UMR 8640 : Chimie Biophysique

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Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Proceedings of the National Academy of Sciences, Volume 113 n°.3, January 2016, Pages 497-502


This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a socalled fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Expanding discriminative dimensions for analysis and imaging

OPTIMAL can discriminate – without any separation or washing step – a targeted photoswitchable probe used as labelling or titration contrast agent among various interfering compounds, photoswitchable or not.

Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging

Non-invasive separation-free protocols are attractive to analyze complex mixtures. To increase selectivity, we propose to perform analysis under kinetic control upon exploiting the photochemical reactivity of labeling contrast agents. Our simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering as well as spectral crowding presently bring limitations. We introduce OPIOM (Out-of-Phase Imaging after Optical Modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase-sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, opening attractive perspectives for multiplexed observations in biological samples.


Photochemical properties of Spinach and its use in selective imaging

We propose a dynamic model that accounts for the photochemical behavior of the Spinach system, a recently described fluorescent probe for RNA imaging. We exploit the dynamic fluorogen exchange and the unprecedented photoconversion properties in a non-covalent fluorescence turn-on system to significantly improve signal-to-background ratio during long-term microscopy imaging.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

Cell fate decisions and cellular functions are dictated by the spatiotemporal dynamics of molecular signaling networks. However, the techniques available to examine the spatiotemporal properties of these intracellular processes remain limited. Here we report a method to artificially control in space and time such signaling pathways using magnetic nanoparticles conjugated to key regulatory proteins.