The overarching goal of this proposal is to establish the mechanisms responsible for proper segregation of chromosomes during cell division. High vs. low fidelity of chromosome segregation is a characteristic difference between normal vs. cancer cells and understanding the mechanisms responsible for this difference is an essential step towards developing novel anti-cancer therapeutics and treatments strategies. For proper segregation, each chromosome must attach to the microtubules of mitotic apparatus (termed the 'spindle') and this attachment is mediated by special macromolecular assemblies on the chromosome's body known as the 'kinetochores'. The project is enabled by recent demonstrations that geometric constraints of microtubule/kinetochore interactions play a vital role in ensuring proper formation of the mitotic spindle essential for faithful chromosome segregation. These geometric constraints are established by the distribution and orientation of molecular complexes within the kinetochore and this project is designed to reveal how the architecture of the kinetochore, and the orientation of individual molecular complexes within that architecture, adapt to rapidly changing conditions within the mitotic spindle.
We aim to dissect the nature of structural reorganizations known to occur within the kinetochore during different stages of mitosis and different physiological states of the kinetochore. To achieve these goals we use a unique approach in which high- resolution electron-microscopy analyses are conducted on the kinetochores whose behavior and movements were followed in live cells up to the moment of fixation. This approach, termed correlative LM/EM allows us evaluate the structure of kinetochores that are in known functional state thus revealing the exact structural changes that occur during transitions between various physiological states. We will also perform comparative structural analyses of kinetochores upon two functionally-characterized experimental conditions: 1) treatment with low concentration of taxol, a condition known to allow satisfaction of the mitotic checkpoint despite the lack of centromere tension vs. 2) treatment with low concentration of nocodazole, a condition known to prevent satisfaction of the SAC despite highly-stretched centromeres. Together, these studies will allow us to create an accurate model for the structure of the kinetochore and determine how the architecture and the distribution of molecular components within the kinetochore adapt in response to various types of microtubule attachment and various types of chromosome movements.

Public Health Relevance

This project aims to reveal the mechanisms that ensure proper segregation of chromosomes during cell division (mitosis). We use sophisticated light and electron microscopy to investigate how molecular architecture of the kinetochore, an organelle that attaches chromosomes to the spindle microtubules, adapts to various types of microtubule attachment and various types of chromosome movements. Detailed understanding of these adaptations is essential for finding new means to prevent or repair problems in chromosome segregation that are a hallmark feature of cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM059363-14S1
Application #
8448918
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Gindhart, Joseph G
Project Start
1999-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
14
Fiscal Year
2013
Total Cost
$39,203
Indirect Cost
$12,535
Name
Wadsworth Center
Department
Type
DUNS #
153695478
City
Menands
State
NY
Country
United States
Zip Code
12204
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
Magidson, Valentin; Khodjakov, Alexey (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545-60
Tikhonenko, Irina; Magidson, Valentin; Graf, Ralph et al. (2013) A kinesin-mediated mechanism that couples centrosomes to nuclei. Cell Mol Life Sci 70:1285-96
Schilling, Z; Frank, E; Magidson, V et al. (2012) Predictive-focus illumination for reducing photodamage in live-cell microscopy. J Microsc 246:160-7
Yang, Feikun; Hu, Liyan; Chen, Cheng et al. (2012) BubR1 is modified by sumoylation during mitotic progression. J Biol Chem 287:4875-82
Vinogradova, Tatiana; Paul, Raja; Grimaldi, Ashley D et al. (2012) Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance. Mol Biol Cell 23:820-33
O'Connell, Christopher B; Khodjakov, Alexey; McEwen, Bruce F (2012) Kinetochore flexibility: creating a dynamic chromosome-spindle interface. Curr Opin Cell Biol 24:40-7
Magidson, Valentin; O'Connell, Christopher B; Loncarek, Jadranka et al. (2011) The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. Cell 146:555-67
Walczak, Claire E; Cai, Shang; Khodjakov, Alexey (2010) Mechanisms of chromosome behaviour during mitosis. Nat Rev Mol Cell Biol 11:91-102
Colello, Diane; Reverte, Carlos G; Ward, Rachel et al. (2010) Androgen and Src signaling regulate centrosome activity. J Cell Sci 123:2094-102

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