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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BOURGEOIS Dominique

Pixel Presentation

Responsable: Dominique Bourgeois

Presentation

Pixel is a multidisciplinary team that works at the frontier between structural and cell biology. The team was created in 2010, motivated by the evolution of modern structural biology towards integration into the cellular context. The Pixel team has a strong methodological focus based on two main objectives:

- Mechanistic investigation and engineering of fluorescent proteins
- Development of Single Molecule Localization Microscopy (SMLM), notably PhotoActivatable Localization Microscopy (PALM super-resolution) and single particle tracking PALM (sptPALM).
To foster these developments, the Pixel team closely collaborates with other biology-oriented IBS teams, to address biologically relevant projects, mainly in the field of microbiology, and it also maintains strong links with the M4D imaging platform. Recently, with the creation of the I2SR group, the team added its own biological focus, and now investigates the nanoscale organization of the phagocytic synapse.

One of our central themes concerns photophysical studies of phototransformable fluorescent proteins (PTFPs). These genetically encoded fluorescence markers are fascinating because their fluorescence state can be tuned by illumination with proper laser light. For example, the emission color of some PTFPs can be changed from green to red upon illumination with violet light (so-called photoconvertible FPs, PCFPs). Other PTFPs can be reversibly switched from on states to off states by alternating cyan and violet light (so-called reversibly photoswitchable FPs, RSFPs). PTFPs are fundamental players in super-resolution microscopy as well as other advanced fluorescence methods such as pulse-chase imaging, modulated-illumination imaging, photochromic Förster Resonance Energy Transfer (FRET) or biological data storage. Phototransformation properties like photoactivation, photoconversion and photoswitching need to be understood and optimized for each application. In addition, photoblinking (the stochastic and transient loss of fluorescence) and photobleaching (the irreversible loss of fluorescence) are crucial photophysical properties that apply to all fluorescent proteins and that we investigate in great details, although they still hide a lot of mysteries.

Many groups worldwide study and develop fluorescent proteins to create ever more performing markers or sensors. We contribute to this field of research by investigating PTFPs using an extensive set of tools: we typically employ a combination of kinetic crystallography (including XFEL in collaboration with the Weik group), in crystallo spectroscopy, single molecule imaging in vitro and in cellulo and modelling in silico (collaboration I. Demachy), and recently NMR (collaboration IBS, B. Brutscher).

In the last years, we have largely focused our attention on photoblinking and photobleaching in popular PCFPs such as mEos4b. These photophysical processes seem to exhibit a never ending complexity, but we discover more and more about them. For example, in collaboration with the team of P. Dedecker (KUL, Belgium) we recently deciphered a major mechanism of blinking in red mEos4b, typically causing serious trouble in the quality and interpretation of quantitative PALM (qPALM) or sptPALM data, and showed that this mechanism very closely relates to the reversible photoswitching mechanisms at play in RSFPs. This allowed us to propose a trick to reduce blinking in sptPALM, so as to enable the reconstruction of significantly longer tracks. We also turned our attention to green state photophysics in PCFPs. Indeed, although PCFPs are only detected once photoconverted to the red state, what happens before photoconversion considerably affects their performance as markers for SMLM. Recently, in collaboration with the teams of J.B Sibarita and M. Sainlos (IINS, Bordeaux), we started a project aiming at further improving the photostability of PCFPs, by combining structural studies with high-content-screening single-molecule imaging approaches to achieve efficient semi-rational engineering.
More and more, we realize the important link between PTFPs protein dynamics and PTFPs photophysical behavior, so that NMR, which can address the dynamical behavior of proteins with great detail, is also becoming a central tool for our investigations.

In parallel with these photophysical studies, we develop PALM super-resolution microscopy, a single-molecule-based localization method now used worldwide to overcome the diffraction limit. PALM and its derivatives qPALM and sptPALM are almost entirely based on the proper manipulation of PTFP’s photophysics, which brings coherence to our activities. Our PALM microscope is used to study PTFPs in cellulo (or in vitro) at the single molecule level. Recently, in collaboration with the german team of Jörg Enderlein (Göttingen University), we have started the development of PALM microscopy at cryogenic temperatures (cryo-PALM) which constitutes one of the major developments in the field in the years to come, especially for applications in integrated structural biology based on correlative microscopy (cryo-CLEM). Our role in this project is to investigate PTFP’s photoswitching capabilities at low temperature.
On the biological side, we address fundamental questions in microbiology in collaboration with other IBS teams. Examples include the cell division machinery in S. Pneumoniae (collaboration C. Morlot), the nucleoid dynamics and the DNA repair mechanisms in D. radiodurans (collaboration J. Timmins), a collaboration that played a major role in the creation of the I2SR group), and the stress response mechanisms by LDCI, RavA et ViaA proteins in E. coli (collaboration I. Gutsche).
Our own biological project, led by P. Frachet, concerns the investigation of the molecular organization of the phagocytic synapse during the clearance of apoptotic cells or cancer cells, using super resolution imaging (SR). This fundamental cellular process is directly linked to the programmed cellular pathways that control regular cellular renewal, inflammation, and immunity.
All these biological projects are carried out on our home-built PALM microscope, which was integrated into the M4D platform, (collaboration J.P. Kleman). A new microscope has recently been purchased to match the increasing demand for SMLM-based super resolution microscopy at IBS.

Collaborations

External collaborations :

- Georg-August-University Göttingen, Germany
- University of Leuven, Belgium
- Institute for Interdisciplinary Neuroscience, France, France
- Université Paris XI, France

Internal Collaborations (Super-resolution microscopy: biological projects):
- DNA Damage and Repair team (Nucleoid dynamics and DNA repair in D. radiodurans, J. Wulffele)
- Pneumococcus group (Cell division and cell wall synthesis, J. Trouvé)
- MICA group (E. coli stress response)

Collaborations internes (Fluorescent proteins photophysics):
- Structural protein dynamics team (M. Weik: Fluorescent protein excited state dynamics studied by XFEL)
- Protein & RNA Folding and Methods development (Photophysics of PTFPs investigated by NMR, Nina Christou)

Staff

Permanent Staff :
- Dominique Bourgeois (CNRS - DR1)
- Virgile Adam (CNRS - CR1)
- Martin Byrdin (CEA staff scientist, 30%)
- Philippe Frachet (UGA MCF HCex )
- Pascale Tacnet (CNRS IR1)

Non-Permanent Staff :
- Oleksandr Glushonkov

PhD Students :
- Angela Mantovanelli
- Nina Christou (50% NMR group)
- Jennyfer Trouvé (100% PG group , co-supervision Pixel)
- Samy Dufour
- Jip Wulffele

former Postdocs :
- Joel Beaudouin
- Sergiy Avilov
- Delphine Arcizet

Former PhD Students :
- Daniel Thedie
- Romain Berardozzi
- Chenxi Duan
- Aline Regis-Faro

Research topics

- Fluorescent proteins photophysics. Characterization of fluorescence mechanisms. Photoactivation, photoconversion, photoswitching, blinking and photobleaching mechanisms. Design of improved fluorescent protein variants
- Super resolution microscopy: PALM/TIRF microscopy; methodological developments, cryonanoscopy. Collaborations for microbiology applications
- Photoactivated protein dynamics. Coupled kinetic crystallography, in crystallo spectroscopy, single molecule imaging and theoretical modeling. Trapping of intermediates.
- Biotechnological application of fluorescent proteins. Efferosynapse, apoptosis.

Key words

Fluorescent proteins; super resolution fluorescence microscopy; TIRF microscopy, single molecule imaging; photoactivation; blinking, photobleaching microspectrophotometry; kinetic crystallography; structural dynamics ; photophysics; Protein dynamics and mechanisms; Macromolecular nanomachines; Methods developments; Protein design and engineering.

Specialized techniques

- Super-resolution microscopy : PhotoActivated Localization Microscopy (PALM); TIRF microscopy; single molecule imaging.
- Optical spectroscopy: Temperature-controlled UV-visible and fluorescence microspectrophotometry
- Kinetic crystallography : Combined in crystallo spectroscopy and crystallography, trapping of intermediate states.
- Molecular and cell biology: Fluorescent protein fusion constructs.

Highlights

- IrisFP structure
- Eos FP photoconversion
- IrisFP X blinking
- Padron cryoswitching
- IrisFP Vis blinking
- IrisFP bleaching
- role of Arg66
- rsFolder
- Iris FP SFX
- rsEGFP2 onswitching by XFEL
- mEos2 dark states
- EGFP triplet state
- mEos4b red state blinking
- mEos4b green state blinking