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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 is strongly methodology oriented and follows two main objectives:

- Mechanistic investigation and engineering of fluorescent proteins
- Development of super-resolution fluorescence microscopy to address biologically relevant projects, mainly in the field of microbiology and in collaboration with other IBS teams.

Our central theme 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. Other PTFPs can be reversibly switched from on states to off states by alternating cyan and violet light. PTFPs are fundamental players in super-resolution microscopy as well as other advanced fluorescence methods such as photochromic Förster Resonance Energy Transfer (FRET) or biological data storage. Phototransformation properties like photoactivation, photoconversion and photoswitching need to be understood and optimized for special applications. 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 remain poorly characterized.
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 photoswitching and photobleaching in PTFPs. Whereas photoswitching offers many exciting applications, photobleaching constitutes one of the most fundamental drawbacks of fluorescent proteins. Currently, we are also turning our attention to the study of photoblinking of photoconvertible PTFPs which is caused by rather mysterious processes creating trouble in single-molecule based quantitative super-resolution microscopy applications and for single particle tracking.

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 does not involve sophisticated microscope optics, and is 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 will mainly be the elaboration of PTFPs functioning at low temperature.
Importantly, we also address fundamental biological questions in microbiology in collaboration with other IBS teams. Examples include the cell division machinery in S. Pneumoniae (collaboration C. Morlot), the DNA repair mechanisms in D. radiodurans (collaboration J. Timmins), stress response mechanisms by LDCI, RavA et ViaA proteins in E. coli (collaboration I. Gutsche). A new projet has been started on the study of the phagocytary synapsis (efferocytose) of macrophages (collaboration P. Frachet). These biological projects are carried out on our PALM microscope, which was recently integrated into the M4D platform, (collaboration J.P. Kleman).

Collaborations

External collaborations :

- Georg-August-University Göttingen, Germany
- Max Planck Institute for terrestrial microbiology Marburg, Germany
- University of Leuven, Belgium
- Bordeaux Imaging Center, France
- Université Paris XI, France
- i2BC-JOLIOT, Saclay, France

Internal Collaborations (Super-resolution microscopy: biological projects):
- SAGAG group (The heparan sulfate synthesis machinery , J. Beaudouin)
- DNA Damage and Repair team (Dynamique dans D. radiodurans, K. Floch)
- Pneumococcus group (Cell division and cell wall synthesis, J. Trouvé)
- MMBV team (E. coli stress response, C. Liesche; Development of cryoCLEM, A. Burt)
- IRPAS group (The efferocytose synaptic machinery, P. Frachet)

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%)

Non-Permanent Staff :
- Joel Beaudouin
- Oleksandr Glushonkov

PhD Students :
- Daniel Thédié
- Kevin Floc’h (50% VIC group)
- Nina Christou (50% NMR group)
- Jennyfer Trouvé (100% PG group , co-supervision Pixel)
- Samy Dufour (100% IRPAS group, co-supervision Pixel)

former Postdocs :
- Sergiy Avilov
- Delphine Arcizet

Former PhD Students :
- 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

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