Models for Motility Assay Simulations. 

 

In recent years variety of in vitro motility assay has been developed to study the sliding of actin filaments by myosin bound to a coverslips. Typical assay images movement of fluorescent (rhodamine) labeled actin filaments myosin over coated surfaces or over fixed myosin filaments.

Motility Assays – labelled actin glides over myosin (HMM) coated surface to determine how speed is affected by ATP, its hydrolysis products and       .

The actin filaments, with or without regulatory proteins (cTn, Tm), are loaded into flow cells and movement and filament is recorded with video microscopy. These experiments include different conditions, for example concentration of calcium, or different geometrical relationships between an actin filament sliding over lawn of myosin molecules or over fixed myosin filament. In order to extract kinetic of regulated actin-MyBP-C interactions we are developing modules, associated with MUSICO platform, for simulation of these systems. As an example of this approach, we have developed a simple model for assessing affinity of myosin binding protein C (MyBP-C) to actin (Previs et al., 2012).

Effect of Mutations in MyBP-C on Sarcomere Mechanical Function. Recent measurements in motility assays provided measurements of the interactions kinetics between cMyBP-C, serving as a nonproducing crossbridge between myosin and actin filaments and as an inhibitor of myosin binding. Thus, these data suggested that actin-myosin transition rates are also significantly altered too. In order to assess the effect cMyBP-C mutations on affinity for cMyBP-C to actin we have developed a computational model for simulation of the Previs et al, 2012 experiments. These simulations include calculations of history of displacement of a short (250 nm) actin filament over myosin filament with intact cMyBP-C.

Simulations of sliding actin filament over regions of myosin filament with cMyBP-C (marked red in the far right figure) or without.  In absence of cMyBP-C (cMyBP-C null) the sliding speed is constant (about 1.85 µm) (blue line) as observed (insert C). In presence of cMyBP-C (WT, red line), the sliding velocity is the same as in previous case (~1.85 µm) until the tip of actin filament reaches the C-region, then slows down to (~1.28 µm), due to cMyBP-C binding to actin filament, mimicking the observations (insert B).

From fitting the data to observations of Previs et al., 2012, we estimated            in (         ) and equilibrium constant  of binding cMyBP-C to actin.

The predicted C-zone velocities closely matches observations for WT and cMyBP-C null (mutant) under different conditions. The best fits to data provided estimates of              in (        ) and equilibrium constant      of binding MyBP-C to actn. The model also predicted number of attached crossbridges and the number of cMyBP-C per Factin.

The predicted evolutions of displacements of actin filaments mimicking observations for WT and cMyBP-C null (mutant) under different conditions.

Further development of the motility assay model will include thin filament regulation and loading condition in variety of motility assay and experimental conditions. When fully developed this model will be suitable for studying the effect of mutations in the cMyBP-C gene relevant to a significant number of cardiac myopathies in children and adults. The estimated affinities of MyBP-C can be used in MUSICO 3D fibers simulations of disease.