INSECT FLIGHT MUSCLE: STRETCH-ACTIVATION & FORCE GENERATION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的计划将活跃工作肌肉的时间分辨X射线衍射与来自快速冷冻/冷冻替代电子显微镜的肌肉的3D重建相关联。 我们的目的是描述横纹肌中运动蛋白（肌动蛋白上的肌球蛋白）和调节蛋白（肌钙蛋白和原肌球蛋白）之间的结构和动态编排。 通过拉伸的机械触发对于人类心肌的生理特性（Frank-Starling行为）是重要的，并且对于大多数昆虫（>所有已知动物物种的一半！）的飞行是必不可少的，但其潜在机制尚不清楚。 最近发表的3D重建从我们的实验室确定了一类新认识的肌球蛋白和肌钙蛋白（'肌钙蛋白桥'）之间的连接，并提出了一种分子机制，我们已经测试了实时X射线衍射'电影'收缩昆虫飞行肌肉正弦拉伸激活。 结果是一致的假设，肌钙蛋白桥机械拖船原肌球蛋白在拉伸激活过程中，暴露肌动蛋白上的肌球蛋白结合位点，并允许随后延迟力的产生，但只有在存在钙。 在没有钙的情况下，拉伸松弛的肌肉引起可观察到的肌钙蛋白桥的运动，但仅限制原肌球蛋白的运动。 X光片还显示了螺旋肌球蛋白丝的意外扭曲，这可能是昆虫飞行肌肉所独有的特征。 目前的实验旨在剖析拉伸和钙之间的相互作用，并更具体地确定构成肌钙蛋白桥的确切化学伙伴，并着眼于探索类似的机械联系如何在其他肌肉中运作，例如哺乳动物的心肌。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our program correlates time-resolved X-ray diffraction of actively working muscle with 3-D reconstructions from muscles quick-frozen/freeze-substituted for electron microscopy. We aim to characterize the structure and dynamic choreography between motor proteins (myosin on actin) and the regulatory proteins (troponin and tropomyosin) in striated muscle. Mechanical triggering by stretch is important to the physiological properties (Frank-Starling behavior) of human cardiac muscle, and is essential to flight in most insects (> half of all known animal species!), but the underlying mechanism has been unknown. Recently published 3D reconstructions from our lab identified a newly recognized class of connections between myosin and troponin ('troponin bridges'), and suggested a molecular mechanism which we have tested with real-time X-ray diffraction 'movies' of contracting insect flight muscle that were sinusoidally stretch-activated. Results were consistent with the hypothesis that troponin bridges mechanically tug tropomyosin aside during stretch activation, exposing myosin-binding sites on actin and allowing subsequent delayed force generation, but only in the presence of calcium. In the absence of calcium, stretching relaxed muscles caused observable movements of the troponin bridges but only restricted movement of tropomyosin. The X-ray movies also revealed an unexpected twisting of the helical myosin filaments, a feature which may be unique to insect flight muscle. Current experiments are aimed at dissecting the interplay between stretch and calcium, and identifying more specifically the exact chemical partners that make up troponin bridges, with an eye towards exploring how analogous mechanical linkages might operate in other muscles, such as mammalian cardiac muscle.