This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In yeast cells, endocytosis is entirely dependent upon the actin cytoskeleton. A major current area of our research is to elucidate the endocytic roles of actin. We have isolated numerous mutants that block endocytosis in yeast at discrete steps in the process, and we tagged some 20-30 endocytic and cytoskeletal proteins with GFP. We will use x-ray tomography to fully elucidate the spatial organization of the endocytic pathway and to relate this pathway with a variety of other cellular features. We will determine how these features change through the cell cycle in response to endocytic induction of the Ste2p receptor protein endocytosis and in response to mutation of specific endocytic and cytoskeletal proteins