MYOSIN HINGE REGION AND CONTRACTION

本项目的主要目标是利用果蝇突变体来研究 肌球蛋白“铰链”区域在肌肉结构和收缩中的作用。 果蝇肌球蛋白重链基因是独一无二的，因为 该基因通过选择性编码所有肌肉肌球蛋白重链。 剪接和影响肌肉功能的突变体可以很容易地分离和 分析过了。体外突变的基因可以稳定地插入到生殖系中。 此外，突变形式的蛋白质在某些肌肉中的表达 (飞行和跳跃)可以在不考虑功能改变的情况下进行研究 可能会导致致命的表型。该项目将重点研究 当这些肌肉的肌球蛋白含有替代蛋白时，这些肌肉的表型 用铰链代替了野生型。可选铰链通常是 在机械和超微结构截然不同的肌肉中表达 比飞行和跳跃肌肉更有特性。我们将确定 这种替代对成年生物体的飞行和跳跃能力。我们会 研究肌原纤维超微结构的改变是否由 肌球蛋白铰链的变化。我们将使用针对铰链多肽的抗体 以确定肌球蛋白是否有不同的定位 肌肉中表达肌球蛋白的替代铰链具有这两种类型 铰链。使用与上面概述的方法类似的方法，我们将检查 肌球蛋白棒的替代C末端“尾部”是否会影响 果蝇肌肉的结构和功能特征。如果更改 在生物体内检测到的功能是转换铰链或 尾部区域，未来的研究将涉及分析：L)点 旨在测试关键氨基酸残基的突变体 功能，2)解剖跳跃和跳跃肌肉的力学性质 肌纤维和单个分离的肌原纤维，以及3)体外运动 蛋白质之间的机械相互作用可以直接研究。此外, 基因抑制分析将用于确定肌球蛋白的区域 和其他与肌球蛋白铰链相互作用的蛋白质。总的来说，通过使用 与结构和功能分析相结合的分子遗传学方法， 我们将直接确定肌球蛋白的替代区域的影响 肌肉生理学方面的研究。

The major objective of this project is to use Drosophila mutants to study the role of the myosin "hinge" region in muscle structure and contraction. The Drosophila myosin heavy chain gene is unique in that a single copy of the gene encodes all muscle myosin heavy chains through alternative splicing, and mutants affecting muscle function can be easily isolated and analyzed. Genes mutated in vitro can be stably inserted into the germline. Furthermore, expression of mutant forms of the protein in certain muscles (flight, and jump) can be studied without concern that altered function might cause lethal phenotypes. The project will focus on the study of phenotypes of these muscles when their myosins contain an alternative hinge substituted for the wild type. The alternative hinge is normally expressed in muscles with very different mechanical and ultrastructural properties than flight and jump muscles. We will determine the effects of this substitution on flight and jump ability of adult organisms. We will study whether changes in myofibril ultrastructure are brought about by changes in the myosin hinge. We will use antibodies against hinge peptides to determine whether there is differential localization of myosins with alternative hinges in muscles that express myosins having both types of hinge. Using similar approaches to those outlined above, we will examine whether alternative C-terminal "tailpieces" of the myosin rod affect structural and functional characteristics of Drosophila muscle. If altered function is detected in the organisms that have switched hinge or tailpiece regions, future studies will involve analysis of: l) point mutants designed to test critical amino acid residues imparting altered function, 2) mechanical properties of dissected flight and jump muscle myofibers and single isolated myofibrils, and 3) in vitro motility where mechanical interaction of proteins can be studied directly. In addition, genetic suppression analysis will be used to determine regions of myosin and other proteins that interact with the myosin hinge. Overall, by using a molecular genetic approach coupled to structural and functional assays, we will directly determine the impact of alternative regions of the myosin rod on muscle physiology.