Proteolytic Regulation of Centrosome Assembly

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Program Director/Principal Investigator (Last, First, Middle): Song, Mi Hye Summary Centrosomes, as the primary microtubule-organizing center, establish bipolar spindles that ensure accurate transmission of genetic contents into two daughter cells. To maintain genomic integrity, centrosome number must be strictly regulated by duplicating only once per cell cycle. Abnormal centrosomes are associated with human disorders, including cancers, microcephaly and other developmental defects. Our long-term goal is to understand the genetic mechanisms of centrosome function and assembly using the early C. elegans embryo as an in vivo model. The overall objective is to investigate how the ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C) and the coactivator FZR-1 (Cdh1 in human), contributes to centrosome assembly through proteasomal degradation of centrosome factors. FZR-1/Cdh1 (an APC/C cofactor) confers a substrate- binding through recognizing highly conserved degron motifs (KEN- and D-boxes). Cdh1-deficient mice exhibit embryonic lethality, genomic instability, and higher susceptibility to tumorigenesis and defective brain development. The abundance of centrosome components directly influences centrosome number: Blocking degradation of centrosome factors causes extra centrosomes, while depletion inhibits centrosome duplication. While we realize the great impact of the APC/C complex for regulating levels of centrosome factors, its substrate repertoire and regulatory mechanisms remain elusive. We propose to investigate SAS-7 as a potential substrate of APC/CFZR-1: SAS-7 functions most upstream in centrosome assembly and SAS-7 protein contains conserved degron motifs at multiple sites. We hypothesize that SAS-7 is another centrosome factor that is directly targeted APC/CFZR-1. Inhibiting APC/CFZR-1 blocks SAS-7 degradation, leading to hyper-stabilization of SAS-7 and compensating for a partial loss of ZYG-1 function in zyg-1 mutants. Our rationale is that its substrate repertoire of APC/CFZR-1 in centrosome assembly and defining their role will reveal the regulatory mechanisms of APC/CFZR-1 required for the fidelity of cell division. The aims of the project are to (1) determine if SAS-7 levels are affected by APC/CFZR-1-dependent proteolysis, (2) identify functional degron motifs within SAS-7, mediating APC/CFZR-1 targeting, and (3) understand how APC/CFZR-1-dependent proteolytic regulation of SAS-7 contributes to centrosome assembly and function. The use of in vivo-based genetics in a model system C. elegans will lay the groundwork for understanding human systems. The proposed project should contribute to advances in fundamental understandings of centrosome biology in humans and therapeutic interventions for human diseases and conditions such as cancers and microcephaly associated with abnormal centrosomes.