Regulation of selective endocytic transport

该项目解决了细胞如何调节从周围环境中选择性摄取分子和物质的基本问题。内吞作用是细胞内化表面成分的过程。在内吞过程中，一部分质膜被拉入细胞内部，形成一个小的球形结构，称为内吞囊泡。本研究将探讨进行内吞作用的细胞机制及其是如何被调节的。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，其中许多学生属于在科学领域代表性不足的群体。这个项目的本科生和研究生都将参与动手酵母研究，细胞区室的概念，以及荧光显微镜进入中学教室。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和研究生课程中。本研究将探讨内吞作用的基本和突出问题。目标1将解决该领域的一个持续问题：是否存在内吞检查点，如果有，它是如何调节的？它将验证这样的假设，即机械组件结合整体膜蛋白货物是内吞机械组装的中心事件，并解决有关内吞部位膜弯曲中称为网格蛋白的脚手架蛋白功能的争议。目的2和3将探讨该实验室最近发现的两种新的内吞机制调节因子的活性。目的2将研究一种新的内吞蛋白调节肌动蛋白细胞骨架的概念，肌动蛋白细胞骨架提供了将囊泡拉入细胞的力量。目的3将测试第二种新蛋白通过泛素化-去泛素化调节内吞机制动力学的想法，泛素化-去泛素化是内吞蛋白的重要但鲜为人知的翻译后修饰。将系统地测试其他候选的内吞机制新蛋白质，以验证它们是否确实在内吞作用中起作用，并了解它们是如何起作用的。强大的酿酒酵母系统将允许在体内和体外研究内吞调节因子。这项研究将是多学科的，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、平衡结合分析和动力学分析，以及建模方法。

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.