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Inchingolo, A. V.

Publications and source records attributed to Inchingolo, A. V..

2 recordsLinked to original sources
bioRxiv2018

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

Cardiac 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.

biophysics↗
bioRxiv2018

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

Regulated 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.

biophysics↗