Rab7 and Accessory Protein Function in Endocytosis

这个项目涉及三个具体目标，以机械细节了解晚期内吞运输和分选。这项工作对于了解膜转运机制和受体转运的调控具有重要意义。Wandinger-Ness博士实验室的研究已经确定Rab7及其相互作用的伙伴(脂蛋白激酶和磷酸酶以及蛋白酶体的一个亚单位)是晚期内吞运输和分选的关键调节因子。他们还证明了微管上的晚期内体运动是双向的，并依赖于功能上的Rab7。由于单个Rab GTP酶在很大程度上具有不同的效应器，而不是共享的效应器，因此全面了解膜运输将需要详细研究涉及的个别运输步骤和分子。在MCB9982161提供的支持下，确定了晚期内吞运输和分选的几个关键组件后，她的计划是从机械的角度确定这些组件如何共同发挥作用，以协调和调节晚期内吞体内运输和分选。计划中的研究将检查磷脂酰肌醇的代谢、蛋白酶体功能和微管运输，所有这些都将在Rab7及其相互作用的伙伴在晚期内小体上如何协调调节的背景下进行。根据hVPS34脂蛋白激酶及其P150接头与肌管蛋白1脂磷酸酶或Rab7形成互斥复合体的证据，Wandinger-Ness博士假设，这些选择性相互作用有助于协调调节局部磷脂酰肌醇3-磷酸(PI3P)水平。在Aim1中，她将使用体外和体内试验来测试这一假设，并建立控制晚期内体磷脂酰肌醇代谢的机制。由于磷脂酰肌醇通过作为膜平台招募促进膜融合和腔内囊泡形成的分子，在运输中发挥核心作用，因此研究磷脂酰肌醇是如何合成和翻转的，将为这一方面的运输如何调控提供新的见解。XAPC7是蛋白酶体的一个α亚基，Wandinger-Ness博士已经证明它与Rab7形成复合体。根据她最初发表的数据，蛋白酶体似乎负向调节运输，但蛋白酶体对晚期内切体的确切功能尚不清楚。AIM2旨在确定蛋白酶体在运输调节中的功能。预计这些研究将与越来越多的文献相结合，这些文献涉及泛素化和蛋白酶体活性在膜信号受体的适当运输和下调中的作用。最后，来自Wandinger-Ness博士和其他人的研究数据表明，功能性Rab7及其相互作用的伙伴RILP对微管上中心体定向的晚期内体运动非常重要。AIM3将探索Dynactin相关/RILP复合体和KIF1Bb运动蛋白马达如何协同调节双向晚期内体运动。总之，这些研究有望提供一个更全面的理解晚期内体运输和分选，这是真核细胞生命的一个基本方面。此外，国家科学基金会对该项目的资助将继续促进资源开发，过去在试剂、教育培训和资源开发方面的捐款就是例证。这些研究将继续扩大Wandinger-Ness博士已经免费传播给100多名研究人员的试剂库，导致大量引用。这项研究为跨学科研究生培养计划中的教育创新奠定了基础。之前的NSF资金为在新墨西哥大学建立最先进的显微镜设施提供了种子资金，现在联邦政府资助的各种研究项目的学生和研究员大量使用该设施。最后，在先前的支持下，该项目对总共21名本科生、研究生和博士后研究员的培训起到了核心作用，其中一半以上是妇女和代表性不足的少数群体。预计通过此次续签支持，这一广泛的培训记录将得到扩展。

This project addresses three specific aims relevant to understanding late endocytic transport and sorting in mechanistic detail. This work is relevant for understanding membrane transport mechanisms and the regulation of receptor trafficking. Studies in Dr. Wandinger-Ness's laboratory have identified Rab7 and its interacting partners (lipid kinases and phosphatases and a proteasome subunit) as a key regulators of late endocytic transport and sorting. They have also demonstrated that late endosomal motility on microtubules is bidirectional and dependent on functional Rab7. Since individual Rab GTPases have largely distinct rather than shared effectors, a comprehensive understanding of membrane trafficking will require the detailed study of individual transport steps and molecules involved. Having identified several key components in late endocytic transport and sorting with support provided by MCB9982161, her plan is to establish in mechanistic terms how these components function together to orchestrate and regulate late endosomal transport and sorting. Planned studies will examine phophoinositide metabolism, proteasome function and microtubule transport all in the context of how these are coordinately regulated by Rab7 and its interacting partners on late endosomes. Based on evidence that the hVPS34 lipid kinase and its p150 adapter form mutually exclusive complexes with the myotubularin 1 lipid phosphatase or with Rab7, Dr. Wandinger-Ness hypothesizes that these selective interactions serve to coordinately regulate local phosphatidyl inositol 3-phosphate (PI3P) levels. In Aim1, she will use in vitro and in vivo assays to test the postulate and establish the mechanisms governing late endosomal phosphoinositide metabolism. Because phosphatidyl inositides play a central role in transport by serving as membrane platforms for recruiting molecules that facilitate membrane fusion and intraluminal vesicle formation, addressing how phosphoinositides are synthesized and turned over will provide new insights into how this aspect of transport is regulated. XAPC7 is an alpha subunit of the proteasome that has been shown by Dr. Wandinger-Ness to complex with Rab7. Based on her initial published data, the proteasome seems to negatively regulate transport, but the exact function of the proteasome on late endosomes remains unclear. Aim2 is geared toward determining the function of the proteasome in the regulation of transport. It is expected that these studies will interface with the growing literature implicating ubiquitination and proteasome activity in the proper trafficking and downregulation of membrane signalling receptors. Finally, data from Dr. Wandinger-Ness' own work and others demonstrate that functional Rab7 and its interacting partner RILP are important for centrosome-directed late endosomal motility on microtubules. Aim3 will explore how the dynactin associated/RILP complex and the KIF1Bb kinesin motor may cooperate to regulate bidirectional late endosomal motility. Together, these studies promise to offer a more comprehensive understanding of late endosomal transport and sorting that is a fundamental facet of the life of eukaryotic cells. In addition, NSF funding of this project will continue to facilitate resource development as exemplified by past contributions in reagents, educational training and resource development. The studies will continue to expand the repertoire of reagents that Dr. Wandinger-Ness has already freely disseminated to over 100 investigators, resulting in numerous citations. The research serves as an underpinning for educational innovation in an interdisciplinary graduate training program. Prior NSF funding provided seed monies for the establishment of a state-of-the-art microscopy facility at the University of New Mexico that is now heavily utilized by students and fellows in various Federally funded research projects. Finally, the project has with prior support been central to the training of a total of 21 undergraduate and graduate students and postdoctoral fellows, more than half of which are women and underrepresented minorities. It is expected this extensive training track record will be expanded with this renewal support.