Regulation of selective endocytic transport

选择性内吞转运的调节

• 批准号: 1616775
• 负责人: Santiago Di Pietro
• 金额: $ 99.07万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616775&HistoricalAwards=false
• 关键词: Regulation; selective; endocytic; transport

This project addresses the fundamental question of how cells regulate the selective uptake of molecules and material from the surrounding environment. Endocytosis is the process by which cells internalize surface components. During endocytosis, a portion of the plasma membrane is pulled into the inside of the cell forming a small, spherical structure known as an endocytic vesicle. This research will investigate the cellular machinery that carries out endocytosis and how it is regulated. This project will support diversity in science and education through training of undergraduate and graduate students, many of whom belong to groups underrepresented in the sciences. Both undergraduate and graduate students in this project will be involved in taking hands-on yeast research, concepts in cellular compartments, and fluorescence microscopy into middle school classrooms. In addition, knowledge gained through this research will be integrated in undergraduate and graduate courses taught by the principal investigator.This research will explore fundamental and outstanding questions in endocytosis. Aim 1 will tackle one of the ongoing problems in the field: is there an endocytic checkpoint and if so how is it regulated? It will test the hypothesis that binding of integral membrane protein cargo by components of the machinery is a central event in the assembly of the endocytic machinery and address a controversy regarding the function of a scaffolding protein called clathrin in membrane bending at endocytic sites. Aims 2 and 3 will explore the activity of two new regulators of the endocytic machinery recently discovered by this laboratory. Aim 2 will investigate the concept that one of the new endocytic proteins regulates the actin cytoskeleton, which provides force to pull the vesicle into the cell. Aim 3 will test the idea that the second new protein regulates dynamics of the endocytic machinery through ubiquitination-deubiquitination, an important but poorly understood posttranslational modification of endocytic proteins. Additional candidate new proteins of the endocytic machinery will be systematically tested to verify if they indeed work in endocytosis and to understand how they work. The powerful Saccharomyces cerevisiae system will allow for the study of endocytosis regulators both in vivo and in vitro. The research will be multidisciplinary including yeast genetics, live cell fluorescence microscopy, electron microscopy and electron tomography, biochemistry, X-ray protein crystallography, equilibrium binding assays and kinetic assays, as well as modeling approaches.

该项目解决了细胞如何调节从周围环境中选择性吸收分子和材料的基本问题。内吞作用是细胞内化表面成分的过程。在内吞作用期间，质膜的一部分被拉入细胞内部，形成称为内吞囊泡的小的球形结构。本研究将探讨进行内吞作用的细胞机制及其调节方式。该项目将通过培训本科生和研究生来支持科学和教育的多样性，其中许多人属于科学界代表性不足的群体。本项目的本科生和研究生将参与实际酵母研究，细胞室概念和荧光显微镜进入中学课堂。此外，通过本研究获得的知识将被整合到由首席研究员教授的本科生和研究生课程中。本研究将探索内吞作用的基本和突出问题。目标1将解决该领域中一个持续存在的问题：是否存在内吞检查点，如果存在，它是如何调节的？它将测试的假设，即结合的组成部分的机器的完整的膜蛋白货物是一个中心事件的组装的内吞机械和解决一个有争议的功能称为网格蛋白的支架蛋白在膜弯曲内吞网站。目的2和3将探讨本实验室最近发现的两种新的内吞机制调节剂的活性。目的二是探讨一种新的内吞蛋白调节肌动蛋白细胞骨架，提供将囊泡拉入细胞的力。目的3将测试的想法，第二个新的蛋白质调节动态的内吞机制，通过泛素化-去泛素化，一个重要的，但了解甚少的翻译后修饰的内吞蛋白。将系统地测试内吞机制的其他候选新蛋白质，以验证它们是否确实在内吞作用中起作用，并了解它们如何起作用。强大的酿酒酵母系统将允许在体内和体外的内吞调节剂的研究。该研究将是多学科的，包括酵母遗传学，活细胞荧光显微镜，电子显微镜和电子断层扫描，生物化学，X射线蛋白质晶体学，平衡结合测定和动力学测定，以及建模方法。