The long-term goal of our research is to understand the molecular mechanisms that control centrosome biogenesis. Centrosomes are microtubule-nucleating organelles that play a central role in most microtubule- related functions, including cell motility, intracellular transport and chromosome segregation. In addition, the centrosome forms the basis of the basal body, which is required for the formation of cilia and flagella, and i therefore crucial for cells to both sense their environment and transduce signals. Centrosomes are present as a single copy at the beginning of the cycle and duplicate once during S phase to ensure only two copies are present to organize the poles of the mitotic spindle. Abnormalities in centrosome number are commonly observed in human cancer cells, where extra centrosomes lead to chromosome segregation errors that are thought to drive tumor formation. Understanding the mechanism by which cells achieve the once per cycle duplication of the centrosome is therefore an important fundamental question of considerable relevance to human health. Polo-like kinase 4 (Plk4) has emerged as the central, dose-dependent regulator of centrosome duplication. Suppressing Plk4 inhibits centrosome duplication, while Plk4 overexpression leads to the production of too many centrosomes. However, we understand little about how this kinase functions; and in particular, the critical Plk4 targets that control centrosome biogenesis remain to be identified. Our proposed research seeks to establish the mechanisms through which Plk4 orchestrates and coordinates centrosome biogenesis. Previous efforts to study Plk4 have been hampered because tools to specifically and rapidly manipulate Plk4 function have not been available. In this application we have overcome this limitation by developing two complementary methodologies that allow us to regulate Plk4 levels and activity in living cells. Using these tools we will establish the direct effect of altering Plk4 levels/actiity and distinguish between kinase-dependent and scaffolding functions of Plk4 in centrosome biogenesis.
In Aim 1, we will study the effect of rapid loss/inhibition of Plk4 on cell growth and centrosome structure. In our preliminary data we have identified a highly conserved centrosome protein as a key Plk4 substrate required for centrosome duplication.
In Aim 2, we propose to characterize how Plk4-mediated phosphorylation of this substrate contributes to centrosome assembly. These studies are relevant for understanding the normal regulation of centrosome assembly and for furthering ongoing efforts to target Plk4 in cancer therapy.

Public Health Relevance

Centrosomes are microtubule-organizing centers critical for faithful cell division. Abnormalities in centrosome number are commonly observed in human cancers where they to contribute to cell division errors that are thought to drive malignant transformation. The work we propose here seeks to define how centrosome copy number is faithfully controlled, with the goal of elucidating how centrosome abnormalities impact human health and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Gindhart, Joseph G
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Johns Hopkins University
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Levine, Michelle S; Holland, Andrew J (2018) The impact of mitotic errors on cell proliferation and tumorigenesis. Genes Dev 32:620-638
Nigg, Erich A; Holland, Andrew J (2018) Once and only once: mechanisms of centriole duplication and their deregulation in disease. Nat Rev Mol Cell Biol 19:297-312
Leda, Marcin; Holland, Andrew J; Goryachev, Andrew B (2018) Autoamplification and Competition Drive Symmetry Breaking: Initiation of Centriole Duplication by the PLK4-STIL Network. iScience 8:222-235
Lambrus, Bramwell G; Moyer, Tyler C; Holland, Andrew J (2018) Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells. Methods Cell Biol 144:107-135
Evans, Lauren T; Holland, Andrew J (2018) Pushed out of a tough crowd: centrosome aberrations promote invasiveness. EMBO J 37:
Lambrus, Bramwell G; Holland, Andrew J (2017) A New Mode of Mitotic Surveillance. Trends Cell Biol 27:314-321
Levine, Michelle S; Bakker, Bjorn; Boeckx, Bram et al. (2017) Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals. Dev Cell 40:313-322.e5
Levine, Michelle; Holland, Andrew (2017) Cell cycle proteins moonlight in multiciliogenesis. Science 358:716-718
Zitouni, Sihem; Francia, Maria E; Leal, Filipe et al. (2016) CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis. Curr Biol 26:1127-37
Lambrus, Bramwell G; Daggubati, Vikas; Uetake, Yumi et al. (2016) A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis. J Cell Biol 214:143-53

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