In-depth molecular studies of dynein transport in the RPE

摘要 吞噬小体清除是视网膜色素上皮(RPE)的一项主要功能，这是 视网膜的健康，因此也就是我们的视觉。吞噬小体在RPE中的清除依赖于 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 分娩涉及微管加端马达，Kinesin-1。然而，尽管它们在 在顺行方向，吞噬小体在RPE的微管上表现出双向运动。这表明 逆行马达Dynein的作用，它将真核细胞中的各种货物移动到负端 微管。动力蛋白在吞噬小体在RPE中的运输中的作用尚不清楚。我目前的情况 研究将集中在动力蛋白对吞噬小体运动的贡献，从而促进吞噬小体的效率 RPE的许可。在目标1中，我将利用最先进的活细胞成像技术在人类RPE细胞上测试 动力蛋白在介导吞噬小体与降解内切酶瞬间相互作用中的作用 车厢。在目标2中，我将对动力蛋白的调节机制进行深入的分子研究- 通过研究分子中间体，称为适配器蛋白，它连接了RPE中的 把马达装到货物上。这将为动力蛋白微管马达如何通过 接头蛋白的组合组合。它还将阐明接头蛋白是如何参与 调节相对的微管马达之间的协调，实现精确的细胞内 运输。在申请这个奖项时，我的目标是开发一个独立的研究项目，在 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 先进的人类RPE活细胞成像(与加州大学洛杉矶分校David Williams博士合作)和深入的分子培训 动力蛋白马达(与加州大学圣迭戈分校萨玛拉·雷克-彼得森博士合作)。除了我的研究培训，我还将参加 参加视觉科学和普通细胞生物学的各种会议，以及参加加州大学洛杉矶分校赞助的 帮助我向独立首席调查员过渡的项目。最后，我的合作导师们将 为我提供实验室管理、研究人员培训和拨款准备方面的指导 申请几种类型的筹资机制。总的来说，这项发展计划将为我做好准备 在研究机构获得并在一个独立的职位上出类拔萃。

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.