scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V. Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

AbstractProper 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.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

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.

scholarly journals Looking at the Functional State of Proteins Inside Cells

1999 ◽  
Vol 7 (8) ◽  
pp. 3-4
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Functional State ◽  
Spatial Data ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Cellular Functions ◽  
Lifetime Imaging ◽  
Intracellular Proteins

Many intracellular proteins are catalysts that regulate cellular functions. These catalysts can be assayed to determine their functional state, but untii now it was not possible to simultaneously obtain a functional analysis and spatial data. Tony Ng, Anthony Squire, and others, working in the laboratories of Phillippe Bastiaens and Peter Parker, have combined Fluorescence Lifetime Imaging Microscopy (FLIM) with Fluorescence Resonance Energy Transfer (FRET) to spatially resolve the activation of a protein catalyst within living cells. Their technique was also applied to fixed cells.

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