Dual observation of the ATP-evoked small GTPase activation and Ca2+ transient in astrocytes using a dark red fluorescent protein.

Junichi Nabekura,Hideji Murakoshi,Yoshihisa Nakahata

doi:10.1038/srep39564

Abstract

Intracellular signal transduction involves a number of biochemical reactions, which largely consist of protein-protein interactions and protein conformational changes. Monitoring Förster resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy (FLIM), called FLIM-FRET, is one of the best ways to visualize such protein dynamics. Here, we attempted to apply dark red fluorescent proteins with significantly smaller quantum yields. Application of the dark mCherry mutants to single-molecule FRET sensors revealed that these dark mCherry mutants are a good acceptor in a pair with mRuby2. Because the FRET measurement between mRuby2 and dark mCherry requires only the red region of wavelengths, it facilitates dual observation with other signaling sensors such as genetically encoded Ca2+ sensors. Taking advantage of this approach, we attempted dual observation of Ca2+ and Rho GTPase (RhoA and Cdc42) activities in astrocytes and found that ATP triggers both RhoA and Cdc42 activation. In early phase, while Cdc42 activity is independent of Ca2+ transient evoked by ATP, RhoA activity is Ca2+ dependent. Moreover, the transient Ca2+ upregulation triggers long-lasting Cdc42 and RhoA activities, thereby converting short-term Ca2+ signaling to long-term signaling. Thus, the new FRET pair should be useful for dual observation of intracellular biochemical reactions.

Highlights

Intracellular signal transduction involves a number of signaling proteins such as kinases[1] and small GTPases[2]
We successfully demonstrated that the pair “mRuby[2] and a dark red fluorescent protein” is suitable for fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) measurement (Figs 2 and S1 and S2)
Chromoprotein named Ultramarine was reported as an acceptor for FLIM-FRET, and its spectral properties are similar to those of dark mCherrys[12], the reduced cell-to-cell variability of dark mCherrys represents a big advantage over Ultramarine (Fig. 2e)

Summary

Introduction

Intracellular signal transduction involves a number of signaling proteins such as kinases[1] and small GTPases[2]. Because the pair “mRuby[2] with dark mCherry” requires only narrow bandwidth (550–650 nm) for FLIM-FRET measurements, we employed this pair with a green fluorescent protein-based calcium sensor G-GECO, which uses the bandwidth (500–550 nm) for dual observation of Ca2+ and Rho GTPase activity in astrocytes. Using a newly developed FRET pair, we monitored the temporal activity pattern of Ca2+ and the activities of cell division cycle 42 (Cdc42) or Ras homolog A (RhoA) These are members of the Rho family of small GTPases and are known to play pivotal roles in morphological changes and migration of cells by regulating actin polymerization[2]. An ATP-evoked calcium transient temporally regulates Cdc[42] and RhoA activities in astrocytes

Objectives

Methods

Results

Conclusion