Mechanisms of Kinesin Regulation

分子马达Kinesin-1执行大量的转运任务，其调节机制 管理这些过程至关重要。Kinesin-1的错误调节或Kinesin-1货物的错误定位可能 与多种疾病有关，如帕金森氏症、S病、神经纤维瘤病、精神分裂症和 夏科-玛丽-图斯病。运动蛋白-1？S的运动机制是众所周知的，我们现在也知道了 当货物不需要马达时，S C末端的尾巴直接与头部相互作用并抑制头部 运输。然而，我们不知道Kinesin-1调节器如何启动或停止货物移动。尾巴是 当然涉及，因为它与头部、微管和几个不同的Kinesin-1激活剂结合在一起，这些激动素-1在 不同的运输复合体。与尾巴分离，Miro蛋白具有直接的、依赖于钙的相互作用 与激动素-1的S酶头域和MIRO是钙依赖的抑制所必需的 线粒体的运动性。我们假设尾巴是一个本质上无序的区域，具有结构 促进参与Kinesin-1自动抑制和/或的多个结合伙伴相互作用的灵活性 激活，而MIRO有一个明显的机制，直接抑制Kinesin-1头部的酶机制 来抑制线粒体的运动。为了解决这一假设，我们将首先在体外获得详细的信息。 用核磁共振和电子顺磁共振研究Kinesin-1尾巴的结构及其与结合伴侣的相互作用 光谱学。我们将确定MIRO是否是一种直接的、钙离子可切换的激动素-1抑制剂？S酶 活性，用EPR评估其对Kinesin-1机制的影响，并通过以下方式定位其与Kinesin-1 Heads的相互作用 交联性。在获得关于尾巴和Miro的结构和机械信息后，我们将 通过成像，确定它们是否以及如何通过控制体内的Kinesin-1来影响线粒体的运动 活的果蝇S2细胞的线粒体。这些目标结合在一起，将在 关于分子马达运输机制的体外生物物理和细胞生物学研究。此外，由于米罗和 其他Kinesin-1调节因子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，将加快开发治疗的努力。

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.