Calcium regulation of thin filament movement in an in vitro motility assay

Homsher, Earl; Kim, Bibiana; Bobkova, A. F.; Tobacman, Larry S.

doi:10.1016/s0006-3495(96)79753-9

Cited by 129 publications

(177 citation statements)

References 33 publications

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“…The free Ca 2ϩ concentration in the ATP/Tm⅐Tn solution was controlled using a Ca 2ϩ -EGTA buffer system containing 2 mM EGTA and 44 M to 2 mM CaCl 2 . Individual filaments were classified into the following three groups based on their motility, according to the criteria of Homsher et al (30): moving at a uniform speed, moving erratically, and not moving. Filaments were judged not to be moving if the measured velocity was below a cutoff value, 0.5 m/s, which is the mean ϩ 2 of the measured "velocities" of control filaments immobilized to the HMM-coated surface in the absence of ATP.…”

Section: Methodsmentioning

confidence: 99%

“…The differences of velocities and percentages of filaments moving at uniform speeds at pCa 6.7 between each pair of WT filaments and R95C filaments or cofilaments were significant (p Ͻ 0.05, t test). A significant fraction of filaments was not moving at intermediate pCa, and to avoid very large S.D., those stalled filaments were excluded from velocity analysis (30). However, this procedure resulted in excessive high velocity at high pCa if a very small number of filaments moved at velocities above the cutoff, even though the vast majority of the filaments had stalled.…”

Section: R95c E226k G268dmentioning

confidence: 99%

“…In the R95C mutant, a positive charge is removed, so that Tm may tend to shift away from the blocked state. With respect to the second mechanism, it is known that the number of cross-bridges per unit length of the thin filament alters Ca 2ϩ sensitivity (30). Because R95C filaments have a higher affinity for myosin than WT filaments, a larger number of HMM molecules may interact with R95C filaments, so that FIGURE 9.…”

Section: Velocitymentioning

confidence: 99%

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Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Noguchi

Gomibuchi

Murakami

et al. 2010

Journal of Biological Chemistry

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Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin⅐gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca 2؉ . Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca 2؉ sensitivity in the presence of tropomyosin-troponin.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

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Section: Methodsmentioning

confidence: 99%

Section: R95c E226k G268dmentioning

confidence: 99%

Section: Velocitymentioning

confidence: 99%

See 1 more Smart Citation

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Noguchi

Gomibuchi

Murakami

et al. 2010

Journal of Biological Chemistry

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“…Up to 50 filaments per heart sample were tracked within a field in which Ͼ90% of the filaments were moving. For each filament, its mean velocity and SD were determined, and filaments having a SD of the mean velocity of 30% or better were accepted, which was indicative of a continuous and smoothly moving actin filament (39). The velocity data in Table 3 are reported for multiple hearts as the mean and SD based on the mean velocity of the recorded filaments for each heart.…”

Section: )mentioning

confidence: 99%

Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function

Schmitt

Debold²,

Ahmad³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

108

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Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the ␣-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin (V actin) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower V actin in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated.animal models ͉ biophysics ͉ genetics ͉ single molecule ͉ laser trap D ilated cardiomyopathy (DCM) is an early step in the pathway leading to heart failure, the leading cause of human morbidity and mortality (1, 2). Although etiological factors such as ischemia and diabetes can result in DCM, at least 30% of cases are genetic in origin, with mutations found in sarcomeric proteins associated with the cytoskeletal and contractile systems (3). Clinically, DCM is characterized by myocardial hypertrophy, thin-walled ventricles, and myocyte hypoplasia (2, 4). Dilated hearts are hypocontractile, with systolic dysfunction leading to a reduced ejection fraction: a hallmark of a failing heart (2, 3). Only recently have autosomal dominant missense mutations to the ␤-cardiac myosin heavy chain (MHC) been identified that result in DCM (5, 6). Because the primary defect resides within the heart's molecular motor, we hypothesized that altered cardiac contractile function in DCM hearts might reflect changes in the mutant myosin's ability to generate force and motion as it cyclically interacts with actin during its hydrolysis of ATP.To investigate the impact of two distinct DCM-causing MHC mutations (S532P and F764L) on cardiac myosin's molecular performance, we have genetically engineered these missense mutations separately into the murine ␣-cardiac MHC gene, the human ␤-MHC homolog, to generate two separate DCM knockin mouse models. In addition to cardiac dilation and dysfunction, defects in isolated myocyte contractility were observed that are consistent with DCM. Interestingly, defects at the whole-heart and cellular levels were cor...

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“…Different technical approaches to measuring troponin control of filament motility have produced differing patterns of results in some experimental circumstances (these are discussed in Ref. 28); however, all the published analyses agree that reduction in the proportion of filaments that are motile is a characteristic feature of actin-tropomyosin filaments switched off by troponin or other inhibitory proteins (22,32,46,47). At pCa5 troponin did not alter the fraction of filaments motile or the velocity.…”

Section: Comparison Of Rabbit Skeletal and Wild Type Drosophilamentioning

confidence: 99%

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Bing¹,

Razzaq²,

Sparrow³

et al. 1998

Journal of Biological Chemistry

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In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin؉troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of the filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament regulation.

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Calcium regulation of thin filament movement in an in vitro motility assay

Cited by 129 publications

References 33 publications

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

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