Stepwise translation of myofilaments

描述（由申请人提供）：这项竞争性更新的目的是继续探索肌丝的逐步滑动。滑动动力学将在申请人实验室开发的两种制剂中进行探索：一种单一肌原纤维制剂，其中研究单个肌节的缩短和延长动力学；一种由单个肌动蛋白丝在单个肌凝蛋白或粗丝上滑动组成的制剂。两种制备中的初步证据表明，滑动是分步发生的，步长是2.7 nm的整数倍（即n x 2.7）。后续的实验被设计来检验这种范式是否真实。为此，将对两种制剂进行控制实验，并实施各种技术改进以提高分辨率。一个问题是，步长和步短是否表现出相同的步进范式。另一个问题是n > 1的值是否是有限分辨率的工件，以及在更高分辨率下，所有步骤是否都具有不同的值。暂停的持续时间（即步骤之间的间隔）也将被探讨。将探讨暂停时间对负载的函数依赖性。最后，将实施几种策略（例如，减少单灯丝对的重叠）以减少交叉桥的数量，使其接近1。以这种方式，滑动动力学与很少的交叉桥梁将被检查。虽然这可能不是一个“单分子”实验，但其优点是与肌动蛋白相互作用的肌球蛋白分子保持在其自然结构中。因此，实验应该能提供有关收缩过程的近分子性质的有价值的信息。

DESCRIPTION (provided by applicant): 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 applicant: 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.