Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States; Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, United States
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, United States; Center for Biological Systems Engineering, Washington University in St Louis, St Louis, United States
William Conway
Department of Physics, Harvard University, Cambridge, United States
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, United States; Center for Biological Systems Engineering, Washington University in St Louis, St Louis, United States
Daniel J Needleman
Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States; Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, United States; John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, United States
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.
