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

2018 ◽  
Author(s):  
A. V. Inchingolo ◽  
M. Mihailescu ◽  
D. Hongsheng ◽  
N. M. Kad
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Calcium Binding ◽  
Thin Filament ◽  
Striated Muscle ◽  
Monte Carlo Model ◽  
Local Concentration ◽  
Single Molecule Imaging ◽  
Thin Filaments ◽  
Myosin Binding

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.

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

eLife ◽  
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.

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

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.

Regulation of Contraction in Striated Muscle

2000 ◽  
Vol 80 (2) ◽  
pp. 853-924 ◽  
Author(s):  
A. M. Gordon ◽  
E. Homsher ◽  
M. Regnier
Keyword(s):  
Binding Sites ◽  
Thin Filament ◽  
Striated Muscle ◽  
Force Development ◽  
Cross Bridge ◽  
Hydrolysis Products ◽  
Strong Binding ◽  
Regulation Of Contraction ◽  
Myosin Binding ◽  
Cross Bridges

Ca2+ regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A7TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca2+binding sites on TnC, conformational changes resulting from Ca2+ binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca2+ binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1–2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca2+-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the “open” probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A7TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca2+ binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca2+ regulates the strong binding of M·ADP·Pi to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca2+ activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A7TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca2+]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca2+ binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. These results and conclusions suggest possible explanations for differences between skeletal and cardiac muscle regulation and delineate the paths future research may take toward a better understanding of striated muscle regulation.

High-Resolution Measurements of Subsarcomere Calcium-Binding Distributions from EPXMA Images

1996 ◽  
Vol 54 ◽  
pp. 292-293
Author(s):  
M. E. Cantino ◽  
J. G. Eichen
Keyword(s):  
Calcium Binding ◽  
Thin Filament ◽  
Direct Evidence ◽  
Striated Muscle ◽  
Regulatory Protein ◽  
Psoas Muscle ◽  
Thin Filaments ◽  
Regulation Of Contraction

The distribution of calcium binding to thin filaments in vertebrate striated muscle provides clues about the role of calcium in regulation of contraction. A number of studies have suggested that the extent of Ca binding to the thin filament regulatory protein, troponin, is enhanced by the attachment of crossbridges. Electron probe X-ray microanalysis (EPXMA) has provided direct evidence of this enhancement by showing that more Ca is found in the overlap than in the nonoverlap region of sarcomeres in skinned rabbit psoas muscle in rigor. In that study, characterization of the spatial extent of this enhancement was limited by the pixel width of bands (4 pixels) within which Ca counts were averaged at each position across the sarcomere. In order to improve the resolution with which data are extracted from the image we have developed an automated routine for averaging calcium counts from successive columns of pixels across each half sarcomere in the image.

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

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.

Modulation of Striated Muscle Function is Reflected by Thick Filament Structure.

2000 ◽  
Vol 6 (S2) ◽  
pp. 76-77
Author(s):  
Rhea J.C. Levine ◽  
Irina Kulakovskaya ◽  
H. Lee Sweeney ◽  
Saul Winegrad ◽  
Zhaohui Yang
Keyword(s):  
Structural Changes ◽  
Striated Muscle ◽  
Contractile Function ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Thin Filaments ◽  
Calcium Dependent ◽  
Regulation Of Activity ◽  
Myosin Binding ◽  
Tissue Specimens

In mammalian skeletal and cardiac muscles, regulation of activity occurs when calcium binds to troponin on thin filaments, which ultimately results in exposure of myosin-binding sites on actin. However, modulation of contractile function, affecting such parameters as calcium sensitivity, the rate of rise of tension, the expression of maximum tension and/or the rate of onset of relaxation, is also calcium dependent. It is, in part, a property of the thick filament itself and its component myosin and/or accessory proteins. Among these are phosphorylation of myosin regulatory light chains or light chain 2 (RLCs; LC2) and in cardiac, but not skeletal fibers, phosphorylation of myosin-binding protein C (MyBP-C).Gentle methods of separating thick filaments from small tissue specimens, subjected to various experimental protocols designed to explore the functional parameters of such modulatory activities, allow examination of any accompanying structural changes.

scholarly journals Molecular-scale visualization of sarcomere contraction within native cardiomyocytes

2020 ◽  
Author(s):  
Laura Burbaum ◽  
Jonathan Schneider ◽  
Sarah Scholze ◽  
Ralph T Böttcher ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Thin Filament ◽  
Striated Muscle ◽  
Hexagonal Lattice ◽  
Muscular Contraction ◽  
Neonatal Rat ◽  
Molecular Architecture ◽  
Thin Filaments ◽  
Rat Cardiomyocytes ◽  
Thick Filaments ◽  
Activated State

Sarcomeres, the basic contractile units of striated muscle, produce the forces driving muscular contraction through cross-bridge interactions between actin-containing thin filaments and myosin II-based thick filaments. Until now, direct visualization of the molecular architecture underlying sarcomere contractility has remained elusive. Here, we use in situ cryo-electron to-mography to unveil sarcomere contraction in frozen-hydrated neonatal rat cardiomyocytes. We show that the hexagonal lattice of the thick filaments is already established at the neonatal stage, with an excess of thin filaments outside the trigonal positions. Structural assessment of actin polarity by subtomogram averaging reveals that thin filaments in the fully activated state form overlapping arrays of opposite polarity in the center of the sarcomere. Our approach provides direct evidence for thin filament sliding during muscle contraction and may serve as a basis for structural understanding of thin filament activation and actomyosin interactions inside unperturbed cellular environments.

scholarly journals CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mert Colpan ◽  
Jessika Iwanski ◽  
Carol C. Gregorio
Keyword(s):  
Muscle Contraction ◽  
Cardiac Muscle ◽  
Adenylyl Cyclase ◽  
Thin Filament ◽  
Essential Role ◽  
Striated Muscle ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Thin Filaments ◽  
Pointed End

AbstractThe precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

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