Biophysics of Kinetochore-Microtubule Interaction: Quantitative Measurement of NDC80 Binding in Cell Using FLIM-FRET

Abstract

Chromosome motion during cell division is driven by coupling the dynamic ends of microtubules to the kinetochore. Erroneous kinetochore-microtubule attachments frequently occur in early mitosis, but are corrected to prevent chromosome mis-segregation. However, the mechanisms of kinetochore-microtubule coupling and error correction remain poorly understood. The NDC80 complex is the predominant coupler of the kinetochore to microtubules, and is thus directly implicated in these mechanisms. The lack of techniques to quantify the attachment of the NDC80 complex to microtubules in vivo has been a major obstacle to investigate this possibility. Here, I present a method that utilizes on cell engineering, Förster resonance energy transfer measurement by fluorescence lifetime imaging microscopy (FLIM-FRET), and Bayesian analysis, for quantitative measurement of the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living cells. Using this method, I found that Aurora B kinase modulates the attachment of NDC80 to kinetochore microtubules (kMTs) in a graded fashion in vivo, and that the NDC80 attachment increases from prometaphase to metaphase in the course of error correction. Measurements of NDC80 binding at the kinetochores oscillating around metaphase plate demonstrated that NDC80 complex preferentially associates with polymerizing kMTs, and that the fraction of NDC80 bound increases with the distance between sister kinetochores, a proxy for centromere tension. The positive correlation was dependent on the haspin-dependent localization of Aurora B kinase at the centromere. Taken together, these results argue that tension-dependent phosphoregulation of NDC80 by centromere-localized Aurora B is the basis of chromosome autonomous error correction. We believe that this FLIM-FRET technique is a powerful tool to further dissect the molecular mechanisms of kinetochore function.