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Abstract
Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.
Highlights
Chromosome segregation errors lead to aneuploidy and micronuclei formation, which are closely associated with cancer, infertility, and birth defects (Santaguida and Amon, 2015)
We developed a method to quantitatively measure the binding of the NDC80 complex to microtubules at individual kinetochores in human tissue culture cells
We observed that NDC80-kinetochore microtubules (kMTs) binding is strongly correlated to centromere tension, to an extent which is sufficient to account for the changes in NDC80-kMT binding over the course of prometaphase and metaphase
Summary
Chromosome segregation errors lead to aneuploidy and micronuclei formation, which are closely associated with cancer, infertility, and birth defects (Santaguida and Amon, 2015). Yoo et al further showed that Aurora B needs to be properly placed between two kinetochore hands to make NDC80-microtubule binding dependent on tension Without this tension dependency, chromosomes could segregate unevenly into the newly formed cells – a problem that can lead to cancer, infertility and birth defects. In vitro experiments showed that the binding affinity of NDC80 for microtubules decreases upon the phosphorylation of the N-terminal tail of Ndc80/Hec protein by Aurora B kinase (Cheeseman et al, 2006; Zaytsev et al, 2014, 2015), which may explain the contribution of Aurora B to error correction (Tanaka et al, 2002) It is unclear how the biochemical activities of NDC80 and Aurora B result in tension-dependent kMT detachment. The lack of techniques to measure the binding of the NDC80 to kMTs in vivo has been a major obstacle to investigate this
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