Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

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

doi:10.1038/srep04219

Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

Sci Rep. 2014 Feb 27:4:4219. doi: 10.1038/srep04219.

Authors

Elizabeth Hinde¹, Kyoko Yokomori², Katharina Gaus³, Klaus M Hahn⁴, Enrico Gratton⁵

Affiliations

¹ 1] Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA [2] Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia.
² Department of Biological Chemistry, University of California, Irvine, USA.
³ Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia.
⁴ Department of Pharmacology and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, USA.
⁵ Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA.

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.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Cell Nucleus / metabolism
Cytoplasm / metabolism
DNA Damage
Enzyme Activation
Fluorescence Resonance Energy Transfer*
Mice
Microscopy, Fluorescence*
Mutation
NIH 3T3 Cells
Protein Binding
Protein Multimerization*
Protein Transport
alpha Karyopherins / metabolism
rac1 GTP-Binding Protein / chemistry*
rac1 GTP-Binding Protein / genetics
rac1 GTP-Binding Protein / metabolism*

Substances

alpha Karyopherins
karyopherin alpha 2
rac1 GTP-Binding Protein

Abstract

Publication types

MeSH terms

Substances

Grants and funding