Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET article

Daria M. Shcherbakova, Natasha Cox Cammer, Tsipora M. Huisman, Vladislav V. Verkhusha, Louis Hodgson

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Original languageEnglish (US)
Pages (from-to)591-600
Number of pages10
JournalNature Chemical Biology
Volume14
Issue number6
DOIs
StatePublished - Jun 1 2018

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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