Troponin I and troponin T interact with troponin C to produce different Ca2+-dependent effects on actin–tropomyosin filament motility

We have developed an in vitro motility assay to make a detailed quantitative analysis of Ca2+ control of skeletal-muscle troponin-tropomyosin control of actin-filament movement over immobilized myosin. Ca2+ regulates both filament velocity and the fraction of filaments that are motile. We have demonstrated that the two effects are due to separate interactions of troponin C with troponin I and troponin T. When 64 nM of the complex actin-tropomyosin-troponin I-troponin C was added at pCa 5, more than 80% of filaments were moving and their velocity did not change. At pCa 9, more than 20% of the filaments were moving. When 20 nM of the complex actin-tropomyosin-troponin T+troponin I+troponin C was added at pCa 5, filament motility remained high, whereas velocity increased. The 30% increase in velocity observed when troponin T was present was also observed when heavy meromyosin fragment 1 labelled with N-ethylmaleimide (NEM S-1) was added after actin-tropomyosin filaments. The NEM S-1 effect was not additive with the troponin T-dependent velocity increase. The pattern of motile behaviour is characteristic of myosin on silicone-treated glass and different from the behaviour on nitrocellulose-coated glass.

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Function of skeletal muscle myosin heavy and light chain isoforms by an in vitro motility assay.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80744-3 ◽

1993 ◽

Vol 268 (27) ◽

pp. 20414-20418

Author(s):

S Lowey ◽

G.S. Waller ◽

K.M. Trybus

Keyword(s):

Skeletal Muscle ◽

Light Chain ◽

Motility Assay ◽

In Vitro Motility Assay ◽

Skeletal Muscle Myosin ◽

In Vitro Motility ◽

Muscle Myosin

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Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around each other

Nature Communications ◽

10.1038/s41467-020-16328-z ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 2

Author(s):

Aniruddha Mitra ◽

Laura Meißner ◽

Rojapriyadharshini Gandhimathi ◽

Roman Renger ◽

Felix Ruhnow ◽

...

Keyword(s):

Mitotic Spindle ◽

Three Dimensional ◽

Helical Motion ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Torque Generation ◽

Free Microtubules ◽

20 Nm

Abstract Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

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Unidirectional Movement Of Fascin-induced Actin Bundle On Heavy Meromyosin In An In Vitro Motility Assay

Medicine & Science in Sports & Exercise ◽

10.1249/01.mss.0000323614.30214.85 ◽

2008 ◽

Vol 40 (Supplement) ◽

pp. S297

Author(s):

Hideyo Takatsuki ◽

Kevin M. Rice ◽

Shinichi Asano ◽

Devashish Desai ◽

Madhukar Kolli ◽

...

Keyword(s):

Motility Assay ◽

Heavy Meromyosin ◽

In Vitro Motility Assay ◽

Actin Bundle ◽

In Vitro Motility ◽

Unidirectional Movement

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A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0082 ◽

2021 ◽

pp. mbc.E21-02-0082

Author(s):

Samantha K. Barrick ◽

Lina Greenberg ◽

Michael J. Greenberg

Keyword(s):

Dilated Cardiomyopathy ◽

Calcium Binding ◽

Thin Filament ◽

Troponin T ◽

Calcium Sensitivity ◽

Cellular Level ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Fluorescence Measurements

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

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The binding sites of rabbit skeletal troponin-I on troponin-T

Canadian Journal of Biochemistry ◽

10.1139/o80-090 ◽

1980 ◽

Vol 58 (8) ◽

pp. 649-654 ◽

Cited By ~ 31

Author(s):

Joyce R. Pearlstone ◽

Lawrence B. Smillie

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Binding Sites ◽

Troponin I ◽

Troponin T ◽

Gel Filtration ◽

Troponin C ◽

Separate Site

Various fragments derived from rabbit skeletal muscle troponin-T (Tn-T) by chemical and (or) proteolytic cleavage were mixed with whole troponin-I (Tn-I) and applied to a Sephadex G-75 gel filtration column in order to determine the binding site of Tn-I on Tn-T. This site of interaction was found to span two distinct regions of Tn-T. The first site involves the highly acidic NH2-terminal fragment CB3 (residues 1–70 of Tn-T). A second separate site is located in the region of residues 152–209 of Tn-T. The present study, in conjunction with our earlier work on tropomyosin – Tn-T binding and Tn-T – troponin-C binding, depicts Tn-T as being a functionally efficient molecule composed of several distinct domains of specialized amino acid sequence, each of which carries out a role in the binding of a different protein.

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Glutathione reverses early effects of glycation on myosin function

AJP Cell Physiology ◽

10.1152/ajpcell.00502.2002 ◽

2003 ◽

Vol 285 (2) ◽

pp. C419-C424 ◽

Cited By ~ 24

Author(s):

B. Ramamurthy ◽

A. Daniel Jones ◽

L. Larsson

Keyword(s):

Skeletal Muscle ◽

Extracellular Proteins ◽

Dependent Manner ◽

Skeletal Muscle Function ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Glucose Exposure ◽

And Function ◽

Dose Dependent

Nonenzymatic glycosylation (glycation) has been recognized as an important posttranslational modification underlying alterations of structure and function of extracellular proteins during aging and diabetes. Intracellular proteins may also be affected by this modification, and glycation has been suggested to contribute to aging-related impairment in skeletal muscle function. Glycation is the chemical reaction of reducing sugars with primary amino groups resulting in the formation of irreversible advanced glycation end products. Glutathione is an abundant tripeptide in skeletal muscle. To understand the effect of glutathione on glycated myosin function, we used a single-fiber in vitro motility assay in which myosin is extracted from a single muscle fiber segment to propel fluorescent-labeled actin filaments. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of preincubation values after 30-min exposure to 1, 3, and 6 mM glucose, respectively. The 30-min 6 mM glucose incubation was followed by a 20-min 10 mM glutathione incubation. Glutathione treatment restored motility (0.98 ± 0.06 μm/s, n = 3; P < 0.001) after glucose exposure (0.10 ± 0.07 μm/s, n = 3), close to preincubation levels (1.12 ± 0.06 μm/s, n = 3). It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function.

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