The objective of our research is to understand the molecular mechanisms that regulate centrosome number. The centrosome is the main microtubule-organizing center of the cell. It comprises microtubule-based cylindrical structures termed centrioles, which are embedded in a complex and highly structured proteinaceous pericentriolar material (PCM). In the absence of centrioles the PCM is unstable and centrosomes disassemble, meaning that the number of centrosomes is determined by the number of centrioles. If a cell enters mitosis with more than two centrosomes, the formation of a multipolar spindle is very likely to occur, increasing the probability of chromosome miss segregation. Supernumerary centrosomes may also lead to the formation of multiple primary cilia, disrupting cilia-based signaling pathways and cellular homeostasis. Recent data strongly indicate that experimentally perturbed centrosome numbers may directly lead to cellular transformation. Therefore, it is important that a cell stably maintain proper number of centrosomes. Supernumerary centrioles are a hallmark of tumors, especially the most aggressive ones. A cycling cell limits centrosome number to two per cell by controlling the process of centriole duplication. Centriole duplication conforms to two stringent roles: 1. each of two preexisting (mother) centrioles must duplicate only once per cell cycle, and 2. only one daughter centriole forms per mother centriole. Molecular mechanisms responsible for the stringency of centriole duplication are still largely unknown. It has been demonstrated that a specific close orthogonal orientation between mother and daughter centriole within a centrosome, centriole engagement, serves as an intrinsic block to centrosome reduplication within the same cell cycle. Premature resolution of this rigid orthogonal orientation between the centrioles (disengagement) has emerged as the principal event that allows a new round of centriole duplication. Despite its importance in promotion of centrosome reduplication, the process of centriole disengagement is mechanistically the most obscure part of the centriole cycle. Our studies have identified a serine/threonine kinase Polo-like kinase1 (Plk1) as a key promoter of premature procentriole disengagement and reduplication during interphase arrest. The cell's ability to aberrantly reduplicate centrioles during interphase arrest directly correlates with the presence of an active Plk1 (pThr210) on the centrosomes and with precocious accumulation of maturation markers at normally immature procentrioles. In agreement with these findings, inhibition of Plk1 activity either during later stages of the cell cycle or during interphase, prevents subsequent centriole disengagement. We hypothesize that timely Plk1 activity provides a powerful synchronization mechanism between cell and centriole cycle, revealing a novel interphase function of Plk1. Our data strongly indicate that activation of Plk1 on the centrosome initiates a series of irreversible intra-centrosomal molecular and architectural changes, leading to centriole disengagement. Our goal now is to describe these changes, to mechanistically explain the process of centriole disengagement, and to understand the proposed concept of centriole-block-to-reduplication. Using biochemical methods and comparative proteomics we aim to identify Plk1-dependent quantitative and qualitative changes in the centrosome proteome that are specific to the process of centriole disengagement. In addition, we combine biochemical methods with live and fixed cell microscopy, super resolution microscopy, and correlative light/electron microscopy to study centrosomal ultra-structural changes that occur within the centrosome during centriole cycle. Plk1 kinase has been associated with tumor invasion, higher pathological grade, poor prognosis and elevated centrosome number and aneuploidy, making Plk1 an important target for the development of anticancer agents. Therefore it is very important to elucidate molecular mechanisms underlying the function of Plk1 on the centrosome. Our project further aims to analyze in detail the consequences of elevated acute and prolonged Plk1 activity in a series of transformed and non-transformed human cell lines. Understanding the mechanisms that regulate centriole biogenesis is not only an interesting basic biological question but has significant implications for cancer biology and centrosome-associated diseases. Our studies are pertaining to the mission of the NIH: to use basic science for improvement of human condition, and for understanding the basis for development, and hopefully treatment, of centrosome-related diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011459-02
Application #
8763538
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$656,681
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code