Myosin-based kinetic differences between muscle types

描述（由申请人提供）：横纹肌的一个独特特征是其纤维类型的巨大多样性。大部分功能多样性是由于一系列肌球蛋白亚型，有助于确定肌肉速度。分子水平的序列比较和功能研究表明，肌球蛋白重链的特定结构域调节可能影响肌肉速度的过桥循环的动力学步骤。我们将利用果蝇系统的独特功能，研究肌球蛋白头部结构和肌肉动力学的异构体差异之间的关系。一个主要的进步将是使用肌原纤维制剂，允许精确操纵肌节长度和离子浓度（钙，MgATP，MgADP和磷酸盐），从中我们可以推导出高精度的肌动蛋白-肌球蛋白跨桥循环的动力学常数。该研究解决了3个基本问题：（i）什么步骤（S）的过桥周期建立了一个非常快的肌肉和一个非常慢的肌肉之间的主要动力学差异？(ii)具有中间速度的纤维类型是否具有在极端速度之间的交叉桥速率常数？和（iii）肌球蛋白跨桥循环的哪些步骤受到肌球蛋白同种型之间特定可变区变化的影响？为了回答这些问题（框架作为假设），我们将对比动力学方案获得的肌原纤维的间接飞行肌（IFM，一个非常快的肌肉），跳跃肌（快速肌肉），IFM肌原纤维转基因表达肌球蛋白从胚胎体壁肌（EMB，一个非常慢的肌肉），IFM肌原纤维表达嵌合体的IFM和EMB肌球蛋白。通过将动力学差异与特定结构区域（包括转换器区域）相关联，我们将基于肌球蛋白的最新结构模型推导分子机制。这项研究的主要优势是完全集成的方法（从单个分子到整个动物），使之成为可能，通过采用果蝇，并使用肌原纤维的桥梁实验与分离肌球蛋白和皮肤纤维实验之间的差距。

DESCRIPTION (provided by applicant): A unique feature of striated muscle is its enormous diversity of fiber types. Much of the functional diversity is due to a range of myosin isoforms that help determine muscle speed. Sequence comparisons and functional studies at the molecular level have suggested specific structural domains of the myosin heavy chain modulate kinetic steps of the crossbridge cycle that presumably affect muscle speed. We will exploit unique features of the Drosophila system for investigating relationships between isoform differences in myosin head structure and muscle kinetics. A major advance will be the use of myofibrillar preparations that allow precise manipulation of sarcomere length and ion concentrations (calcium, MgATP, MgADP and phosphate), from which we can deduce kinetic constants of the acto-myosin cross-bridge cycle with high precision. The research addresses 3 basic questions: (i) what step(s) of the crossbridge cycle establishes the major kinetic differences between a very fast muscle and a very slow muscle? (ii) do fiber types with intermediate speeds have crossbridge rate constants between those of the extremes? and (iii) what steps of the myosin crossbridge cycle are affected by changes in specific variable region(s) between myosin isoforms? To answer these questions (framed as hypotheses), we will contrast kinetic schemes obtained from myofibrils of indirect flight muscle (IFM, an extremely fast muscle), jump muscle (a fast muscle), IFM myofibrils transgenically expressing myosin from embryonic body wall muscle (EMB, a very slow muscle), and IFM myofibrils expressing chimeras of IFM and EMB myosin. By correlating kinetic differences to specific structural regions (including the converter region), we will deduce molecular mechanisms based on the latest structural models of myosin. The major strengths of this investigation are the fully integrated approach (from single molecules to whole animal) made possible by employing Drosophila, and the use of myofibrils which bridges a gap between experiments with isolated myosin and skinned fiber experiments.