Dynamic multicolor protein labeling in living cells

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Dynamic multicolor protein labeling in living cells, Chem. Sci.2017, Advance Article 

 

Imaging the dynamics of proteins in living cells is essential for deciphering biological processes. A common strategy for imaging proteins is to fuse them to peptide or protein sequences that provide fluorescence, such as autofluorescent proteins or self-labeling tags (such as SNAP-tag/CLIP-tag and Halo-tag9) that can be labeled specifically with chemical probes. A related strategy is to use protein tags that can generate fluorescence upon interaction and activation of fluorogenic compounds (so-called fluorogens). Fluorogenactivating proteins (FAPs) able to specifically bind fluorogens such as malachite green and thiazole orange and activate their fluorescence have been developed from single-chain antibodies. Because fluorogens exhibit very low fluorescence background in cells, proteins can be imaged with high contrast without the need for washing the fluorogen excess.

 

 

This study presents fluorogenic dyes expanding the spectral properties of FAST to the red edge of the visible spectrum. Originally, FAST emitted green-yellow light upon blue light excitation when using HMBR as fluorogenic partner. Here, we show that FAST can fluoresce red light upon green light excitation using HBR-3,5DOM instead of HMBR. With FAST, it is thus now possible to adapt the tag color to the spectral conditions of the experiment without changing the protein tag, providing an experimental versatility that is not encountered with autofluorescent proteins. In addition, the ability to change the color by changing the dye renders possible to switch color in a single experiment. In this work, we show that the fast exchange dynamics characterizing FAST and the high permeability of the designed fluorogens allow color swapping on the second timescale. We used this unique kinetic signature to develop a method for the selective imaging of intracellular FAST-tagged proteins in spectrally crowded environments. A major obstacle to multiple observations is the spectral overlap between fluorophores, which limits most experiments to the observation of three or four targets. By measuring the degree of temporal anti-correlation of the two fluorescent states of FAST during dynamic color switching, we successfully imaged FAST-tagged proteins in presence of green and red fluorescent species despite spectral crowding. Imaging three proteins with only two observation channels is a promising step towards overcoming spectral crowding and opens exciting prospects for pushing the frontiers of multiplexed imaging.

 

Consultez le communiqué de presse associé à cet article : Observer les cellules avec un arc-en-ciel fluorescent !

 

Résumé: 

Chem. Sci.2017, Advance Article 

 

Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14-kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). Here, we report a collection of fluorogens enabling to tune the fluorescence color of FAST from greenyellow to orange and red. Beyond allowing multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST’s reversible labeling. This unprecedented behavior allows selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color crosscorrelation, opening exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

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Chimie Biophysique
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Dynamic multicolor protein labeling in living cells

 

Chenge Li, Marie-Aude Plamont, Hanna L. Sladitschek, Vanessa Rodrigues, Isabelle Aujard, Pierre Neveu, Thomas Le Saux, Ludovic Jullien and Arnaud Gautier

 

Chem. Sci., 2017, Advance Article 

 

DOI: 10.1039/C7SC01364G