Abstract
In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads.
Highlights
For an evaluation of the ensemble behaviour of myosin II molecules, we have taken advantage of actin filament breakage and have quantified the number of motors involved in processive sliding in the classical IVMA at various heavy meromyosin (HMM) densities and ATP concentrations
We find that only those filaments that are able to interact with Nmin ≤ N ≤ Nmax HMM molecules slide continuously in the assay
Breakage can occur due to inherent defects of the IVMA, e.g. presence of dead heads or random orientation of motors, here we show that breakage of actin filament can be induced by only changing the ATP concentration in a step exchange of flow chamber solution
Summary
For an evaluation of the ensemble behaviour of myosin II molecules, we have taken advantage of actin filament breakage and have quantified the number of motors involved in processive sliding in the classical IVMA at various heavy meromyosin (HMM) densities and ATP concentrations. We find that only those filaments that are able to interact with Nmin ≤ N ≤ Nmax HMM molecules slide continuously in the assay. If the number of interacting molecules per actin is less than the minimum limit, Nmin, the actin filament is unable to sustain processive sliding. When the number of available HMM molecules per actin filament is greater than the maximum limit, Nmax, the filaments are not able to sustain processive sliding and undergo breakage into smaller fragments. Actin filament breakage is consistent with a model in which strong binding of ‘Na’ HMM molecules on actin leads to a build-up of compressive stress between force-bearing heads and resisting rigor heads that eventually cause actin filament breakage by buckling
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