cytoplasm viscosity, which correlated with a delay in initiating human cell phagocytosis. To investigate which myosin IB domains sustain cell viscosity changes, we overexpressed truncated forms of the protein. Our results demonstrate that both actin-binding sites that are present in the heavy chain but not the SH3 domain are required to modulate the density of the actin network. These data suggested that, as well as the motor activity, myosin IB in E. histolytica plays a structural role on the actin network owing to its ability to cross-link filaments. The gelation state of cell cytoplasm and the dynamics of cortical F-actin during phagocytosis seem to be modulated by the myosin IB structuring cytoskeleton activity. PMID:15226399

Marion, Sabrina; Wilhelm, Claire; Voigt, Heike; Bacri, Jean-Claude; Guillén, Nancy

2004-07-01

344

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

PubMed Central

Myosin VI supports movement toward the (?) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120° redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779–785]. However, this redirection alone is insufficient to account for the large (?)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (+)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (+)-end-directed motion, whereas longer constructs move toward the (?) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (?) end. We suggest that the lever arm rotates ?180° between pre- and poststroke conformations.

Bryant, Zev; Altman, David; Spudich, James A.

2007-01-01

345

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

PubMed Central

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

Naber, Nariman; Cooke, Roger

2011-01-01

346

Identification and characterization of multiple novel Rab-myosin Va interactions.  

PubMed

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

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

2013-11-01

347

Identification and characterization of multiple novel Rab-myosin Va interactions  

PubMed Central

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

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

2013-01-01

348

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.

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

1999-01-01

349

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.

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

2013-01-01

350

Rap1 Activation in Collagen Phagocytosis Is Dependent on Nonmuscle Myosin II-A  

PubMed Central

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or ?1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.

Arora, Pamela D.; Conti, Mary Anne; Ravid, Shoshana; Sacks, David B.; Kapus, Andras; Adelstein, Robert S.; Bresnick, Anne R.

2008-01-01

351

The role of PLK1-phosphorylated SVIL in myosin II activation and cytokinetic furrowing.  

PubMed

Polo-like kinase 1 (PLK1) is a widely conserved serine/threonine kinase that regulates progression of multiple stages of mitosis. Although extensive studies about PLK1 functions during cell division have been performed, it is still not known how PLK1 regulates myosin II activation at the equatorial cortex and ingression of the cleavage furrow. In this report, we show that an actin/myosin-II-binding protein, supervillin (SVIL), is a substrate of PLK1. PLK1 phosphorylates Ser238 of SVIL, which can promote the localization of SVIL to the central spindle and association with PRC1. Expression of a PLK1 phosphorylation site mutant, S238A-SVIL, inhibited myosin II activation at the equatorial cortex and induced aberrant furrowing. SVIL has both actin- and myosin-II-binding regions in the N-terminus. Expression of ?Myo-SVIL (deleted of the myosin-II-binding region), but not of ?Act-SVIL (deleted of actin-binding region), reduced myosin II activation and caused defects in furrowing. Our study indicates a possible role of phosphorylated SVIL as a molecular link between the central spindle and the contractile ring to coordinate the activation of myosin II for the ingression of the cleavage furrow. PMID:23750008

Hasegawa, Hitoki; Hyodo, Toshinori; Asano, Eri; Ito, Satoko; Maeda, Masao; Kuribayashi, Hirokazu; Natsume, Atsushi; Wakabayashi, Toshihiko; Hamaguchi, Michinari; Senga, Takeshi

2013-08-15

352

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.

1976-01-01

353

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis.  

PubMed

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. Here, we examine a collection of genes that cause defects in cytokinesis and reveal with live cell imaging two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. Our findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex. PMID:16174742

Dean, Sara O; Rogers, Stephen L; Stuurman, Nico; Vale, Ronald D; Spudich, James A

2005-09-20

354

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.

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

2009-01-01

355

Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection.  

PubMed

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin-actin) or mixed polarity (?-actinin-actin) bundles. The stepping dynamics of individual Qdot-labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor. PMID:23046080

Ali, M Yusuf; Previs, Samantha B; Trybus, Kathleen M; Sweeney, H Lee; Warshaw, David M

2013-01-01

356

Effects of shaker-1 mutations on myosin-VIIa protein and mRNA expression.  

PubMed

Numerous mammalian diseases have been found to be due to mutations in components of the actin cytoskeleton. Recently, mutations in the gene for an unconventional myosin, myosin-VIIa, were found to be the basis for the deafness and vestibular dysfunction observed in shaker-1 (sh1) mice and for a human deafness-blindness syndrome, Usher syndrome type 1B. Seven alleles of sh1 mice were analyzed to assess the affects of different myosin-VIIa mutations on both gene expression and tissue function. Myosin-VIIa is expressed in the inner ear and the retina, as well as the kidney, lung, and testis. Northern blot analysis indicated that myosin-VIIa mRNA expression, size, and stability were unaffected in the seven sh1 alleles. Immunoblot analysis showed that all seven alleles expressed some full-length myosin-VIIa protein. The range of expression, however, ran from sh1 [original], which expressed wild-type levels of protein, to two strains, sh1(4494SB) and sh1(4626SB), which expressed less than 1% of the normal level of myosin-VIIa protein. For the three alleles of sh1 that have been characterized and that have mutations in the motor domain, sh1 [original], sh1(816SB) and sh1(6J), the level of protein expression observed in these sh1 alleles correlated well with the predicted effects of the mutations on motor function. No change in retinal or testicular structure was observed at the light microscopic level during the life span of the seven sh1 alleles. Myosin-VIIa protein, when detectable, was observed to locate properly in the sh1 mice. On the basis of these results, we propose that the mutations in myosin-VIIa in the sh1 alleles leads to both motor dysfunction and to a protein destabilization phenotype. PMID:9186010

Hasson, T; Walsh, J; Cable, J; Mooseker, M S; Brown, S D; Steel, K P

1997-01-01

357

Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells  

PubMed Central

Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (?)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (?)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening.

Bhat, Purnima

2009-01-01

358

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain  

PubMed Central

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

2011-01-01

359

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

PubMed

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

2013-01-01

360

Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid.  

PubMed Central

The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.

Nwagwu, M

1975-01-01

361

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.

Rivard, Maxime; Couture, Charles-Andre; Miri, Amir K.; Laliberte, Mathieu; Bertrand-Grenier, Antony; Mongeau, Luc; Legare, Francois

2013-01-01

362

Spontaneous Detachment of the Leading Head Contributes to Myosin VI Backward Steps  

PubMed Central

Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.

Ikezaki, Keigo; Komori, Tomotaka; Yanagida, Toshio

2013-01-01

363

Myosin heavy chain isoform expression in rat smooth muscle development.  

PubMed

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms. PMID:9688613

White, S L; Zhou, M Y; Low, R B; Periasamy, M

1998-08-01

364

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.

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

2011-01-01

365

Chaperones of F[subscript 1]-ATPase  

SciTech Connect

Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11p for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.

Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas; Xu, Xingjue; Gatti, Domenico L.; Ackerman, Sharon H.; (WSU-MED); (NWU)

2009-09-25

366

Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions  

PubMed Central

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q? mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q? mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II.

Mondal, Subhanjan; Bakthavatsalam, Deenadayalan; Steimle, Paul; Gassen, Berthold; Rivero, Francisco; Noegel, Angelika A.

2008-01-01

367

On Myosin II dynamics in the presence of external loads.  

PubMed

We address the controversial hot question concerning the validity of the loose coupling versus the lever-arm theories in the actomyosin dynamics by re-interpreting and extending the phenomenological washboard potential model proposed by some of us in a previous paper. In this new model a Brownian motion harnessing thermal energy is assumed to co-exist with the deterministic swing of the lever-arm, to yield an excellent fit of the set of data obtained by some of us on the sliding of Myosin II heads on immobilized actin filaments under various load conditions. Our theoretical arguments are complemented by accurate numerical simulations, and the robustness of the model is tested via different choices of parameters and potential profiles. PMID:15946790

Buonocore, A; Caputo, L; Ishii, Y; Pirozzi, E; Yanagida, T; Ricciardi, L M

2005-08-01

368

Nonmuscle myosin II isoforms coassemble in living cells.  

PubMed

Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms. PMID:24814144

Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

2014-05-19

369

Sarcomere lattice geometry influences cooperative myosin binding in muscle.  

PubMed

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca(2+) activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge bind