Thin Filament Regulation Blends Thermodynamic and Mechanical Mechanisms

Abstract

A simple thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson process. However, we find that frequencies of run time intervals do not support a simple exponential distribution expected for the time-dependent Poisson probability of an uninterrupted run. Each assay consists of fluorescently labeled native thin filaments (TF) on a surface of skeletal muscle HMM in a flow cell containing fixed -log free calcium (pCa). Whereas unregulated actin filaments move continuously, TF alternate randomly between time intervals of runs and pauses. We construct histograms of run times estimated visually from video recordings of assays with the most frequent events (pCa: 6.6, 6.2, 5.8, and 5.4) by unbiased observers. Whereas, at low calcium (pCa 6.6), the distribution appears indistinguishable from exponential, short run times are increasingly suppressed as pCa is decreased in the assay. The pCa 5.4 histogram most resembles gamma rather than exponential distribution. Goodness of fit of the expected and observed frequencies using chi square statistic confirms the calcium-dependent trend from exponential to gamma distribution. Contrary to our expectations, adding tropomyosin-troponin complex to same flow cell decreases rather than increases the proportion of short run times, thereby shifting the distributions away from exponential and toward gamma at all pCa. We can explain these observations mathematically if events during the run delay the rate that pause events arrive by a thermodynamic process. Given the constraint of constant free energy, we propose that the delay events arise by changes in mechanical force acting on the position of tropomyosin during the run. These results motivate further investigation of motion-dependent fluctuations and run-lengthening agents. Work supported by NIH R01-HL128683 (JRP) and leave supported by UWG (HGZ).