Dual mechanism of kinetochore microtubule detachment

Abstract

During eukaryotic cell division, microtubules connect to chromosomes by attaching to the kinetochore via the NDC80 complex (NDC80c). Improperly attached kinetochore microtubules (KMTs) are selective detached from the kinetochore. While the proper regulation of KMT detachments is crucial for accurate chromosome segregation, the manner by which KMT detachments are controlled by the biophysical properties of NDC80 and KMTs remains poorly understood. Here, we investigate the mechanism of KMT detachment by combining quantitative measurements of NCD80c-KMT binding and KMT detachments, with FLIM-FRET and photoconversion, and mathematical modeling. We find that individual NDC80c at kinetochores bind noncooperatively to KMTs, with a free energy difference between bound and unbound NDC80c of -1.0 {+/-} 0.1kBT, and we determine that each phosphorylated residue of NDC80c decreases the NDC80c-KMT binding energy by 0.35 {+/-} 0.03kBT. We show that the energetics of NDC80c binding can be related to KMT detachment by a dual detachment mechanism in which KMTs detach from kinetochores when either 1) all NDC80c spontaneously unbind from the KMT or 2) following KMT catastrophe. We find that the affinity of NDC80c for KMTs is reduced at low-tension, non-bioriented kinetochores due to centromere-localized Aurora B phosphorylating the NDC80c, resulting in an elevated detachment rate for the associated KMTs. Taken together, this work leads to a multiscale, bottom-up biophysical model for how the energetics of NDC80c-KMT interactions impact KMT detachments and provides an understanding of the molecular basis of their regulation during mitotic error correction. SIGNIFICANCE STATEMENTChromosome segregation errors can lead to aneuploidy, birth defects and cancer. Accurate cellular division, where one copy of each chromosome is segregated to each daughter cell, depends on regulation of attachments between chromosomes and microtubules in the mitotic spindle. While extensive work has identified proteins involved in suppressing mitotic errors, it is still unclear how incorrect chromosome-microtubule attachments which result in mitotic errors are selectively removed. Here, we employed a combination of quantitative live-cell fluorescence imaging, molecular perturbations, and mathematical modeling to elucidate the biophysical mechanism of how microtubules are selectively detached from misaligned mitotic chromosomes. This work leads to quantitative understanding of how chromosome attachments are regulated and mitotic errors are corrected.