There is a fundamental gap in our knowledge to explain the intrinsic ability of centrioles to duplicate and function as microtubule-organizing centers. To fill this gap, we require a thorough molecular characterization 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. During mitosis, centriole function is activated and their duplication initiated by of the kinases, Polo and Polo-like kinase 4 (Plk4), respectively. Normally, upon mitotic entry, cells contain two centrosomes, each containing a mother-daughter centriole pair that undergoes Polo-dependent `maturation', the process of recruiting additional PCM, allowing them to facilitate spindle assembly. As cells exit mitosis, each daughter cell inherits a centriole pair which has been modified by Plk4 to duplicate. Thus, Polo kinases control both the formation and duplication of functional centrosomes, and consequently, alterations in Polo 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 centrosome function and duplication to ensure accurate distribution of the genome during division. The objective of this application is to understand how Polo and Plk4 are activated on mitotic centrioles to promote PCM recruitment and centriole duplication. Drawn from our preliminary data, our central hypothesis is that maturation and duplication are intrinsic behaviors of centrioles because Sas4, a centriole-surface protein, controls the activities of both master-regulators, Polo and Plk4. The rationale for the proposed research is that understanding the evolutionarily conserved fundamental mechanisms of centriole biology has the potential to translate into therapeutic strategies to address centriole-linked human disease. This hypothesis will be tested in three specific aims: 1) Examine a mechanism of centrosome maturation whereby Sas4 activates Polo kinase to generate a mitotic platform for PCM recruitment; 2) Determine if Sas4 regulates Plk4 stability, and whether Sas4 phosphorylation by Plk4 controls the Plk4/Sas4 centriolar pattern to restrict daughter centriole assembly to a single site; and 3) Determine if phosphorylation of Asl and Sas4 by Plk4 induces formation of a daughter centriole initiation complex to recruit Ana2. The approach is innovative, in the applicant's opinion, because it represents a new and substantive departure of the status quo by shifting focus to the role of the centriole surface protein, Sas4, as a key activator and substrate for both Polo and Plk4. The proposed research is significant because a thorough understanding of centriole biogenesis and function is intimately linked to our success in preventing/treating centriole-associated diseases (including ciliopathy, birth defects, neurodevelopmental disorders, and cancer).
The proposed research is relevant to public health because it focuses on dissecting evolutionarily conserved mechanisms that control the behaviors of centrosomes -- processes that, when dysfunctional, contribute to ciliopathy, birth defects and tumorigenesis. Thus, the project is relevant to a part of the NIH's mission because it will enhance our knowledge as to the etiology of numerous centrosome-related human diseases.
|Xie, Shuwei; Reinecke, James B; Farmer, Trey et al. (2018) Vesicular trafficking plays a role in centriole disengagement and duplication. Mol Biol Cell 29:2622-2631|
|Boese, Cody J; Nye, Jonathan; Buster, Daniel W et al. (2018) Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state. Mol Biol Cell 29:2874-2886|