Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

Elizabeth Hinde,Katharina Gaus,Kyoko Yokomori,Enrico Gratton,Klaus M Hahn

doi:10.1038/srep04219

Abstract

Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-α2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.

Highlights

We describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein
We discovered from simultaneous phasor and Number and Brightness analysis of the Rac[1] FLARE dual chain biosensor, a nucleus-wide activation of Rac[1] upon induction of DNA damage that resulted in a redistribution of Rac[1] between the nucleus and cytoplasm based on its oligomeric state
Here we establish a method to assess how protein stoichiometry modulates signalling protein activity that is based on the acquisition of fluorescence lifetime imaging microscopy (FLIM) data in a format that enables concomitant phasor and Number and Brightness (N&B) analysis

Summary

Introduction

We describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By FLIM imaging of a FRET biosensor in a format that enables concomitant phasor and N&B analysis in each pixel of an image, we demonstrate that this method can determine the oligomeric state of the biosensor FRET donor, FRET acceptor and subsequent stoichiometry upon forming a complex, as well as verify the degree of signalling activity detected by FLIM-FRET or probe interaction with an entirely different binding partner through a cross variance analysis We use this multiplexed method to investigate the role of Rac[1] activation in the DNA damage response pathway and how the oligomeric state of Rac[1] affects the profile of this activation. This multiplexed approach to in vivo detection of protein activation is a powerful approach to reveal the oligomeric state a molecular complex must form in order to achieve correct regulation of a signalling pathway

Methods

Results

Conclusion