Mechanisms of Kinesin Regulation

分子运动驱动蛋白-1执行大量的运输任务，并且其调节机制 管理这些进程至关重要。驱动蛋白-1的错误调节或驱动蛋白-1货物的错误定位可能 与帕金森病、神经纤维瘤病、精神分裂症等多种疾病有关， 腓骨肌萎缩症驱动蛋白-1的运动机制已经很清楚，我们现在也知道， 驱动蛋白-1的C末端尾部直接与头部相互作用，并在马达不需要运输货物时抑制头部 运输然而，我们不知道驱动蛋白-1调节剂如何启动或停止货物运输。尾部 当然参与，因为它结合到头部，微管，和几个不同的驱动蛋白-1激活剂，功能在 不同的运输系统。与尾部分开，Miro蛋白具有直接的Ca2+依赖性相互作用 与驱动蛋白-1 s酶的头部结构域，和米罗是需要钙依赖性抑制 线粒体运动我们假设尾部是一个内在无序的结构域， - 促进参与驱动蛋白-1自身抑制的多个结合配偶体相互作用的灵活性，和/或 激活，而Miro具有独特的机制，直接抑制驱动蛋白-1头部的酶促机制 来抑制线粒体运动为了解决这一假设，我们将首先在体外获得详细的信息， 关于驱动蛋白1尾部的结构及其与结合伴侣的相互作用，通过NMR和EPR 谱我们将确定Miro是否是一种直接的、Ca2+可转换的驱动蛋白-1酶抑制剂， 活性，使用EPR评估其对驱动蛋白-1机制的影响，并通过以下方法绘制其与驱动蛋白-1头部的相互作用： 交联。在获得关于尾巴和米罗的结构和机制信息之后，我们将 通过在体内控制驱动蛋白-1，通过成像， 果蝇S2细胞中的线粒体。这些目标将共同提供一个令人兴奋的新的桥梁， 体外生物物理学和细胞生物学工作的分子马达运输机制。此外，作为米罗和 其他驱动蛋白-1调节剂与几种神经系统疾病有关，我们的工作将提供 详细的，相关的生化信息和试剂，将加速努力开发治疗。

The molecular motor kinesin-1 performs a large number of transport tasks, and the regulatory mechanisms governing those processes are critical. Mis-regulation of kinesin-1 or mis-localization of kinesin-1 cargoes may be implicated in several diseases such as Parkinson¿s disease, neurofibromatosis, schizophrenia, and Charcot-Marie-Tooth disease. Kinesin-1¿s motile mechanism is well understood, and we now also know that kinesin-1¿s C-terminal tail interacts directly with and inhibits the heads when the motor is not needed for cargo transport. However, we do not know how kinesin-1 regulators initiate or stop cargo movement. The tail is certainly involved, as it binds to heads, microtubules, and several distinct kinesin-1 activators that function in different transport complexes. Separate from the tail, the Miro protein has a direct, Ca2+-dependent interaction with kinesin-1¿s enzymatic head domains, and Miro is required for Ca2+-dependent suppression of mitochondrial motility. We hypothesize that the tail is an intrinsically disordered domain, having structural flexibility that facilitates multiple binding partner interactions involved in kinesin-1 auto-inhibition and/or activation, while Miro has a distinct mechanism, directly inhibiting the enzymatic mechanism of kinesin-1 heads to suppress mitochondrial movement. To address this hypothesis, we will first gain detailed information in vitro about the structure of the kinesin-1 tail and its interactions with binding partners, by NMR and EPR spectroscopy. We will determine whether Miro is a direct, Ca2+-switchable inhibitor of kinesin-1¿s enzymatic activity, assess its effects on kinesin-1 mechanism using EPR, and map its interaction with kinesin-1 heads by cross-linking. After obtaining this structural and mechanistic information on both the tails and Miro, we will determine whether and how they influence mitochondrial movement by controlling kinesin-1 in vivo, by imaging mitochondria in live Drosophila S2 cells. These Aims together will provide an exciting new bridge between in vitro biophysical and cell biological work on molecular motor transport mechanisms. Furthermore, as Miro and other kinesin-1 regulators have been implicated in several neurological diseases, our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.