Regulation of Cytoplasmic Dynein Motility in Neuronal Transport

 描述（由申请人提供）：这是K99独立之路奖的重新提交申请。分子马达蛋白以高度调节的方式主动转运细胞内货物。细胞质动力蛋白是最大的，最复杂的，和最不了解的微管运动蛋白家族。由于动力蛋白被细胞用于许多不同的功能，其活性受到外部蛋白质因子的复杂网络的高度调节，这些因子影响马达的基本机械化学。作为一名研究生，我在哥伦比亚大学接受了Richard Vallee博士的培训，研究神经发育疾病蛋白LIS 1和NudE/L对细胞质动力蛋白调节的生物化学和生物物理学。在加州大学弗朗西斯科分校罗纳德Vale博士实验室的博士后生涯中，我开发了荧光单分子显微镜的新技能。我利用这些新的技能发现了一种新的动力蛋白运动模式，用于将反平行微管分开，这是有丝分裂纺锤体组装过程中的一个关键功能。最近，我利用这些技能和培训，分离和表征一个稳定的超复合体的动力蛋白，动力肌动蛋白，和衔接蛋白BicD 2的大小超过2 MDa。我已经在单分子水平上对这种复合物进行了第一次生物物理测量，揭示了意想不到的新运动特性。这个建议的目的是了解动力蛋白调节途径如何在分子和细胞水平上对马达进行适当的控制。在指导K99阶段，这个多学科的建议旨在：1）阐明由dynactin-BicD 2和Lis 1-NudE/L组成的不同调节途径的动力蛋白运动调节的分子机制，和2）探测这种调节活性在活的神经元系统中生理重要货物的运输中的作用。随着在指导阶段获得的新培训和技能，我将在独立R 00阶段扩展我的研究范围，以了解动力蛋白持续合成调节如何用于相反极性电机的协调，以及人类神经退行性疾病突变对这种协调的影响。对于本申请中提出的实验，我将获得纳米精度，多色单分子显微镜和数据分析，果蝇遗传学，DNA纳米技术，基因组工程和活体动物共聚焦显微镜的额外培训。作为共同导师，罗恩瓦尔和Yuh-Nung Jan将提供先进成像技术、果蝇神经生物学和遗传学方面的专业知识。我的合作者将提供光学捕获显微镜和新型果蝇基因组编辑技术的必要经验和支持。这些新的技能和培训将为我提供最好的机会，实现我的职业目标，在两年内建立一个独立和成功的研究实验室。总的来说，这一建议的实施将回答长期存在的问题，在分子转运领域，并提供新的见解人类神经退行性疾病的机制，导致受损的细胞内运输。

 DESCRIPTION (provided by applicant): This is a resubmission application for the K99 Pathway to Independence Award. Molecular motor proteins actively transport intracellular cargos in a highly regulated fashion. Cytoplasmic dynein is the largest, most complex, and least understood of the microtubule motor protein families. Because dynein is utilized by the cell for many diverse functions, its activity is highly regulated by a complex web of external protein factors that impinge on the basic mechanochemistry of the motor. As a graduate student, I trained with Dr. Richard Vallee at Columbia University to study the biochemistry and biophysics of cytoplasmic dynein regulation by the neurodevelopmental disease proteins LIS1 and NudE/L. During my postdoctoral career in the lab of Dr. Ronald Vale at the University of California, San Francisco, I have developed new skills in fluorescence single-molecule microscopy. I have used these new skills to discover a novel mode of dynein motility used to slide anti-parallel microtubules apart, a function critical during mitotic spindle assembly. Recently, I have utilized these skills and training to isolate and characterize a stable super-complex of dynein, dynactin, and the adapter protein BicD2 that is over 2MDa in size. I have made the first biophysical measurements of this complex at the single molecule level, revealing unanticipated new motile properties. The goal of this proposal is to understand how dynein regulatory pathways exert proper control of the motor at both the molecular and cellular level. During the mentored K99 phase, this multi-disciplinary proposal aims to: 1) Elucidate the molecular mechanism of dynein motor regulation by divergent regulatory pathways made up of dynactin-BicD2, and Lis1-NudE/L, and 2) Probe the roles of this regulatory activity in the transport of physiological important cargo in a living neuronal system. With the new training and skills acquired in the mentored phase, I will then extend the scope of my research in the independent R00 phase in an effort to understand how dynein processivity regulation is utilized in the coordination of opposite polarity motors, and what effects human neurodegenerative disease mutations have on this coordination. For the experiments proposed in this application, I will acquire additional training in nanometer-precision, multi-color single molecule microscopy and data analysis, Drosophila genetics, DNA nanotechnology, genome engineering, and confocal microscopy in live animals. As co-mentors, Ron Vale and Yuh-Nung Jan will provide expertise in advanced imaging techniques, Drosophila neurobiology and genetics. My collaborators will provide the necessary experience and support in optical trapping microscopy and novel Drosophila genome editing techniques. These new skills and training will afford me the best opportunity to achieve my career goal to launch an independent and successful research laboratory within two years. Overall, the implementation of this proposal will answer long-standing questions in the molecular transport field, and provide novel insight into the mechanism of human neurodegenerative diseases that result from impaired intracellular transport.