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ANR funded project

Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques (Blanc SVSE 5)
Edition 2011


NOBLEACH


Controlling photobleaching of fluorescent proteins for super-resolution microscopy.

Why are fluorescent proteins so light-sensitive ?
This research will serve the development of modern biological fluorescence microscopy by engineering better fluorescence markers.

Investigating photobleaching mechanisms of fluorescent proteins to rationally engineer photoresistant variants.
Fluorescence microscopy plays a growing role in life sciences. Recently, super-resolution microscopy (nanoscopy) has been developed, which provides fluorescence images of live samples with ~20 nm spatial resolution, thus opening a huge research field. Nanoscopy will undoubtedly impact the understanding of diseases and contribute to improve public health. Some special markers, called “phototransformable” fluorescent proteins (PTFPs) are required for nanoscopy. They are homologous to the well known Green Fluorescent Protein (GFP), and, as GFP, exhibit a high susceptibility to photobleaching, that is, a rapid loss of fluorescence upon illumination. In this project, we propose to investigate the photobleaching mechanisms of PTFPs, understand the role of environmental factors, and design photoresistant variants.

From single-crystal to single-molecule studies.
We use a vast panel of techniques, extending from single-crystal to single-molecule studies. Charge (electron and proton) transfer mechanisms, at the heart of bleaching processes, will be studied in depth. In addition to studies at the molecular scale obtained by crystallography, spectroscopy, and modeling, we will put emphasis on single-molecule investigations in vitro and in cellulo. Eukaryotic and prokariotic benchmark-cells will allow us validating the performances of fluorescent proteins of interest for super-resolution imaging. In a feedback loop manner, we will aim at proposing rationally designed FP variants with better phototransformation and photobleaching properties.

Results

After 18 months of work, our main result is a detailed structural view of the photobleaching mechanism in the photo transformable fluorescent protein IrisFP. Our work have allowed showing that this mechanism considerably depends on the light intensity used to excite fluorescence: at high-intensity, a photoinduced electron transfer mechanism completely destabilizes the protein chromophore. At low intensity, a buildup of singlet oxygen results in the sulfoxidation of protein residues located nearby the chromophore. The first process, that does not generate reactive oxygen species, is expected to be less cytotoxic for live cells. There would therefore be an advantage for microscopists to use relatively intense laser powers (for a shorter time) when they work with fluorescent proteins markers like IrisFP.

Outlook

These results suggest ideas to attempt the rational design of photoresistant IrisFP mutants. Other fluorescent proteins are under study, and their photophysical behavior are being investigated from a mechanistic standpoint as well as in the cellular context at the single molecule level. This fundamental biophysical project is linked to other biologically relevant projects, in particular the study of bacterial cell division.

Scientific outputs and patents

Dominique Bourgeois, Aline Regis-Faro & Virgile Adam. “Photoactivated structural dynamics of fluorescent proteins” Biochem. Soc. Trans., (2012), 40, 531-538

Dominique Bourgeois & Virgile Adam. “Reversible photoswitching in fluorescent proteins: A mechanistic view” IUBMB Life, (2012), 10.1002/iub.1023

Partners

LCP CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR SUD

IBS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

LPCV CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

ANR grant: 503 119 euros
Beginning and duration: - 36 mois

Submission abstract

Fluorescence-based imaging techniques play an ever growing role in life sciences. In the last five years, the most spectacular development has concerned super-resolution microscopy (nanoscopy), which provides fluorescence images with ~20 nm spatial resolution. By allowing dissecting the cell at the level of individual macromolecules, nanoscopy opens a huge research field, integrating structural and cell biology. Although still in its infancy, the technique will undoubtedly impact our understanding of diseases and contribute to improving public health.
Single-molecule based localization methods (PALM/STORM ) are the most versatile and promising nanoscopy techniques. These approaches are almost entirely based on manipulating the photophysical properties of the fluorescent label, typically employing photoactivatable markers that can be selectively activated from an off-state to an on-state upon proper illumination. Genetically encoded GFP-like photoactivatable fluorescent proteins (FPs) are thus fundamental players in PALM super-resolution microscopy.
We have acquired in the last few years a state-of-the-art expertise in fluorescent protein’s photophysics. We developed and characterized new photoactivatable FPs using a combination of kinetic crystallography, in crystallo spectroscopy, and modelling, in the context of the ANR “DSPF” project.
One key problem of fluorescent proteins concerns their high susceptibility to photobleaching, that is, their rapid and non-reversible loss of fluorescence upon illumination. Photobleaching affects practically all fluorescence microscopy techniques and seriously limits the achievable resolution in PALM nanoscopy. Yet, the mechanisms underlying photobleaching in FPs remain largely mysterious. We now propose to extend our investigations of fluorescent proteins to the question of photobleaching, in order to characterize the involved mechanisms, understand the role of environmental factors (redox potential, pH, oxygen), and design photoresistant candidates. As photobleaching mechanisms are intertwined with those of photoactivation, our studies will combine interrogations of both processes.
We will use a vast panel of techniques, extending from single-crystal to single-molecule studies. Charge (electron and proton) transfer mechanisms, at the heart of bleaching processes, will be studied in depth. In addition to studies at the molecular scale obtained by crystallography, spectroscopy, and modelling, we will put emphasis on single-molecule investigations in vitro and in cellulo. Benchmark-cells will allow us validating the performances of fluorescent proteins of interest for PALM imaging. In a feedback loop manner, we will aim at proposing rationally designed FP variants with better photoactivation and photobleaching properties. In addition to serving the development of super-resolution microscopy and increasing knowledge on fluorescent proteins photophysics, this fundamental research may also benefit the biotechnology community and lead to the development of new biosensors.
The project will be conducted in the context of a new research team, named “Pixel”. This team uniquely bridges two Grenoble institutes, IBS and iRTSV, with the aim of developing super-resolution fluorescence microscopy at the frontier between structural and cell biology. The proposal is in line with a strong evolution of the activities of the coordinator towards imaging and integrated structural cell biology. The strategic position of the Pixel team will be optimal to directly import the outcoming results into the cell biology community.

 

ANR Programme: Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques (Blanc SVSE 5) 2011

Project ID: ANR-11-BSV5-0012

Project coordinator:
Monsieur Dominique Bourgeois (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES)
dominique.bourgeois@nullibs.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.