The centrosome is essential for many cellular processes, including (i) formation of the mitotic spindle, (ii) cell polarity and asymmetric cell division, (iii) assembly of cilia and flagella, and (iv) suppression of parthenogenesis. In each case, strict control over centrosome biogenesis is required. The life cycle of the centrosome, from birth to death, is mechanistically not clear. Similar to the control of DNA replication, the centrosome copy number is highly regulated, and in cycling cells, the centrosome duplicates exactly once per cell cycle. During meiosis, the centrosome copy number is reduced (centriole degeneration) so that upon fertilization, the proper number can be restored. These patterns are conserved from algae to human. Centrosome amplification occurs frequently in almost all types of cancer. For example, centrosome amplification strongly correlates with loss of tumor suppressor proteins (such as p53 and pRB), and is induced after expression of viral oncoproteins (such as adenovirus E1A, HPV16 E6/E7, and human T-lymphotropic virus Tax proteins) [1]. The underlying mechanisms, however, are not known. The goal of my research is to understand how the centrosome number is maintained during cell division and development. We use several experimental systems to address this, including C. elegans, which provides strong genetic tools, Xenopus egg extracts, which allow detailed biochemical analysis, and tissue culture cells, which are relevant to our interest in understanding human centrosomes in normal cell division and in disease. The following two specific aims are proposed: 1) Determine the "licensing" mechanism for centrosome duplication. We have developed a powerful system using Xenopus egg extract and purified human centrosomes that recapitulates the centrosome duplication cycle in vitro. This has allowed us to determine that the licensing event for centrosome duplication occurs in anaphase, mediated in part by the separase protease. We now propose to identify additional players involved in the licensing process, examine their in vivo role in centrosome duplication, characterize the molecular nature of their action, and identify the relevant substrate(s) of separase involved in the process. 2) Determine the "blocking" mechanisms that prevent centrosome amplification There are at least three negative regulatory pathways work together to block centrosomes amplification in cells: 1) Centrosome-intrinsic block to re-duplication, 2) Suppression of de novo centrosome assembly, 3) Centrosome degeneration during sexual reproduction. We have developed an in vitro assay using Xenopus egg extracts to characterize proteins and activities that negatively regulate centrosome biogenesis. PUBLIC HEALTH RELEVANCE: Project Narrative The centrosome is the major microtubule organization center in most animal cells and strongly influences spindle assembly during mitosis. As a consequence, centrosome number is precisely regulated to ensure proper cell division. The goal of my research is to understand how the centrosome number is maintained during cell division and development.
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