Stepwise translation of myofilaments

描述(由申请人提供)：本次竞赛更新的目的是继续探索肌丝的渐进式滑动。滑动的动力学将在申请人的实验室开发的两种制剂中进行探索：一种是单一的肌原纤维制剂，其中研究了单个肌节的缩短和延长的动力学；另一种是由单一肌动蛋白细丝滑动在单一肌球蛋白或粗丝上的制剂。初步地，两种制剂中的证据表明滑动是以阶梯状发生的，并且阶梯大小是2.7 nm(即，n×2.7)的整数倍。后续实验旨在测试这一范式是否真实。为此，将对两种制剂进行对照实验，并实施各种技术改进以提高分辨率。一个问题是，延长步幅和缩短步幅是否表现出相同的步幅范式。另一个问题是n&gt；1的值是否是有限分辨率的伪像，以及在分辨率较高的情况下，所有步骤是否具有不同的值。还将探讨暂停的持续时间(即步骤之间的时间间隔)。将探索暂停持续时间与负载的函数关系。最后，将实施几种策略(例如，减少单丝对的重叠)，以将交叉桥的数量减少到接近一个。以这种方式，将检查在极少跨桥的情况下的滑动动力学。尽管这可能不是一个“单分子”实验，但优势在于与肌动蛋白相互作用的肌球蛋白分子保持其自然构型。因此，这些实验应该会产生关于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.