MYOSIN MOTOR IN ACTION

本节使用超微结构方法来测试 肌球蛋白头，从而提供了建模的基础， 肌球蛋白跨桥活动周期的过渡。执行 快速冷冻技术，结合笼状化合物， 为时间分辨结构分析提供了难得的机会 在完整肌原纤维的背景下，肌球蛋白马达的作用。我们建议使用 快速冷冻结合快速拉伸和释放方案， 同步青蛙纤维中交叉桥并关联它们的结构 状态和倾向与紧张发展。图像分析将 用于解剖肌球蛋白头的各个区域对 在张力发展期间形状改变。我们还将利用 昆虫飞行肌（IFM）的无与伦比的次晶秩序， 对肌球蛋白跨桥的合作探索， 导致三维电磁层析成像和三维分类的交叉桥梁， 对应分析（与M.K. Reedy博士合著）。跨桥将是 被困在松弛（MgATP松弛），早期的“预力”状态（乙二醇， AMPPNP冷或与Ca 2 +; ADP-AlF/4与Ca 2+），强结合状态 （僵硬）或在完全激活的纤维的快速拉伸之后。 该项目的第二个组成部分探讨了 肌球蛋白重链同种型和调节轻链在 果蝇肌原纤维的组装和收缩过程。 项目内的合作允许以下方面的富有成效的结合： 基因和转基因方法与功能和结构分析。 一种新的肌球蛋白、肌球蛋白杆蛋白或MRP，其中催化和 肌动蛋白结合头部区域被N末端延伸取代 与轻链同源，提供了独特的机会， 测试此扩展是否允许直接的网络共享交互， 肌动蛋白。遗传和转基因操作的结构效应， 改变在特殊飞行肌肉中正常表达的MRP的量， 通过一系列技术进行评估，从完整的 肌肉旋转阴影和负染色的孤立丝， 辅以衍射分析。肌原纤维超微结构和 转基因果蝇的间接飞行肌（IFM）的粗丝 将作为评估各种错误表达的影响的基础， 牵张激活反应和特定肌原纤维蛋白 这些肌肉的结构。IFM将被诱导表达肌球蛋白 具有选择性外显子11 S的重链属于其他肌肉， 拉伸激活属性，在空背景上。

This sections uses ultrastructural approaches to test for motions of the myosin head and thus provides the basis for modeling the force bearing transition in the myosin cross bridge activity cycle. Implementation of rapid freezing techniques, in combination with caged compounds has provided the exceptional opportunity for time resolved structural analysis of myosin motors in the context of the intact myofibril. We propose to use rapid freezing in combination with rapid stretch and release protocols to synchronize cross bridges in frog fibers and to correlate their structural states and disposition with tension development. Image analysis will be used to dissect the contribution of various regions of the myosin head to the shape changes during tension development. We will also take advantage of the unexcelled paracrystalline order of insect flight muscle (IFM) in a collaborative exploration of myosin cross bridges, that will eventually lead to 3-D EM tomography and 3-D classification of cross-bridges by correspondence analysis (with Dr. M.K Reedy). Cross bridges will be either trapped in relaxed (MgATP-relaxed), early "pre-force" states (glycol- AMPPNP cold or with Ca2+; ADP-AlF/4 with Ca2+), strong binding states (rigor) or following a rapid stretch of fully activated fibers. A second component of the project explores structure and function of myosin heavy chain isoforms and of the regulatory light chain in the processes of myofibril assembly and contraction in Drosophila. Collaborations within the program project allow a fruitful combination of genetic and transgenic approaches with functional and structural assays. A novel myosin, myosin rod protein or MRP, in which the catalytic and actin binding head region is substituted by an N terminal extension homologous to that of a light chain, offers a unique opportunity for tested whether this extension allows a direct, tethering interaction with actin. The structural effects of genetic and transgenic manipulations that vary the amount of MRP normally expressed in special flight muscle will be assessed by a battery of techniques, from thin sectioning of intact muscles to rotary shadowing and negative staining of isolated filaments, complemented by diffraction analysis. Ultrastructure of myofibrils and thick filaments from the indirect flight muscle (IFM) of transgenic flies will serve as basis for assessing effects of misexpression of various proteins on the stretch activation responses and on the specific myofibril architecture of these muscles. The IFMs will be induced to express myosin heavy chains with alternative exon 11S belonging to other muscles without stretch activation properties, over a null background.