Stress-induced nucleoid remodeling in Deinococcus radiodurans is associated with major changes in Heat Unstable (HU) protein 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. Nucleoid associated proteins (NAPs) are believed to be major actors in nucleoid remodeling, but the details of this process remain poorly understood. Here, using the radiation resistant bacterium D. radiodurans as a model, and advanced fluorescence microscopy, we examined the changes in nucleoid morphology and volume induced by either entry into stationary phase or exposure to UV-C light, and characterized the associated changes in mobility of the major NAP in D. radiodurans, the heat-unstable (HU) protein. While both types of stress induced nucleoid compaction, HU diffusion was reduced in stationary phase cells, but was instead increased following exposure to UV-C, suggesting distinct underlying mechanisms. Furthermore, we show that UV-C-induced nucleoid remodeling involves a rapid nucleoid condensation step associated with increased HU diffusion, followed by a slower decompaction phase to restore normal nucleoid morphology and HU dynamics, before cell division can resume. These findings shed light on the diversity of nucleoid remodeling processes in bacteria and underline the key role of HU in regulating this process through changes in its mode of assembly on DNA.

Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties.

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62,  altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

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 cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins 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 two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins.

Light-Induced Forward and Reverse Intersystem Crossing in Green Fluorescent Proteins at Cryogenic Temperatures.

Combining fluorescence and phosphorescence kinetics, we characterize forward and reverse intersystem crossing (FISC and RISC, respectively) between the singlet and triplet manifolds S ↔ T in photoswitchable (rsEGFP2) and non-photoswitchable (EGFP) green fluorescent proteins upon continuous 488 nm laser excitation at cryogenic temperatures (CTs). Both proteins behave very similarly, with T1 absorption spectra showing a visible peak at 490 nm (10 mM-1 cm-1) and a vibrational progression in the near-infrared (720 to 905 nm). The dark lifetime of T1 is 21-24 ms at 100 K and very weakly temperature-dependent up to 180 K. Above 180 K, T1 lifetimes reduce rapidly to few milliseconds as found at room temperature (RT). FISC and RISC quantum yields are 0.3 and 0.1%, respectively, for both proteins. The light-induced RISC channel becomes faster than the dark reversal at power densities as low as 20 W cm-2. We discuss implications for fluorescence (super resolution-) microscopy at CT and RT.

Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition.

CD47 recognized by its macrophage receptor SIRPα serves as a "don't eat-me" signal protecting viable cells from phagocytosis. How this is abrogated by apoptosis-induced changes in the plasma membrane, concomitantly with exposure of phosphatidylserine and calreticulin "eat-me" signals, is not well understood. Using STORM imaging and single-particle tracking, we interrogate how the distribution of these molecules on the cell surface correlates with plasma membrane alteration, SIRPα binding, and cell engulfment by macrophages. Apoptosis induces calreticulin clustering into blebs and CD47 mobility. Modulation of integrin affinity impacts CD47 mobility on the plasma membrane but not the SIRPα binding, whereas CD47/SIRPα interaction is suppressed by cholesterol destabilization. SIRPα no longer recognizes CD47 localized on apoptotic blebs. Overall, the data suggest that disorganization of the lipid bilayer at the plasma membrane, by inducing inaccessibility of CD47 possibly due to a conformational change, is central to the phagocytosis process.

Single molecule imaging simulations with advanced 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 Single-Molecule Imaging Simulator (SMIS) 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.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

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 lead 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.

Understanding the new BRD4-related syndrome: Clinical and genomic delineation with an international cohort study.

BRD4 is part of a multiprotein complex involved in loading the cohesin complex onto DNA, a fundamental process required for cohesin-mediated loop extrusion and formation of Topologically Associating Domains. Pathogenic variations in this complex have been associated with a growing number of syndromes, collectively known as cohesinopathies, the most classic being Cornelia de Lange syndrome. However, no cohort study has been conducted to delineate the clinical and molecular spectrum of BRD4-related disorder. We formed an international collaborative study, and collected 14 new patients, including two fetuses. We performed phenotype and genotype analysis, integrated prenatal findings from fetopathological examinations, phenotypes of pediatric patients and adults. We report the first cohort of patients with BRD4-related disorder and delineate the dysmorphic features at different ages. This work extends the phenotypic spectrum of cohesinopathies and characterize a new clinically relevant and recognizable pattern, distinguishable from the other cohesinopathies.

Extra kinetic dimensions for label discrimination.

Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.

Stankiewicz-Isidor syndrome: expanding the clinical and molecular phenotype.

PURPOSE: Haploinsufficiency of PSMD12 has been reported in individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), facial dysmorphism, and congenital malformations, defined as Stankiewicz-Isidor syndrome (STISS). Investigations showed that pathogenic variants in PSMD12 perturb intracellular protein homeostasis. Our objective was to further explore the clinical and molecular phenotypic spectrum of STISS. METHODS: We report 24 additional unrelated patients with STISS with various truncating single nucleotide variants or copy-number variant deletions involving PSMD12. We explore disease etiology by assessing patient cells and CRISPR/Cas9-engineered cell clones for various cellular pathways and inflammatory status. RESULTS: The expressivity of most clinical features in STISS is highly variable. In addition to previously reported DD/ID, speech delay, cardiac and renal anomalies, we also confirmed preaxial hand abnormalities as a feature of this syndrome. Of note, 2 patients also showed chilblains resembling signs observed in interferonopathy. Remarkably, our data show that STISS patient cells exhibit a profound remodeling of the mTORC1 and mitophagy pathways with an induction of type I interferon-stimulated genes. CONCLUSION: We refine the phenotype of STISS and show that it can be clinically recognizable and biochemically diagnosed by a type I interferon gene signature.

Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy.

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.

Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae.

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis.

Supramolecular assembly of the Escherichia 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 Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization 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-EM and reveal the molecular determinants of LdcI polymerization, 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 organization in the acid stress response.

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State.

Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

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 nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in 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.

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Cell morphology and nucleoid dynamics in dividing Deinococcus 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 with a multipartite genome, constitutes a valuable system for the study of the nucleoid in cocci. Using advanced microscopy, we show that D. radiodurans undergoes coordinated morphological changes at both the cellular and nucleoid level as it progresses through its cell cycle. The nucleoid is highly condensed, but also surprisingly dynamic, adopting multiple configurations and presenting an unusual arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the cell centre. Single-particle tracking and fluorescence recovery after photobleaching studies of the histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression in this organism.

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 longer tracks in sptPALM with minimum effort.