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.

Rac1 Nucleocytoplasmic Shuttling Drives Nuclear Shape Changes and Tumor Invasion

“…Although in the nucleus phalloidin fluorescence cannot detect the canonical actin filaments seen in the cytoplasm, the vesicle-like Rac1-positive regions in the nuclear membrane were positive for the live-cell actin marker LifeAct, coinciding with zones of nuclear Rac1 activity measured by FRET ( Figures 5C and 5D). Our results, together with a recent fluctuation-based imaging study of Rac1 nuclear activation (Hinde et al, 2014), demonstrate that Rac1 can be active inside the nucleus, suggesting that Rac1-mediated nuclear actin polymers likely differ in length and perhaps conformation from their cytoplasmic counterparts, but are nonetheless able to modify nuclear membrane organization. These Rac1-mediated changes in nuclear membrane organization directly compromise the distribution of nuclear LMNA/C-emerin complex.…”

“…However, the exact mechanisms mediating nuclear actin organization remain unsolved. One report suggests that nucleoplasmic Rac1 is inactive (Kraynov et al, 2000), but others detected active nuclear Rac1 (Wong and Isberg, 2005;Hinde et al, 2014), suggesting a possible role of Rac1 GTPase in nuclear actin polymerization and nuclear structure organization.…”

Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

“…Rac1 is a small GTPase that is primarily localized in the cytoplasm, although nuclear Rac1 has been reported252627. It is a multifunctional protein involved in numerous cellular processes that are critical for cell ruffling, adherence junction formation, cell motility, polarity and proliferation28.…”

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos

“…For example, FCS measurements of p53 in single cells reveal that the TF exists in a mixture of oligomeric states; their rapid homo-tetramerization upon DNA damage activates the transcriptional targets of p53 [121]. Similar strategies have also been observed for the activation of the Rac1 signaling pathway [122], interaction between HP1-α and heterochromatin [123], and DNA-binding of STAT3 upon nuclear translocation [124] (Fig. 4b).…”