The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we are using a unique approach in which the centrosome (labeled with GFP) is ablated by laser micro beam. This approach allows us to create cells that lack the centrosome, and compare their behavior with genetically identical cells that possess this organelle. Further, we combine laser microsurgery with inactivation of individual centrosomal proteins by siRNA. This synergistic methodology provides new information not obtainable by other means. Our current research focuses on the two phenomena that we described during past years of the grant. First, we discovered that the presence of the centrosome is required in normal but not in cancerous cells to progress through the cell cycle during interphase. We also demonstrated that cancerous cells born without centrosomes re-form this organelle de novo. We now investigate particular molecular mechanisms that coordinate cell cycle progression and activation of the centrosome de novo formation pathway. For that we ablate centrosomes in human lines at different stages of transformation (e.g., deficient in p53, pRb, or p21) to determine which of these pathways is needed to block cell cycle progression in the absence of centrosomes (Aim 1). We also ablate centrosomes in cells depleted (by siRNA) of different centrosomal proteins to determine how individual proteins affect centrosome de novo formation (Aim 2). Our second line of research is to clarify the role of the centrosome in mitosis. We have demonstrated that centrosomes are not essential for the formation of mitotic spindles, however, mitosis in acentrosomal cells is error-prone. We now investigate the relative contributions of the centrosomal and centrosome-independent pathways for mitotic spindle formation.
Aims 3 and 4 test two specific hypothesis: 1. That the centrosomes are required for efficient correction of synthetic chromosome mal-orientation; and 2. That the initiation of kinetochore fiber formation occurs via capture of short microtubules nucleated in the vicinity of kinetochores by centrosome-independent mechanisms. Since centrosome abnormalities are a hallmark of malignant tumors, the knowledge obtained in these studies will be useful for designing new strategies for the treatment of cancers. ? ? ?
| Sikirzhytski, Vitali; Renda, Fioranna; Tikhonenko, Irina et al. (2018) Microtubules assemble near most kinetochores during early prometaphase in human cells. J Cell Biol 217:2647-2659 |
| Drpic, Danica; Almeida, Ana C; Aguiar, Paulo et al. (2018) Chromosome Segregation Is Biased by Kinetochore Size. Curr Biol 28:1344-1356.e5 |
| Liu, Shiwei; Kwon, Mijung; Mannino, Mark et al. (2018) Nuclear envelope assembly defects link mitotic errors to chromothripsis. Nature 561:551-555 |
| Renda, Fioranna; Pellacani, Claudia; Strunov, Anton et al. (2017) The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure. PLoS Genet 13:e1006784 |
| Magidson, Valentin; He, Jie; Ault, Jeffrey G et al. (2016) Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells. J Cell Biol 212:307-19 |
| Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey et al. (2016) Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1. Cell Mol Life Sci 73:859-68 |
| Heald, Rebecca; Khodjakov, Alexey (2015) Thirty years of search and capture: The complex simplicity of mitotic spindle assembly. J Cell Biol 211:1103-11 |
| Magidson, Valentin; Paul, Raja; Yang, Nachen et al. (2015) Adaptive changes in the kinetochore architecture facilitate proper spindle assembly. Nat Cell Biol 17:1134-44 |
| Atilgan, Erdinc; Magidson, Valentin; Khodjakov, Alexey et al. (2015) Morphogenesis of the Fission Yeast Cell through Cell Wall Expansion. Curr Biol 25:2150-7 |
| Sikirzhytski, Vitali; Magidson, Valentin; Steinman, Jonathan B et al. (2014) Direct kinetochore-spindle pole connections are not required for chromosome segregation. J Cell Biol 206:231-43 |
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