Mechanisms and Regulation of Kinetochore-Microtubule Attachments.

摘要

The accurate segregation of genetic material during cell division is a fundamental process required for life. To segregate DNA, cells build a molecular machine, the bipolar mitotic spindle. The mitotic spindle biorients duplicated chromosome pairs, called sister chromatids, by attaching one sister to microtubules emanating from one pole, and the other sister to microtubules emanating from the opposite pole. A specialized structure on each sister chromatid called the kinetochore mediates its attachment to the tips of the dynamic microtubules. Three major questions in the mitosis field are intimately related to the interface between kinetochores and microtubule tips. First, how does the kinetochore remain attached to a polymer that is constantly assembling and disassembling under its grip, harnessing the energy from these dynamics to drive chromosome movement? Second, how do kinetochores regulate microtubule attachment to ensure that duplicated chromosomes are bioriented? Lastly, how do kinetochores regulate microtubule dynamics to control the positioning and movement of chromosomes? To address these questions I purified kinetochore subcomplexes that mediate microtubule attachment and used advanced biophysical techniques to study their interaction with microtubules.;The Dam1 and Ndc80 complexes are both essential microtubule-binding components of the budding yeast kinetochore. Using total internal reflection fluorescence microscopy, I showed that both complexes make diffusive attachments to microtubules and that their diffusion becomes biased at the microtubule tip. These studies shifted the direction of the field from ring-centric models to a biased diffusion mechanism for stable attachment to dynamic microtubule tips.;The Aurora B kinase has an established role in the release of improper kinetochore-microtubule attachments. I found that Aurora B phosphorylation decreases the affinity of the Dam1 complex for microtubules and for the Ndc80 complex to mediate corrective detachment. I also established a novel function of Aurora B phosphorylation in regulating microtubule dynamics. I showed that the human Ndc80 complex stabilizes microtubules, while a phosphomimetic mutant version of the complex was unable to substantially modify tip dynamics. Altogether, these results suggest mechanisms by which kinetochores control microtubule attachment and tip dynamics to regulate the movement and segregation of chromosomes.