A Reconstituted System for Studying Kinetochore-Microtubule Attachments

Abstract

Before physically dividing into two nascent daughter cells, a cell must first duplicate, organize and segregate its entire genome. During mitosis, chromosomes are aligned along an axis of symmetry and then pulled apart by force-generating elements in the cell. Advances in genetic, biochemical and biophysical tools have enabled us to identify and study key components of this force-generating apparatus. Here, I will focus on interactions between chromosome-bound organelles called kinetochores and microtubules, dynamic protein filaments. My goal was to better understand how kinetochores harness the work generated by dynamic microtubules to drive chromosome movement. To do so, I used purified kinetochore components and employed biophysical techniques adapted from the study of single molecules. With this system I was able to reconstitute fundamental biological phenomena: persistent kinetochore-like attachments to growing and shortening microtubules, cooperative interactions between components that enhance attachment strength and force-dependent regulation of microtubule dynamics. Using a combination of single molecule techniques and modeling, I have also initiated study into the biophysical mechanisms underlying kinetochore-microtubule attachment. This work has given us valuable insights as to how these essential mitotic machines function within living, dividing cells.