title: Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation.
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.
authors.affiliation: 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.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hinde
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, USA.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yokomori
authors.affiliation: Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia.
authors.forename: Katharina
authors.initials: K
authors.lastname: Gaus
authors.affiliation: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D041681:NIH 3T3 Cells; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D028901:alpha Karyopherins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D020830:rac1 GTP-Binding Protein