In Vivo Hexamerization and Characterization of the Arabidopsis AAA ATPase CDC48A Complex Using Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy

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.

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Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

Biochemical Society Transactions ◽

10.1042/bst0311020 ◽

2003 ◽

Vol 31 (5) ◽

pp. 1020-1027 ◽

Cited By ~ 109

Author(s):

D.S. Lidke ◽

P. Nagy ◽

B.G. Barisas ◽

R. Heintzmann ◽

J.N. Post ◽

...

Keyword(s):

Tyrosine Kinases ◽

Laser Scanning ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wide Field ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

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Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

Thrombosis and Haemostasis ◽

10.1055/a-1401-5014 ◽

2021 ◽

Author(s):

Joanne C. Clark ◽

Raluca A. I. Neagoe ◽

Malou Zuidscherwoude ◽

Deirdre M. Kavanagh ◽

Alexandre Slater ◽

...

Keyword(s):

Energy Transfer ◽

Single Molecule ◽

Fluorescence Correlation Spectroscopy ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

Glycoprotein Vi ◽

D2 Domain ◽

Microscopy Techniques

AbstractCollagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

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