Stepwise translation of myofilaments

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The aim of this competing renewal is a continued exploration of stepwise sliding of myofilaments. Dynamics of sliding will be explored in two preparations that have been developed in the laboratory of the PI: a single myofibril preparation in which the dynamics of shortening and lengthening in single sarcomeres is investigated; and, a preparation consisting of a single actin filament sliding over a single myosin or thick filament. Preliminary,evidence in both preparations indicates that sliding occurs in steps and step size is an integer multiple of 2.7 nm, i.e. n x 2.7. Followup experiments are designed to test whether this paradigm is genuine. To this end, control experiments will be carried out on both preparations, and various technical improvements will be implemented to improve resolution. One question is whether lengthening steps and shortening steps show the same stepping paradigm. Another is whether values of n > 1 are artifacts of limited resolution, and whether, with higher resolution, all steps are of a distinct value. Duration of pauses, i.e., periods between steps, will also be explored. The functional dependence of pause duration on load will be explored. Finally, several strategies will be implemented, e.g., diminished overlap of single filament pairs, to reduce the number of cross-bridges to approach one. In such a way, dynamics of sliding with very few cross-bridges will be examined. Although this may not be a "single molecule" experiment, the advantage is that myosin molecules that interact with actin remain in their natural configuration. Hence, the experiments should yield valuable information on the near-molecular nature of the contractile process.