Role of vesicular trafficking proteins in regulating centrosomes PROJECT SUMMARY/ABSTRACT Centrosomes are comprised of a pair of cylindrical centrioles and associated pericentriolar material (PCM). Their major function 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. Previous studies have revealed that a centrosome-associated membrane structure called the centriculum plays a critical role in regulating centrosome function. However, the mechanism by which membranes and membrane-associated proteins regulate centrosomes is unknown. Prior studies have suggested roles for the membrane-associated endocytic proteins clathrin and dynamin in regulating centrosome number and PCM assembly, respectively. However, our understanding of the mechanism by this occurs is unclear. This proposal uses C. elegans as a model system to elucidate how the vesicular trafficking proteins clathrin (clathrin heavy chain is CHC-1 in C. elegans) and dynamin (DYN-1 in C. elegans) regulate centrosomes. Our long-term goal is to understand how membranes and membrane-associated proteins regulate centrosome biology. The overall objectives in this application are to understand how the membrane-associated endocytic proteins CHC-1 and DYN-1 collaborate with the core centrosome proteins ZYG-1 and SPD-2, respectively to regulate centrosome number and PCM assembly. Our central hypothesis is that CHC-1 and DYN-1 regulate ZYG-1 and SPD-2 protein localizations respectively, either in an endocytosis-dependent or in an endocytosis-independent manner to ensure a properly built and functioning centrosome. Our hypothesis will be tested by pursuing two specific aims: 1) Determine the mechanism by which CHC-1 and ZYG-1 interact and elucidate the effect of perturbing this interaction on centrosome duplication and ZYG-1 localization. 2) Test the hypothesis that DYN-1 plays a critical role in PCM assembly by controlling the amount of SDP-2 recruitment via vesicular trafficking. 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 membrane-associated vesicular trafficking proteins CHC-1 and DYN-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 imperative.