Single Molecule Imaging Reveals the Mechanism Ofactin.Tn.Tm Activation and Deactivation

Striated muscle contraction is tightly regulated by the binding and release of calcium to troponin, which permits tropomyosin to move across actin exposing myosin binding sites. The process of activation is cooperative; myosins propagate the binding of further myosins. However unlike other cooperative systems that are limited by the constituent number of subunits, regulated actin filaments (RTFs) are continuous and therefore the cooperative unit size (regulatory unit) needs to be functionally defined. To provide molecular details of this cooperative activation process we devised an assay in which RTFs are suspended between pedestals above a microscope slide surface. GFP tagged myosin-S1 (FL-myosin-S1) binding to these RTFs was used to report activation. By measuring the binding of FL-myosin-S1 as a function of [ATP], [Ca2+] and [myosin] we were able to determine the mechanism of activation. Global fitting of these data revealed excellent agreement to a model described by a Ca2+ followed by a myosin induced activation switch, akin to that previously reported (McKillop and Geeves 1993). Our results indicate that there is little cooperativity in Ca2+ binding (nH ∼ 1.15) and the regulatory unit size is ∼ 11. Dynamic observation of FL-myosin-S1 binding and release offers further insight into activation. In sub-optimal activation conditions FL-myosin-S1 binds in clusters that remain active and are capable of diffusing along the RTF. The unbiased nature of their diffusion suggests that there is no propensity for new FL-myosin-S1s to bind and release from the pointed over the barbed end of an active cluster. In addition, we detect binding events consistent with stepwise loading of myosins; however their probability of detachment appears greater than expected for a stochastic process. We discuss the possibility that this coordinated release may be modulated by Tm.