Research Projects

For proper chromosome segregation, sister kinetochores must interact with microtubules (MTs) from opposite spindle poles; this is called bi-orientation. To establish bi-orientation prior to chromosome segregation, any aberrant kinetochore–microtubule interactions must be resolved (error correction) by Aurora B or Mps1 kinase that phosphorylates outer kinetochore components. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment). In addition, Mps1 and Stu2 also play a role in error correction and stabilizing attachments under tension. However, due to the dense microtubule network within the spindle in cells, the exchange of kinetochore-MT attachments could not be studied further. Recently, I reconstituted the kinetochore–MT interface in vitro and showed that Dam1p phosphorylation by Aurora B specifically weakens end-on attachments in comparison with lateral attachments. The proposed project is to decipher the steps involved and the role of various regulators in error correction leading to bi-orientation. I propose to generate a sister kinetochore pair from purified kinetochore particles (from budding yeast) in vitro and study their interactions with dynamic MTs. This system is closer to the physiological conditions, as we use dynamic MTs and sister kinetochore pair mimicking sister kinetochores geometry on sister chromatids in cells. This system will also give a great flexibility to add or remove regulators and alter the MT density. We will study the role of Aurora B, Mps1 and Stu2 regulators in error correction process using the in vitro system, at molecular detail and at single MT level. This project provides the following mechanistic details and the various steps involved in error correction: i) the mechanism of exchange of end-on vs lateral kinetochore-MT attachments, how each of the sister kinetochores interact with MTs during error correction. ii) how syntelic attachments are resolved and the role of each regulator and twisting force (details of this hypothesis is provided in the “work plan”) or tension between the sister kinetochores in resolving erroneous attachments or stabilizing the bi-orientation. iii) the mechanism of establishment and stabilization of bi-orientation under philologically comparable tension (generated by MT form opposite poles) between sister kinetochores. iv) the electron microscopy analysis of these structures will reveal structural details of the error correction process. Overall, this in vitro reconstitution project will reveal the steps involved in the error correction process during mitosis leading to bi-orientation and the role of the Aurora B, Mps1 kinases and Stu2 in each of these steps. This would be impossible to study in cells owing to high MT density at the spindle. Thus, this would advance our understanding of early mitosis and pave way to our understanding of aneuploidy, Down syndrome and cancers.