Biology subjects

Didier, P.

Publications and source records attributed to Didier, P..

11 recordsLinked to original sources

bioRxiv2026

Rhodamine-derived ratiometric fluorescent molecular rotor for mitochondrial viscosity sensing

Fluorescent molecular rotors for sensing local microviscosity have become indispensable tools for better understanding the mechanisms of living systems. Current probes relay on intensity or lifetime measurements, while ratiometric molecular rotors are lacking. Here, we introduce a new design concept of molecular rotor using xanthene scaffold with freely rotating 9-aryl group. We studied three rhodamine derivatives, where 2-carboxyphenyl group was replaced with 4-methoxyphenyl, 2-thienyl and 2-benzofuryl. We found that five membered 9-aryl ring provides two major effects. First, free rotation of thienyl derivative, confirmed by theoretical calculations, makes it classical molecular rotor with intensity and lifetime-based response to viscosity. Second, benzofuryl derivative exhibits second emission band in near-infrared region and fluorescence ratiometric response to viscosity. Theoretical calculations suggest that the benzofuryl derivative can attain planar conformation in the excited state, which explains appearance of the NIR emitting band. Both new dyes efficiently target mitochondria, while the 9-benzofuryl derivative enable quantitative ratiometric measurement of viscosity of inner mitochondrial membrane. Ratiometric imaging revealed mitochondria heterogeneity with local viscosity values ranging from 250 to 460 cP. Oxidative stress induced significant rise in the local viscosity in mitochondria in addition to their morphological changes. Overall, we propose a concept that converts bright rhodamine dyes into molecules rotors with a valuable ratiometric response to viscosity, which opens a bunch of potential biological applications.

biochemistry↗

bioRxiv2026

Membrane-Promoted J-Aggregation of BODIPY Dimers Enables Spontaneous Blinking in Green and Red Channels for Live-Cell Nanoscopy

BODIPY dyes are widely used in bioimaging, yet their aggregation behavior within the plasma membrane (PM) remains poorly exploited for single-molecule localization microscopy (SMLM). Here, we design a series of green-emitting PM-targeted BODIPY dimers engineered to undergo spontaneous and transient H- and J-aggregation. By tuning the linker length between the fluorophores, we identify dimers that form intramolecular H-aggregates in polar media and emissive J-aggregates ({lambda}em {approx} 535 nm) through membrane-driven intermolecular interactions. In lipid bilayers, all dimers aggregate above a probe/lipid ratio of 1/100, exclusively generating J-aggregates. In live-cell SMLM, the monomeric MB-488 provides high event numbers via diffusion-driven emission, whereas dimers exhibit stable blinking and yield brighter red-shifted J-aggregate events with improved localization precision. Red-channel events localize to specific PM regions, suggesting preferential J-aggregation within distinct membrane microdomains. HaloTag constructs targeted to cell-surface PDGFR further confirm that intramolecular J-aggregation is possible but strongly amplified by the membrane through intermolecular collisions. These results demonstrate that green BODIPYs and their J-aggregates enable robust live SMLM in green and red channels, and reveal the PM as a privileged environment for emissive J-aggregation.

biophysics↗

bioRxiv2026

Fluorescence based microviscosity mapping in membraneless organelles

Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid-liquid phase separation. Their biological functions are intimately linked to their material properties including viscosity. Condensate viscosity is determined by the size, shape, concentration and molecular interactions between MLOs components. It impacts the diffusion of MLOs constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar sub-compartments. Furthermore, nucleolar reorganization induced by inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the microviscosity of stress granules is lower than that of the nucleolus. Overall, presented results demonstrate the strong potential of the BODIPY based molecular rotors as a versatile and powerful tools for probing the material properties of cellular MLOs.

biophysics↗

bioRxiv2025

Fear, anxiety, and the extended amygdala- Absence of evidence for strict functional segregation

Since the time of Freud, the distinction between fear and anxiety has been a hallmark of influential models of emotion and emotional illness, including the Diagnostic and Statistical Manual of Mental Disorders (DSM) and Research Domain Criteria (RDoC). Fear and anxiety disorders are common, debilitating, and challenging to treat, underscoring the importance of developing accurate models of the underlying neurobiology. Although there is consensus that the extended amygdala (EA) plays a central role in orchestrating responses to threat, the respective contributions of its two major subdivisions--the central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST)--remain contentious. To help adjudicate this debate, we performed a harmonized mega-analysis of fMRI data acquired from 295 adults as they completed a well-established threat-anticipation paradigm. Contrary to popular double-dissociation models, results demonstrated that the Ce responds to temporally uncertain threat and the BST responds to certain threat. In direct comparisons, the two regions showed statistically indistinguishable responses, with strong Bayesian evidence of regional equivalence. In contrast, frontocortical regions responded preferentially to uncertain-threat anticipation. Together, these observations underscore the need to reformulate conceptual models that posit a strict segregation of temporally certain and uncertain threat processing in the EA.

neuroscience↗

bioRxiv2024

Lipophilic molecular rotor to assess the viscosity of oil core in nano-emulsion droplets

HypothesisCharacterization of nanoscale formulations is a continuous challenge. Size, morphology and surface properties are the most common characterizations. However, physicochemical properties inside the nanoparticles, like viscosity, cannot be directly measured. Herein, we propose an original approach to measuring dynamic viscosity using a lipidic molecular rotor solubilized in the core of nano-formulations. These molecules undergo conformational changes in response to viscosity variations, leading to observable changes in fluorescence intensity and lifetime, able to sense the volume properties of dispersed nano-domains. ExperimentsThe lipophilic molecular rotor (BOPIDY derivatives) was specifically synthesized and characterized as oil viscosity sensing in large volumes. A second part of the study compares these results with rBDP-Toco in nano-emulsions. The objective is to evaluate the impact of the formulation, droplet size and composition on the viscosity of the droplets core. FindingsThe lipophilic rotor showed a universal behavior whatever the oil composition, giving a master curve. Applied to nano-formulations, it discloses the viscosity in the nano-emulsion droplets, enabling the detection of slight variations between reference oil samples and the nano-formulated ones. This new tool opens the way to the fine characterization of complex colloids and multi-domain nano and micro systems, potentially applied to hybrid materials and biomaterials.

biophysics↗

bioRxiv2024

Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation Induced Activation and its Application to Live Dynamic Super-Resolution Microscopy

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green-emitting photoactivatable furanyl-BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo-oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93-fold fluorescence enhancement at 510 nm, 49% yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super-resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells.

cell biology↗

bioRxiv2024

Piggybacking functionalized DNA nanostructures into live cell nuclei

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

bioengineering↗

bioRxiv2023

Adolescent social anxiety is associated with diminished discrimination of anticipated threat and safety

Social anxiety--which typically emerges in adolescence--lies on a continuum and, when extreme, can be devastating. Socially anxious individuals are prone to heightened fear, anxiety, and the avoidance of contexts associated with potential social scrutiny. Yet most neuroimaging research has focused on acute social threat. Much less attention has been devoted to understanding the neural systems recruited during the uncertain anticipation of potential encounters with social threat. Here we used a novel fMRI paradigm to probe the neural circuitry engaged during the anticipation and acute presentation of threatening faces and voices in a racially diverse sample of 66 adolescents selectively recruited to encompass a range of social anxiety and enriched for clinically significant levels of distress and impairment. Results demonstrated that adolescents with more severe social anxiety symptoms experience heightened distress when anticipating encounters with social threat, and reduced discrimination of uncertain social threat and safety in the bed nucleus of the stria terminalis (BST), a key division of the central extended amygdala (EAc). Although the EAc--including the BST and central nucleus of the amygdala--was robustly engaged by the acute presentation of threatening faces and voices, the degree of EAc engagement was unrelated to the severity of social anxiety. Together, these observations provide a neurobiologically grounded framework for conceptualizing adolescent social anxiety and set the stage for the kinds of prospective-longitudinal and mechanistic research that will be necessary to determine causation and, ultimately, to develop improved interventions for this often-debilitating illness.

neuroscience↗

bioRxiv2023

Targeted Photoconvertible BODIPYs Based on Directed Photooxidation Induced Conversion for Applications in Photoconversion and Live Super Resolution Imaging

Photomodulable fluorescent probes are drawing an increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single molecule localization microscopy to perform super resolution imaging. Herein we shed light on a red and far-red BODIPY, namely BDP-576 and BDP-650 possessing both properties of conversion and switching. Our study demonstrates that theses pyrrolyl-BODIPYs respectively convert towards typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that these pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced and where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes towards different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 minutes in two color imaging with laser scanning confocal microscopy demonstrating its robustness. The switching properties of these photoconverters were studied at the single molecule level and were then successfully used in live Single Molecule Localization Microscopy in epithelial cells and neurons. Both membrane and mitochondria targeted probes could be used to decipher membrane 3D architecture and mitochondria dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in live advanced imaging.

cell biology↗

bioRxiv2020

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group which is mandatory for anchoring the complexes at the MP, was found to be dispensable for the association of Gag with the gRNA in the cytosol. STATEMENT of SIGNIFICANCEFormation of HIV-1 retroviral particles relies on specific interactions between the retroviral Gag precursor and the unspliced genomic RNA (gRNA). During the late phase of replication, Gag orchestrates the assembly of newly formed viruses at the plasma membrane (PM). It has been shown that the intracellular HIV-1 gRNA recognition is governed by the two-zinc finger (ZF) motifs of the nucleocapsid (NC) domain in Gag. Here we provided a clear picture of the role of ZFs in the cellular trafficking of Gag-gRNA complexes to the PM by showing that either ZF was sufficient to efficiently promote these interactions in the cytoplasm, while interestingly, ZF2 played a more prominent role in the relocation of these ribonucleoprotein complexes at the PM assembly sites.

biophysics↗

bioRxiv2018

Therapeutic Treatment of Aerosolized Staphylococcal Enterotoxin B in Nonhuman Primates with two Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a protein exotoxin found on the cell surface of Staphylococcus aureus that is the source for multiple pathologies in man. When purified and concentrated in aerosol form, SEB can cause an acute and often fatal intoxication, and thus is considered a biological threat agent. There are currently no vaccines or treatments approved for human use. Studies in rodent models of SEB intoxication show that antibody therapy may be a promising treatment strategy, however many have used antibodies only prophylactically or well before any clinical signs of intoxication are apparent. We assessed and compared the protective efficacy of two monoclonal antibodies, Ig121 and c19F1, when administered after aerosol exposure in a uniformly lethal nonhuman primate model of SEB intoxication. Rhesus macaques were challenged using small particle aerosols of SEB, and then were infused intravenously with a single dose of either Ig121 or c19F1 (10 mg/kg) at either 0.5, 2 or 4 hours postexposure. Onset of clinical signs, hematological, and cytokine response in untreated controls confirmed the acute onset and potency of the toxin used in the challenge. All animals administered either Ig121 or c19F1 survived SEB challenge, whereas the untreated controls succumbed to SEB intoxication 30-48 hours postexposure. These results represent the successful therapeutic in vivo protection by two investigational drugs against SEB in a severe nonhuman primate disease model and punctuate the therapeutic value of monoclonal antibodies hold when faced with treatment options for SEB-induced toxicity in a postexposure setting.\n\nOne Sentence Summary: Two high-affinity monoclonal antibodies were tested for therapeutic efficacy using a rhesus macaque challenge model of aerosolized SEB

immunology↗