Role of endocytic proteins and membrane trafficking in regulating centrosomes PROJECT SUMMARY/ABSTRACT Centrosomes are comprised of a pair of cylindrical centrioles and associated pericentriolar matrix (PCM). The major function of centrosomes is to organize a bipolar mitotic spindle to mediate accurate chromosome partitioning. Centrosome dysfunction and abnormal centrosome numbers are associated with diseases such as primary microcephaly and cancer. Hence, it is important to understand the cellular mechanisms that regulate centrosome biology. Preliminary data from previous studies suggest roles for membrane trafficking and endocytic proteins in regulating centrosome-related processes. However, our understanding of how endocytic proteins and membrane trafficking orchestrate the numerical and functional control of centrosomes is unknown. This proposal uses C. elegans as a model system to elucidate the mechanisms by which the endocytic proteins dynamin (DYN-1 in C. elegans) and clathrin (clathrin heavy chain is CHC-1 in C. elegans) and the process of vesicular trafficking regulate centrosome function and number. The long-term goal is to understand how centrosomes are assembled and how their function is regulated at the molecular level. The overall objectives in this application are to shed light on the mechanism by which DYN-1, CHC-1, and vesicular trafficking function to mediate proper PCM assembly and centrosome number. Our central hypothesis is that endocytic proteins and vesicular trafficking regulate centrosome protein localizations and/or functions to ensure a properly built and functioning centrosome. The rationale for this project is that although previous studies have established a relationship between endocytosis and centrosome-related processes, our mechanistic understanding of this relationship is limited. Our hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism by which DYN-1 inhibits excess SPD-2 recruitment to the centrosomes to facilitate proper PCM assembly; 2) Determine if CHC-1 and ZYG-1 interact in vivo to mediate proper centrosome duplication; and 3) Determine whether vesicular trafficking regulates the localization of the important centrosome proteins AIR-1, PLK-1 and ZYG-1. We will utilize cell and molecular biology techniques and C. elegans genetics to test our Specific Aims. The research proposed in this application is innovative because we are the first to investigate the contribution of the endocytic proteins DYN-1 and CHC-1 to centrosome biology in an in vivo model system. Developing therapeutic interventions for any disease begins with understanding the fundamental mechanisms underlying disease-associated processes at the level of basic science. The proposed research is significant because the dysregulation of centrosome number and function is associated with a host of diseases, making a more comprehensive understanding of how centrosome processes are regulated at the mechanistic level im...