Kinetochore oscillations in mitotic metaphase

• Applicant: Professorin Iva Marija Tolic, Ph.D.
• Co-Applicant: Dr. Nenad Pavin
• Subject Area: Cell Biology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 240619959

During mitosis, sister chromatids connect to microtubules extending from the opposite spindle poles via kinetochores, protein complexes on the chromosome. When a pair of sister kinetochores establishes this configuration, they oscillate around the equatorial plane of the spindle, and the amplitude of these oscillations depends on kinesin-8 motors. During the oscillations sister kinetochores are separated due to tension, which has been proposed to regulate the spindle assembly checkpoint. The aim of this project is to study the mechanism of kinetochore oscillations. We will use live-cell imaging of fission yeast cells expressing centromere 2 labeled with GFP in order to quantify the movements of one pair of sister kinetochores. The role of kinesin-8 motors in the oscillations will be studied by comparing the kinetochore movements in wild-type cells and in those lacking kinesin-8, as well as by quantification of the dynamics of GFP-tagged kinesin-8 on the microtubules. To study the forces acting on the kinetochores, we will cut the spindle by using laser ablation. We will introduce a theoretical model in which kinetochore movement is driven by microtubule polymerization and depolymerization forces, whereas kinesin-8 regulates the switch from polymerization to depolymerization. Our preliminary experimental and theoretical results show that in wild type kinetochores are found near the spindle center, whereas in the absence of kinesin-8 they frequently pause near the spindle poles, supporting the preliminary assumptions of the model. In addition, the model will identify the conditions required for the oscillations and give predictions, which will be tested experimentally. For example, the model will predict the dynamics of the inter-kinetochore distance, which is a read-out of tension. In conclusion, understanding the mechanism of kinetochore oscillations will elucidate how the cell regulates the tension between sister kinetochores.

• DFG Programme: Research Grants
• International Connection: Croatia