Regulation of selective endocytic transport

Colorado State University, Fort Collins CO

Investigators

Abstract

This project will investigate the cellular machinery that carries out endocytosis, a process in which a portion of the cell membrane is pulled into the cell interior. Endocytosis regulates how cells take up nutrients, communicate with other cells, and adapt to changes in environmental conditions. While this research will be performed using yeast, discoveries will be broadly applicable because the cellular components that mediate endocytosis are conserved among different species. Studying endocytosis is essential to understanding how cells function, and this knowledge could have a number of applications such as improving crop yields and food production. This project will contribute to education and training of high school, undergraduate and graduate students. Both undergraduate and graduate students who work on this project will be involved in outreach at elementary schools where young students will perform hands-on yeast experiments and visualize cellular components using state-of-the-art research microscopes. The main goal of this outreach activity is to excite young students about science. Knowledge gained through this research will be published in wide-ranging scientific journals, presented at both specialized and broad cell biology conferences, and integrated into undergraduate and graduate courses taught by the researchers. During clathrin-mediated endocytosis, branched actin polymerization provides force needed to drive vesicle internalization. This research will explore three essential and unresolved questions regarding the regulation of the actin network during endocytosis. Aim 1 will establish how actin polymerization is initiated at sites of endocytosis. In particular, Aim 1 will elucidate the molecular mechanism that controls initiation of each new branch of the actin network. Aim 2 will determine the mechanism by which actin capping protein is recruited to sites of endocytosis and how two poorly understood protein components of the endocytic machinery actin network (Aim21 and Bsp1) regulate its function. Aim 3 will define the function of Twinfilin at endocytic sites. While Twinfilin has long been known as a component of actin networks, its cellular function is controversial, with recent high-profile articles reaching disparate conclusions. This project will test the premise that Twinfilin functions downstream of capping protein as an actin filament uncapping and disassembly factor at sites of endocytosis. The powerful Saccharomyces cerevisiae system will allow for the study of endocytosis regulators both in vivo and in vitro. The research will be multidisciplinary employing yeast genetics, live cell fluorescence microscopy, electron microscopy and cryo-electron tomography, biochemistry, X-ray protein crystallography, equilibrium binding assays and kinetic assays, as well as modeling approaches. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.