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.
