Biology subjects

Bourgeois, D.

Publications and source records attributed to Bourgeois, D..

9 recordsLinked to original sources

bioRxiv2024

Decoding mEos4b Day-Long Maturation and Engineering Fast Maturing Variants

The maturation speed of fluorescent proteins is a crucial parameter that influences cellular brightness, effective labeling efficiency and temporal resolution in fluorescence microscopy. Green-to-red photoconvertible fluorescent proteins (PCFPs) used in pulse-chase experiments and super-resolution techniques such as Photoactivated Localization Microscopy (PALM), single-particle-tracking PALM (sptPALM) and Minimal Fluorescence Photon Fluxes Microscopy (MINFLUX) may be hampered by slow maturation. We systematically characterized the maturation speed of mEos-derived PCFPs in E. coli and found that, in contrast to pcStar and mEosEM, several variants such as mEos2, mEos3.1, mEos3.2 and mEos4b mature extremely slowly. Strikingly, the oxidation step in those PCFPs is fast and not rate-limiting. Through a rational mutagenesis approach, we developed a strategy to reduce the day-long maturation time of mEos4b by nearly two orders of magnitude without significantly impacting its molecular brightness and photophysical performance under single-molecule imaging conditions.

biophysics↗

bioRxiv2023

Stress-induced nucleoid remodeling in Deinococcus radiodurans is associated with major changes in HU abundance and dynamics

Bacteria have developed a wide range of strategies to respond to stress, one of which is the rapid large-scale reorganization of their nucleoid, which is often associated with a major reprogramming of the gene expression profile. Nucleoid associated proteins (NAPs) are believed to be major actors in this process, but the molecular mechanisms underlying stress-induced nucleoid remodeling remain poorly understood. Here, using the radiation resistant bacterium, D. radiodurans, as a model, and advanced fluorescence microscopy approaches, we examined the changes in nucleoid morphology and compaction induced by either entry into stationary phase or exposure to UV-C light, and characterized the associated changes in abundance and dynamics of the major NAP in D. radiodurans, the heat-unstable (HU) protein. While both types of stress induced a similar macroscopic rearrangement of the nucleoid into a more compact structure, HU diffusion was significantly reduced in stationary phase cells, but was instead dramatically increased following exposure to UV-C, suggesting that the underlying mechanisms of remodeling are distinct. Furthermore, a detailed comparison of the cellular response to sublethal and lethal doses of UV-C light revealed that UV-induced nucleoid remodeling involves a rapid nucleoid condensation step associated with increased HU diffusion and abundance, followed by a slower decompaction phase to restore normal nucleoid morphology and HU dynamics, before cell growth and division can resume. Together, these findings shed light on the diversity and complexity of stressed-induced nucleoid remodeling processes in bacteria.

microbiology↗

bioRxiv2022

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at [~]110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/504779v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1d5ed02org.highwire.dtl.DTLVardef@1bb2359org.highwire.dtl.DTLVardef@10dc6b1org.highwire.dtl.DTLVardef@16fcd94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Single Molecule Imaging Simulations with Full Fluorophore Photophysics

Advanced fluorescence imaging techniques such as single-molecule localization microscopy (SMLM) fundamentally rely on the photophysical behavior of the employed fluorophores. This behavior is generally complex and impacts data quality in a subtle manner. A simulation software named SMIS (Single-Molecule Imaging Simulator) is introduced that simulates a widefield microscope and incorporates fluorophores with their spectral and photophysical properties. With SMIS, data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented. The influence of advanced fluorophore characteristics, imaging conditions and environmental parameters can be evaluated, facilitating the design of real experiments and their proper interpretation.

biophysics↗

bioRxiv2022

mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state. TOC GRAPHICS O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2020

Supramolecular assembly of the E. coli LdcI upon acid stress

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statement Bacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein

The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature, and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in-situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a non-fluorescent state. Our results add a dynamic view to the static structures obtained by X-ray crystallography. Including NMR into the analytical toolbox used for fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins, and their correlation with the observed photochemical and photophysical properties.\n\nSignificancePhoto-transformable Fluorescent Proteins (PTFPs) are essential tools for super-resolution (SR) microscopy. In practical applications, however, researchers often encounter problems when using PTFPs in a particular cellular context, because the environmental conditions (pH, temperature, redox potential, oxygen level, viscosity, ...) affect their brightness, photostability, phototransformation kinetics, etc. Rational fluorescent protein engineering exploits the mechanistic information available from structural studies, mainly X-ray crystallography, in order to design new PTFP variants with improved properties for particular applications. Here we apply NMR spectroscopy in solution to investigate the light-induced changes in conformational dynamics of rsFolder, a reversibly switchable fluorescent protein. The dynamic view offered by NMR highlights protein regions that comprise potentially interesting mutation points for future mutagenesis campaigns.

biophysics↗

bioRxiv2019

Cell morphology and nucleoid dynamics in dividing D. radiodurans

Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here, we reveal that Deinococcus radiodurans, a relatively large, spherical bacterium, possessing a multipartite genome, and well-known for its radioresistance, constitutes a valuable system for the study of nucleoids in cocci. Using advanced microscopy, we show that as D. radiodurans progresses through its cell cycle, it undergoes coordinated morphological changes at both the cellular and nucleoid level. D. radiodurans nucleoids were found to be highly condensed, but also surprisingly dynamic, adopting multiple distinct configurations and presenting a novel chromosomal arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the centre of cells. Single-molecule and ensemble studies of the abundant histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings clearly demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression.

microbiology↗

bioRxiv2018

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort.

biophysics↗