The centrosomes are non-membranous organelles comprised of microtubule-based structures- centrioles embedded in proteinaceous pericentriolar material (PCM). The centrioles organize PCM and thus, the number of centrosomes depends on the number of the centrioles in the cell. Centrosomes are versatile organelles carrying over several important functions in the cell. They serve as the cells main microtubule organizing centers, as the organizers of the mitotic spindles, and as basal bodies for the formation of cilliary axoneme. If the cycling cell contains more than two centrosomes, the spindle is likely to be multi-polar, resulting in randomized distribution of chromosomes, and aneuploidy. More than one mature centriole present in the cell may also allow the formation of multiple primary cilia and disrupt normal signaling processes. Therefore, it is critical that the number of centrosomes is precisely controlled. Supernumerary centrosomes are consistently found in the most invasive forms of tumors and they may arise as a consequence of abortive mitotic events or the loss of the control over centriole biogenesis during interphase. Recent data strongly indicate that experimentally perturbed centrosome numbers directly lead to cellular transformation. In vertebrate cycling cells the process of centriole formation is stringently regulated assuring that only one centriole duplication cycle occurs per one DNA cycle. The molecular mechanisms of this regulation are still obscure. Published genome-wide RNAi screens and centrosome proteome studies provided a large list of centrosomal and centrosome associated proteins implicated in centrosome function and biogenesis. However, the function of many centrosomal proteins is still largely unknown. The same is true for the cell cycle regulators which run centriole cycle (initiation of centriole duplication, centriole elongation, centriole maturation, or disengagement) and which are also mainly unidentified. We have developed an interphase model that allows us to study all steps of the centriole cycle in a population of highly synchronized mammalian cells in the tissue culture. My previous studies have identified a novel role of mitotic kinase Plk1 in synchronization of centriole- and cell- cycle during interphase. Our current effort aims to dissect Plk1-related pathways involved in the process of centriole reduplication. By comparative proteomics we will identify specific Plk1-dependent biochemical modifications. We also study the roles of selected known centrosomal proteins for their specific roles in final stages of the centriole cycle (during centriole maturation and disengagement). Using high throughput RNAi approach we also plan to inhibit the activity of known human kinases to determine the outcome of this inhibition on centriole reduplication process. The centrioles have precise symmetrical nine-fold geometry which reflects their behavior and function. However, the size of a human centriole (200 x 500 nm) is approaching the resolution limit of a classic light microscopy. Therefore, to understand the behavior and regulation of such a small but structurally complex organelle we use an integrated experimental approach combining sophisticated biochemical analytical tools with advanced high-resolution microscopy imaging techniques and electron microscopy. We have already prepared a number of cell lines expressing centrosomal proteins fused with fluorescent tags suitable for live-cell microscopy analysis, and developed the protocols for correlative light microscopy/electron microscopy analysis. Centriole reduplication has been traditionally considered to be a property of transformed cells. However, it has become obvious from my previous and current studies that the centrioles readily reduplicate in untransformed human and mouse cells harboring normal centriole cycle and checkpoint mechanisms. In these cells, centriole reduplication can be experimentally induced with a panel of chemical inhibitors of cell cycle progression. A great number of cell cycle inhibitors are being currently researched and developed for their potential use in anticancer therapy. In addition, I hypothesize that many current anticancer drugs induce cell cycle delays that can be possibly accompanied by aberrant centrosome reduplication. The comprehensive study exploring the consequences of cell cycle perturbations on centriole cycle in tumor and corresponding normal cells is lacking. For that reason one aspect of our project explores the behavior of the centrosomes during various cell cycle insults in different cell types. This study should answer is there a bias in the response of normal, versus transformed cells with respect to centriole reduplication properties? Identification of tumor types that readily reduplicate centrosomes during cell cycle arrest and have poor centrosome clustering during mitosis might allow the development of powerful antitumor strategies in future.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011459-01
Application #
8553203
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2012
Total Cost
$1,448,934
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Kong, Dong; Loncarek, Jadranka (2015) Correlative light and electron microscopy analysis of the centrosome: A step-by-step protocol. Methods Cell Biol 129:1-18
Shukla, Anil; Kong, Dong; Sharma, Meena et al. (2015) Plk1 relieves centriole block to reduplication by promoting daughter centriole maturation. Nat Commun 6:8077
Kong, Dong; Farmer, Veronica; Shukla, Anil et al. (2014) Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles. J Cell Biol 206:855-65