In-depth molecular studies of dynein transport in the RPE

摘要 吞噬体清除是视网膜色素上皮（RPE）的主要功能，是视网膜色素上皮细胞（RPE）的一个基本功能。 视网膜的健康，以及我们的视觉。RPE中吞噬体的清除依赖于 在分子马达上协调它们向溶酶体隔间的运输以进行降解。这 递送涉及微管加端马达，驱动蛋白-1。然而，尽管它们在 在顺行方向上，吞噬体在RPE中的微管上表现出双向运动。这表明 逆行马达动力蛋白的作用，将真核细胞中的各种货物向负端移动 微管。动力蛋白在RPE中吞噬体运输中的作用尚不清楚。我现在的 研究将集中在动力蛋白对吞噬体运动的贡献， 通过RPE。在目标1中，我将利用最先进的人类RPE细胞活细胞成像来测试 动力蛋白在介导吞噬体和降解性内溶酶体间瞬时相互作用中的作用 隔间在目标2中，我将对调节动力蛋白的机制进行深入的分子研究- 通过研究分子中间体，称为衔接蛋白，连接RPE中基于运输的蛋白质。 发动机的货物。这将为动力蛋白微管马达如何通过以下方式控制货物特异性提供答案： 衔接蛋白的组合装配。它还将阐明衔接蛋白如何参与 调节对立微管马达之间的协调， 运输在申请这个奖项，我的目标是发展一个独立的研究计划，在接口， RPE细胞生物学和分子马达蛋白。我的发展计划包括获得以下方面的专业知识 先进的活细胞成像对人类视网膜色素上皮（与博士大卫威廉姆斯，加州大学洛杉矶分校）和深入的分子培训， 动力蛋白发动机（与萨马拉雷克-彼得森博士，加州大学圣地亚哥分校）。除了我的研究培训，我将参加 在视觉科学和一般细胞生物学的各种会议，以及参加加州大学洛杉矶分校主办的 帮助我过渡到独立首席研究员的项目。最后，我的导师们 在管理实验室、培训研究人员和准备拨款方面为我提供指导 对几种筹资机制的申请。总的来说，这个发展计划将使我做好准备 在研究机构获得独立职位并表现出色。

ABSTRACT Phagosome clearance is a major function of the retinal pigment epithelium (RPE), one that is essential for the health of the retina, and therefore our sense of vision. The clearance of phagosomes in the RPE is dependent on molecular motors that orchestrate their transport towards lysosomal compartments for degradation. This delivery involves the microtubule plus-end motor, kinesin-1. However, despite their net movement in the anterograde direction, phagosomes exhibit bidirectional motility on microtubules in the RPE. This indicates the actions of the retrograde motor, dynein, which moves various cargos in eukaryotic cells towards the minus-end of microtubules. The role of dynein in the trafficking of phagosomes in the RPE is not understood. My current research will focus on the contributions of dynein to the motility of phagosomes that promotes their efficient clearance by the RPE. In aim 1, I will utilize state-of-the-art live-cell imaging on human RPE cells to test the role of dynein in mediating transient interactions between phagosomes and degradative endolysosomal compartments. In aim 2, I will perform in-depth molecular studies into the mechanisms that regulate dynein- based transport in the RPE by investigating molecular intermediaries, called adaptor proteins, which link the motor to its cargo. This will provide answers to how the dynein microtubule motor controls cargo specificity by a combinatorial assembly of adaptor proteins. It will also shed light on how adaptor proteins participate in regulating the coordination between opposing microtubule motors that brings about precise intracellular transport. In applying for this award, my goal is to develop an independent research program at the interface of RPE cell biology and molecular motor proteins. My development plan consists of obtaining expertise in advanced live-cell imaging on human RPE (with Dr. David Williams, UCLA) and in-depth molecular training on the dynein motor (with Dr. Samara Reck-Peterson, UCSD). In addition to my research training, I will attend various conferences in vision science and general cell biology, as well as participate in UCLA-sponsored programs that facilitate my transition to an independent principal investigator. Finally, my co-mentors will provide me with guidance on managing a laboratory, training research staff, and preparation of grant applications for several types of funding mechanisms. Collectively, this development plan will prepare me well to obtain and excel in an independent position at a research institution.