PROJECT SUMMARY/ABSTRACTThere is a fundamental gap in our knowledge to explain the intrinsic ability of centrioles to duplicate andfunction as microtubule-organizing centers. To fill this gap we require a thorough molecularcharacterization of the very first steps in centriole assembly and pericentriolar material (PCM)recruitment; this deficit impedes our understanding of the etiology of centrosome-linked diseases. Duringmitosis centriole function is activated and their duplication initiated by of the kinases Polo and Polo-likekinase 4 (Plk4) respectively. Normally upon mitotic entry cells contain two centrosomes eachcontaining a mother-daughter centriole pair that undergoes Polo-dependent `maturation' the process ofrecruiting additional PCM allowing them to facilitate spindle assembly. As cells exit mitosis eachdaughter cell inherits a centriole pair which has been modified by Plk4 to duplicate. Thus Polo kinasescontrol both the formation and duplication of functional centrosomes and consequently alterations inPolo kinase activity can dramatically influence spindle assembly and proper chromosome segregation.Our long-term goal is to better understand how cells couple cell cycle progression with centrosomefunction and duplication to ensure accurate distribution of the genome during division. The objective ofthis application is to understand how Polo and Plk4 are activated on mitotic centrioles to promote PCMrecruitment and centriole duplication. Drawn from our preliminary data our central hypothesis is thatmaturation and duplication are intrinsic behaviors of centrioles because Sas4 a centriole-surface proteincontrols the activities of both master-regulators Polo and Plk4. The rationale for the proposed researchis that understanding the evolutionarily conserved fundamental mechanisms of centriole biology has thepotential to translate into therapeutic strategies to address centriole-linked human disease. Thishypothesis will be tested in three specific aims: 1) Examine a mechanism of centrosome maturationwhereby Sas4 activates Polo kinase to generate a mitotic platform for PCM recruitment; 2) Determine ifSas4 regulates Plk4 stability and whether Sas4 phosphorylation by Plk4 controls the Plk4/Sas4centriolar pattern to restrict daughter centriole assembly to a single site; and 3) Determine ifphosphorylation of Asl and Sas4 by Plk4 induces formation of a daughter centriole initiation complex torecruit Ana2. The approach is innovative in the applicant's opinion because it represents a new andsubstantive departure of the status quo by shifting focus to the role of the centriole surface protein Sas4as a key activator and substrate for both Polo and Plk4. The proposed research is significant because athorough understanding of centriole biogenesis and function is intimately linked to our success inpreventing/treating centriole-associated diseases (including ciliopathy birth defects neurodevelopmentaldisorders and cancer).