Effect of Calcium on Myosin Binding to a Regulated Thin Filament from Single Molecule to Ensemble

AbstractRegulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which in turn shifts the position of tropomyosin on actin to expose myosin binding sites. The binding of the first myosin holds tropomyosin in a position such that more myosin binding sites on actin are available, resulting in cooperative activation. Troponin and tropomyosin also act to turn off the thin filament; however, this is antagonized by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation we use the RTF tightrope assay, in which single RTFs are suspended between pedestals above a microscope coverslip surface. Single molecule imaging of GFP tagged myosin-S1 (S1-GFP) is used to follow the activation of RTF tightropes. In sub-maximal activation conditions, S1-GFP molecules bind forming metastable clusters, from which release and rebinding of S1-GFP leads to prolonged activation in these regions. Because the RTFs are not fully active we are able to directly observe deactivation in real time. Using a Reversible Jump Markov Chain Monte Carlo model we are able to dynamically assess the fate of active regions. This analysis reveals that myosin binding occurs in a stochastic stepwise fashion; however, an unexpectedly large probability of multiple simultaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated, active process.

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Single-molecule imaging reveals the concerted release of myosin from regulated thin filaments

eLife ◽

10.7554/elife.69184 ◽

2021 ◽

Vol 10 ◽

Author(s):

Quentin M Smith ◽

Alessio V Inchingolo ◽

Madalina-Daniela Mihailescu ◽

Hongsheng Dai ◽

Neil M Kad

Keyword(s):

Single Molecule ◽

Binding Sites ◽

Calcium Binding ◽

Thin Filament ◽

Fluorescent Protein ◽

Striated Muscle ◽

Local Concentration ◽

Single Molecule Imaging ◽

Thin Filaments ◽

Myosin Binding

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.

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Single Molecule Visualization of Cardiac Myosin-Binding Protein C N-terminal Fragments Interacting with Thin Filaments: Mechanisms of Calcium Sensitization

10.1101/421776 ◽

2018 ◽

Author(s):

Alessio V. Inchingolo ◽

Samantha Beck Previs ◽

Michael J. Previs ◽

David M. Warshaw ◽

Neil M. Kad

Keyword(s):

Single Molecule ◽

Protein C ◽

Thin Filament ◽

Binding Protein ◽

Binding Mode ◽

Cardiac Myosin ◽

Thin Filaments ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

AbstractCardiac muscle contraction is activated by calcium binding to troponin and the consequent motion of tropomyosin on actin within the sarcomere. These movements permit myosin binding, filament sliding and motion generation. One potential mechanism by which the N-terminal domains of cardiac myosin-binding protein C (cMyBP-C) play a modulatory role in this activation process is by cMyBP-C binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently-labelled cMyBP-C N-terminal fragments and GFP-labelled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound cooperatively, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that utilizes a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium and thus, enhance myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

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Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers

Biophysical Journal ◽

10.1016/s0006-3495(95)80316-4 ◽

1995 ◽

Vol 68 (4) ◽

pp. 1430-1442 ◽

Cited By ~ 60

Author(s):

J.M. Metzger

Keyword(s):

Skeletal Muscle ◽

Cardiac Myocytes ◽

Thin Filament ◽

Muscle Fibers ◽

Skeletal Muscle Fibers ◽

Myosin Binding ◽

Cooperative Activation

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The structure of the native cardiac thin filament at systolic Ca2+ levels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024288118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2024288118

Author(s):

Cristina M. Risi ◽

Ian Pepper ◽

Betty Belknap ◽

Maicon Landim-Vieira ◽

Howard D. White ◽

...

Keyword(s):

Cardiac Muscle ◽

Binding Sites ◽

Short Range ◽

Bound States ◽

Thin Filament ◽

Narrow Range ◽

The Other ◽

Thin Filaments ◽

Troponin Complex ◽

Myosin Binding

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

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Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner

Scientific Reports ◽

10.1038/s41598-018-21053-1 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 13

Author(s):

Brian Leei Lin ◽

Amy Li ◽

Ji Young Mun ◽

Michael J. Previs ◽

Samantha Beck Previs ◽

...

Keyword(s):

Protein C ◽

Thin Filament ◽

Binding Protein ◽

Dependent Manner ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Skeletal Myosin ◽

Myosin Binding

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1P243 Switch action of troponin I on the Ca^-regulated thin filament as revealed by spin-labeling distance measurement using PELDOR(8. Muscle contraction and muscle protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Seibutsu Butsuri ◽

10.2142/biophys.46.s207_3 ◽

2006 ◽

Vol 46 (supplement2) ◽

pp. S207

Author(s):

Tomoki Aihara ◽

Motoyoshi Nakamura ◽

Shoji Ueki ◽

Masao Miki ◽

Toshiaki Arata

Keyword(s):

Muscle Contraction ◽

Thin Filament ◽

Poster Session ◽

Troponin I ◽

Muscle Protein ◽

Spin Labeling ◽

Distance Measurement ◽

Meeting Program ◽

Regulated Thin Filament

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pH dependence of myosin binding-induced activation of the thin filament in cardiac myocytes and skeletal fibers

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.270.3.h1008 ◽

1996 ◽

Vol 270 (3) ◽

pp. H1008-H1014 ◽

Cited By ~ 1

Author(s):

J. M. Metzger

Keyword(s):

Skeletal Muscle ◽

Cardiac Myocytes ◽

Thin Filament ◽

Ph Dependence ◽

Muscle Fibers ◽

Isometric Tension ◽

Experimental Conditions ◽

Skeletal Muscle Fibers ◽

Myosin Binding ◽

Fast Twitch

The pH dependence of myosin binding-induced thin filament activation was determined in permeabilized cardiac myocytes and slow- and fast-twitch single skeletal muscle fibers by experimental lowering of [MgATP] in the Ca(2+)-free solutions bathing the permeabilized preparations. As the pS (where S is [MgATP] and pS is -log[MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pS range of 4.9-5.3. At pH 7.00, the transition from the relaxed to the activated rigor state was steep in cardiac myocytes [Hill value (nH) = 21.2 +/- 3.1 (SE)] and due to the apparent effect of strongly bound cross bridges to cooperatively activate the thin filament in the absence of added Ca2+. At pH 6.20, the steepness of the tension-pS relationship was markedly reduced (nH = 6.1 +/- 1.0) and the midpoint of the relationship (pS50) was shifted to higher pS values in cardiac myocytes. In comparison, reduced pH had no effect on the steepness or position of the tension-pS relationship in single slow- or fast-twitch skeletal muscle fibers. These findings suggest that myosin binding-induced activation of the thin filament is pH dependent in cardiac myocytes but not in skeletal muscle fibers under these experimental conditions in which Ca2+ is absent.

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