MYOSIN MOTOR IN ACTION

本部分使用超微结构方法测试关节的运动 肌球蛋白头部，从而为建立受力模型提供了基础 肌球蛋白跨桥活动周期的转变。实施 快速冷冻技术与笼子里的化合物相结合 为时间分辨结构分析提供了难得的机会 在完整的肌原纤维的背景下的肌球蛋白马达。我们建议使用 快速冷冻与快速拉伸和释放方案相结合 使蛙纤维中的交叉桥同步并将它们的结构关联起来 状态和气质与紧张的发展。图像分析将是 用来解剖肌球蛋白头部的不同区域对 在张力发展的过程中，形状会发生变化。我们还将利用 在昆虫飞行肌肉(IFM)的无与伦比的晶状目中 对肌球蛋白跨桥的合作探索，最终将 导致3-D EM断层扫描和交叉桥的3-D分类 通信分析(与M.K·里迪博士)。跨桥将是 被困在松弛状态(镁ATP松弛)，早期的“力前”状态(乙二醇式) AMPPNP冷或与钙离子；ADP-Alf/4与钙离子)，强结合状态 (僵硬)或跟随着完全激活的纤维的快速伸展。 该项目的第二个组成部分探索了 肌球蛋白重链亚型及其调控轻链的研究 果蝇肌原纤维的组装和收缩过程。 计划项目内的协作允许富有成效的组合 利用功能和结构分析的遗传和转基因方法。 一种新的肌球蛋白，肌球蛋白棒蛋白或MRP，其中催化和 肌动蛋白结合头区被N端延伸部分取代 与轻链同源，提供了一个独特的机会 已测试此扩展是否允许与 肌动蛋白。基因和转基因操作的结构效应 在特殊飞行肌肉中通常表达的MRP的量将有所不同 通过一系列技术评估，从完整的薄层切面 肌肉到旋转阴影和孤立细丝的负染， 并辅之以衍射分析。肌原纤维和肌原纤维的超微结构 转基因果蝇间接飞行肌肉(IFM)的粗丝 将作为评估各种错误表达的影响的基础 拉伸激活反应和特定肌原纤维上的蛋白质 这些肌肉的结构。诱导IFMS表达肌球蛋白 带有外显子11的重链属于其他肌肉，没有 在空背景上扩展激活属性。

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.