Understanding error correction for chromosome bi-orientation using in vitro reconstitution

Human cells store their genetic information in 46 chromosomes. Therefore, to maintain vital genetic information, a complete set of chromosomes must be inherited precisely by each daughter cell when cell division happens. Errors in this chromosome inheritance cause cell death and various human diseases, such as genetic abnormalities and cancers. Our research goal is to understand the fundamental mechanisms that ensure accurate chromosome inheritance when cells divide. These mechanisms involve an efficient and correct interaction between a chromosome and a cellular apparatus called the mitotic spindle, which drives chromosome motions into the new daughter cells. To examine chromosome interaction with the mitotic spindle in fine detail, we will recreate this process outside of the cell, using defined or purified cellular components. By doing so, we can adjust the density of the chromosome interaction with the mitotic spindle in a microscopy field. This will allow us to observe the details of this interaction and to analyse how errors in this interaction are resolved. To validate that the process recreated outside of the cell is physiologically relevant, we will compare it with the actual process inside the cell. Our research should contribute to our understanding of how various human diseases involving errors in chromosome inheritance arise, and how they might be prevented.

Chromosome mis-segregation in mitosis causes various human diseases, such as cancers and congenital disorders, which are characterized by chromosome instability and aneuploidy. To avoid chromosome mis-segregation, kinetochores on sister chromatids must correctly interact with microtubules extending from opposite spindle poles (chromosome bi-orientation). To ensure bi-orientation, any errors in kinetochore-microtubule interactions must be efficiently resolved through the process called error correction. However, the step-by-step process of error correction remains poorly understood, since it is difficult to visualise and analyse individual kinetochore-MT interactions during error correction in live-cell imaging due to an extremely high density of microtubules where error correction takes place. To overcome this issue, we will reconstitute the resolution of erroneous kinetochore-MT interactions in vitro, using kinetochore particles purified from budding yeast. To mimic sister kinetochores, two kinetochore particles, visualised in different fluorescent colours, will be physically connected. We will then analyse how the connected kinetochore particles interact with microtubules generated in vitro. Using the in vitro reconstitution system, we will address how erroneous kinetochore-microtubule interactions are formed, and how they are resolved by the disruption of kinetochore-microtubule interactions. Furthermore, we will study which step in these processes is promoted by regulators required for bi-orientation (Aurora B and Mps1 kinases and Stu2) and by microtubule dynamics and resultant kinetochore twisting. This study will make a significant contribution to our understanding of error correction - the vital process to ensure correct chromosome segregation in mitosis.