Centrioles are small cylindrical organelles composed of an array of stabilized microtubules around a 9-fold symmetric central hub called the cartwheel. Centrioles duplicate once per cell cycle and recruit pericentriolar material (PCM) to form centrosomes that contribute to multiple cellular functions. Over the past decade, we invested significant effort to develop the one-cell C. elegans embryo as a model for mechanistic analysis of centriole and centrosome assembly. In addition to identifying conserved components, this effort revealed functional requirements for distinct steps in the assembly pathways. The experiments in Aim 1 leverage our expertise and existing tool chest in C. elegans to address how two Polo family kinases, Plk4ZYG-1 and Plk1, control cartwheel and PCM assembly, respectively. Specifically, we will address how Plk4ZYG-1 promotes oligomerization of the cartwheel component SAS-6, and employ in vivo perturbations in conjunction with an in vitro reconstitution assay to elucidate phosphoregulation of PCM assembly by Plk1. Over the last four years, we established a major new direction by developing and characterizing centrinone, a specific and potent Plk4 inhibitor that enables routine removal of centrioles/centrosomes from vertebrate cells. Experiments with centrinone revealed that transformed cells continue to proliferate with reduced mitotic fidelity in the absence of centrosomes, but normal human cells arrest in G1 within 1-2 cell cycles after centrosome loss; this arrest requires p53 but is not due to activation of known pathways (such as DNA damage or stress signaling, aneuploidy, Hippo pathway, or extended mitotic duration).
Aim 2 employs centrinone to understand the mechanism that halts normal human cells in G1 in response to centrosome loss. In particular, we will determine how centrosome loss leads to p53 activation and whether the CDKN2A locus, which is deleted or suppressed in many transformed cell types, is required for the G1 arrest. Centrosomes recruit over 100 different components, including many signaling proteins, and have been implicated in many different cellular processes, most notably cell division. The work in Aim 3 takes advantage of centrinone to perform a focused yet unbiased chemical biology screen with a hand-curated 640 compound Cellular Pathway Analysis library to identify small molecules that preferentially affect the proliferation of centrosome-less compared to centrosome- containing cells. This effort should identify pathways that functionally interact with centrosomes and will lead to a broader understanding of the roles that centrosomes play in cell physiology. In addition, I propose combining centrinone and our long-standing interest and expertise in cytokinesis to determine the role of centrosomes in specifying the dimensions and position of the contractile ring. In summary, the work proposed in this grant will elucidate fundamental mechanisms underlying centriole and centrosome assembly and will broaden our understanding of how centrosomes participate in cellular functions. In addition to advancing fundamental understanding of centrosome biology, this work has the potential to influence therapeutic strategies in cancer.

Public Health Relevance

Centrosomes are cellular organelles that are central to the mechanics of cell division and are also required for cells to maintain commitment to proliferation. Normal dividing cells contain precisely two centrosomes, whereas cancer cells frequently have more. The aims of this proposal are twofold: to elucidate the role of centrosomes in cell physiology, which has the potential to inform therapeutic strategies targeting centrosomes in cancer, and to analyze the mechanisms underlying centrosome assembly and duplication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074207-12
Application #
9404455
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2006-09-29
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
12
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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