Biology subjects

Gautier, A.

Publications and source records attributed to Gautier, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Chemogenetic timestamping for the precise tracing of cell history into protein assemblies

Self-assembling protein fibers enable to record events in single cells, bypassing the need for long-term time-lapse imaging. Fluorescent marks introduced within the growing fiber at user-defined times provide timestamps, giving access to the temporal dynamics of the recorded event. Here, we introduce CATCHFiber, a single-color timestamping strategy for tracing cellular events with high temporal resolution into self-assembling protein fibers. Relying on chemically-induced dimerization to precisely and rapidly control the incorporation of fluorescent proteins into the fiber, CATCHFiber allows the introduction of short 30-min spaced timestamps, significantly increasing the precision of event timings compared to existing methods. This increase in temporal resolution expands the use of fiber-based recorders beyond transcriptional activity, allowing to trace the kinetics of faster processes such as protein degradation, protein neosynthesis and kinase activity, and to determine the timing of cell cycle steps.

cell biology↗

bioRxiv2026

Non-covalent reversibly photoconvertible fluorescent tags for wash-free protein labeling

Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, we obtain positive reversible photoswitchers, that increase their brightness under illumination. We also generate a palette of non-covalent reversibly photoconvertible fluorescent proteins changing their fluorescence color upon illumination, a reversible behavior that still remains absent in regular fluorescent proteins. This light-induced color change opens the possibility to discriminate six spectrally similar FAST variants in live cells upon demonstrating the superiority of using multiple spectral channels for exploiting the time dependence of the fluorescence response to illumination.

biophysics↗

bioRxiv2026

The cuticle lamellae responsible for structural coloration in red algae display common features with the metazoan extracellular matrix

Structural coloration is a physical phenomenon observed in many living organisms, including seaweeds, via the formation of highly organized nanostructures. Although the deposition of multilayer thin-film in the extracellular matrix (ECM) is a widespread characterized system, enabling light interference, to date such lamellae have only been described in the cuticle of red algae belonging to the Gigartinaceae family within macroalgae. Here, we determine the composition of the multilayered cuticle components in the model species Chondrus crispus (Rhodophyta), using genomic, transcriptomic, proteomic, metabolomic and analytical profiling of carbohydrates. The complex structural assembly revealed common features with the ECM of animals. The carbohydrate fraction consists of a complex mixture of ECM carrageenans and glycosaminoglycan-like structures. A major von Willerbrand factor A domain (VWA) protein plays a critical role for the adhesion of macromolecules, including protein-protein interactions and to sulfated polysaccharides. We propose the name of Lamellae Cohesive Protein (LCP) regarding the alternate distribution of chemically distinct constituents within the cuticle. For the first time, we identified major proteins of the algal cuticle, providing a framework for addressing the evolutionary origins of the algal cuticle biosynthesis and raise important questions regarding its role, particularly across the life cycle marked by major structural ECM modifications.

biochemistry↗

bioRxiv2025

Early HIV-1 maturation drives Env clustering and fusion competence

Human immunodeficiency virus type 1 (HIV-1) particles are initially released from the cell in an immature state that cannot cause infection. The proteolytic processing of the Gag structural polyprotein into its individual components triggers a dramatic structural reorganisation that produces fully mature and infectious virions. HIV-1 maturation remodels the Gag lattice to enable membrane fusion, but how early proteolytic steps regulate the envelope glycoprotein (Env) organisation is unknown. Although cryo-electron tomography has defined structural intermediates of maturation, it has not resolved how Env conformers relate to specific early maturation states. Here, we show that partial Gag cleavage, prior to capsid formation, allosterically reorganises the membrane-proximal lattice, promoting Env clustering and fusion-competent conformations. Using native virus preparations combined with cryogenic fluorescence-lifetime imaging correlated to cryo-electron tomography, we map Env conformational signatures onto defined Gag-processing intermediates. This reveals an unrecognised early maturation stage in which incomplete Gag processing primes Env for entry, shifting current models by positioning functional Env activation upstream of core maturation. These results establish that early lattice remodelling is a key determinant of HIV-1 fusion competence and expose a previously inaccessible, intervention-sensitive step with implications for therapeutic and vaccine strategies targeting maturation-entry coupling.

microbiology↗

bioRxiv2025

Photoejection turns non-covalent fluorescent tags into negative reversible photoswitchers

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we introduce RSpFAST, a new class of reversibly photoswitchable fluorescent labels that combine a biomolecular host (pFAST protein tag) with a reversibly photoisomerizable guest (fluorogen), allowing fluorescence brightness to be modulated through illumination and molecular complexation. We combine thermokinetic, photochemical, and structural investigations to obtain a comprehensive mechanistic and kinetic understanding of RSpFAST. Building on this theoretical framework, we demonstrate in both live and fixed cells that RSpFAST exhibits an unprecedented dual behavior: a stable and wash-free fluorescent labeling tag turns into a negative reversible photoswitcher by lowering the fluorogen concentration and increasing light intensity. In this photoejection-driven kinetic regime, RSpFAST is shown to be an efficient marker for dynamic contrast and super-resolution microscopy.

biochemistry↗

bioRxiv2025

Chemogenetic modulation of luciferase emission color for imaging and sensing

Bioluminescent luciferases have emerged as powerful tools for bioimaging, enabling to image biological systems without external excitation light, reducing thus phototoxicity and eliminating background autofluorescence. Advanced imaging requires luciferases that deliver high photon output for enhanced sensitivity, tunable emission colors for multicolor imaging, and red-shifted emission for effective deep tissue imaging. Here, we introduce LumiFAST, a small tunable luciferase engineered by fusing the bright blue-light emitting NanoLuc with the tunable chemogenetic fluorescent reporter pFAST. pFAST binds and stabilizes the fluorescent state of a variety of synthetic fluorogenic chromophores (also called fluorogens). Its proximity with NanoLuc leads to efficient bioluminescence resonance energy transfer (BRET), enabling customizable red-shifted emission. Thanks to the small size of pFAST, LumiFAST maintains a compact structure, while its modular design allows emission color to be tuned from cyan to green, yellow, orange and red simply by changing the fluorogen. Systematic optimization of the fusion topology and linker length yielded an optimal variant with apparent BRET efficiencies reaching up to 90 %. The red-shifted emission of LumiFAST enables dual-color microscopy imaging when used alongside NanoLuc and allows imaging through thick scattering media. Beyond imaging, our insights into the structural factors governing efficient BRET allowed us to engineer biosensors based on NanoLuc and pFAST for the visualization of protease activity and protein-protein interactions in live cells.

cell biology↗

bioRxiv2025

Characterizing drug activity with sensitive interactomes in human living cells

A number of human diseases results from abnormal protein-protein interactions (PPIs) involving key regulatory proteins. Therefore, an important strategy in therapeutics consists in developing inhibitory molecules that should ideally be specific for the aberrant PPI. In this context, it is critical to evaluate the number of PPIs that could be affected by the candidate molecule and to analyze the inhibitory potential before and after the formation of the PPI. Surprisingly, these two molecular aspects are rarely considered, due to a lack of appropriate methodological approaches. In this study, we present a novel methodology that captures drug-sensitive PPIs by considering drug-induced cellular functions in live cell conditions. As a proof-of-concept, we identified interactions of the human core signaling protein ERK1 that are specifically affected by two different inhibitory molecules. In addition, we used a complementary set of innovative tools that allowed visualizing the inhibitory effect on ERK1/cofactor protein complexes after their assembly in living cells. Overall, our work establishes a unique methodological approach for deciphering drug activity for potentially any target bait protein of interest.

molecular biology↗

bioRxiv2025

Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei

The insect-specific yellow gene, first described over a century ago, remains enigmatic despite its conserved role in pigmentation. In the malaria vector mosquito Anopheles coluzzii, yellow is strongly expressed during juvenile stages and in adult ovaries, mirroring pigmentation patterns that likely enhance desiccation resistance, camouflage, and reproductive fitness. To dissect its function, we generated a complete knockout allele yellow(-)KI by replacing the first exon with an EGFP reporter, and introgressed it into multiple genetic backgrounds. Loss of yellow abolished black cuticle pigmentation and delayed larval development but left adult lifespan unaffected. Developmental delay likely impaired mating success in competition with wild-type mosquitoes, hastening the decline of the X-linked yellow(-)KI allele. Notably, yellow(-)KI females showed a consistent twofold increase in susceptibility to the rodent malaria parasite Plasmodium berghei, independent of the complement factor TEP1 and melanization, whereas infection with P. falciparum was unchanged. Antibiotic treatment reversed the heightened P. berghei susceptibility, implicating altered gut microbiota as a mediator. Our findings reveal that yellow not only shapes pigmentation but also indirectly modulates vector competence through microbiota-dependent mechanisms, underscoring the complex interplay between cuticle biochemistry, microbial ecology, and pathogen transmission.

microbiology↗

bioRxiv2025

Super-resolution live-cell mapping of protein-protein interactions using chemogenetic split reporters and STED microscopy

The ability to map protein-protein interactions (PPI) within living cells at high spatial resolution is essential for unravelling their roles in cellular biology. Although several super-resolution microscopy techniques are available, visualizing PPI below the diffraction limit of light across entire live cells remains a challenge. Here, we introduce an approach that combines the chemogenetic split fluorescent reporter splitFAST2 with STED microscopy for sub-diffraction imaging of PPI. As the fluorescence of splitFAST2 is activated only where the two proteins interact, this system allows for highly precise and unambiguous localization in live cells, enhanced further by the rapid super-resolution imaging capabilities of STED. The improved spatial resolution of our approach enabled us to precisely map the subcellular localization of inducible interactions or constitutive interactions. Beyond simply detecting PPIs below the diffraction limit in whole living cells, we also demonstrated that splitFAST2 can serve as the basis for fluorescent probes with very low background, enabling effective subdiffraction imaging of repetitive cellular structures like filamentous actin and microtubules.

biophysics↗

bioRxiv2025

Chemically responsive protein switches for the precise control of biological activities

Controlling the proximity or interaction of proteins with small molecules enables researchers to chemically regulate cellular functions. Here, we leveraged CATCHFIRE (chemically assisted tethering of chimera by fluorogenic induced recognition) - a technology enabling to chemically induce dimerization in a reversible manner - to create chemically responsive proteins switches for the precise and reversible control of various biological activities. CATCHFIRE allowed us to chemically induce the assembly and thus function of various split enzymes - including luciferases, proteases, DNA recombinases. We extended this approach to develop CATCH-ON, a chemically inducible gene expression system relying on the chemically induced dimerization of the DNA-binding domain GAL4 and the truncated transcription factor p65{Delta}. CATCH-ON allowed us to precisely regulate the expression of cellular enzymes such as proteases, DNA recombinases, or suicide switches, as well as to control the secretion of therapeutically relevant proteins such as insulin. We showed that the CATCH-ON system is fast-acting, reversible, titratable, non-toxic and compatible with other chemically induced dimerization systems, opening exciting possibilities for its application in basic research, biotechnology and cell therapy.

bioengineering↗

bioRxiv2025

Peptide-functionalized fluorescent polymeric nanoparticles: polysarcosine length defines stealth properties

Stealth properties of nanoparticles are essential for their proper functionalization in biological systems. To address limitations of polyethylene glycol (PEG), commonly used for this purpose, we explore the potential of polysarcosine (PSar) as stealth shell in peptide-functionalized dye-loaded polymeric NPs. To this end, polymeric NPs loaded with rhodamine dye with bulky hydrophobic counterion and bearing azide groups at their surface were grafted with PSar of different lengths ranging from 5 to 19 sarcosine units using strain-promoted cycloaddition. The obtained peptide-functionalized NPs showed remarkable colloidal stability in physiological media. The length of PSar showed a profound effect on stealth properties of NPs. The increase in the length of grafted PSar lead to decrease in the negative surface charge to nearly neutral values and decreased protein adsorption according to fluorescence correlation spectroscopy. The NPs with 19mer PSar showed minimal interactions with live cells and glass surfaces in a complex biological medium, in contrast to its shorter PSar analogues. These stealth NPs bearing HaloTag ligand enabled specific targeting of proteins at the cell surface. The obtained results show that a relatively short PSar peptide can be used for achieving stealth properties in polymeric NPs, allowing specific protein targeting with minimized non-specific interactions. The obtained PSar-functionalized polymeric NPs appear as a powerful platform for the fabrication of the next generation of nanomaterials for bioimaging and biosensing applications.

pharmacology and toxicology↗

bioRxiv2025

Cellular and mitochondrial effects of a gold-N Heterocyclic Carbene on LNCaP and PC3 prostate cancer cell lines

The discovery of cisplatin and its anti-tumor activities but also its side-effects led to the development of new cytotoxic metal complexes. Metal-based compounds and especially gold N-heterocyclic carbene complexes have shown their antitumoral activities and present a great interest of use in medicine. These metallodrugs have been shown to target nuclear DNA, but also other organelles such as mitochondria. The mechanisms of action have been studied and would involve the triggering of cell death, in particular by apoptosis, highlighting mitochondrial dysfunctions. The organometallic compound used in this study has been characterized by its IC50 and GI50 in two prostatic cancer cell lines (LNCaP and PC3). Its capacity to enter the cells and particularly mitochondria was also analyzed. Furthermore, its effects on several parameters (proliferation, cell death, cell cycle) have been determined in vitro. The compound displayed a cytotoxic effect (IC50 < 5{micro}M) confirmed by the MTT assays and cell death analysis by flow cytometry. It also displayed a cytostatic effect confirmed by the determination of the GI50, and induced a brief cell cycle arrest, depending on the cell line and the metallocarbene concentration. Furthermore, the incubation with the gold N-heterocyclic carbene led to mitochondrial membrane potential changes and decreased the accumulation of the mitochondrial protein Rieske. All our results show a global cytotoxic and cytostatic effects of the gold N-heterocyclic carbene that can involve mitochondria.

pharmacology and toxicology↗

bioRxiv2024

Multiplexed in vivo imaging with fluorescence lifetime modulating tags

Fluorescence lifetime imaging opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, we describe a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives. We show that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime modulating tags enabled multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish embryo. The combination of fluorescence lifetime multiplexing with spectral multiplexing allowed us to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

cell biology↗

bioRxiv2024

A tunable and versatile chemogenetic near infrared fluorescent reporter

Near-infrared (NIR) fluorescent reporters provide additional colors for highly multiplexed imaging of cells and organisms, and enable imaging with less toxic light and higher contrast and depth. Here, we present the engineering of nirFAST, a small tunable chemogenetic NIR fluorescent reporter that is brighter than top-performing NIR fluorescent proteins in cultured mammalian cells. nirFAST is a small genetically encoded protein of 14 kDa that binds and stabilizes the fluorescent state of synthetic, highly cell-permeant, fluorogenic chromophores (so-called fluorogens) that are otherwise dark when free. Engineered to emit NIR light, nirFAST can also emit far-red or red lights through change of chromophore. nirFAST allows the imaging of proteins in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its near infrared fluorescence provides an additional color for high spectral multiplexing. We showed that nirFAST is well-suited for stimulated emission depletion (STED) nanoscopy, allowing the efficient imaging of proteins with subdiffraction resolution in live cells. nirFAST enabled the design of a chemogenetic green-NIR fluorescent ubiquitination-based cell cycle indicator (FUCCI) for the monitoring of the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a fluorogenic chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

cell biology↗

bioRxiv2023

Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters

Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here designed a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and studied the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrated that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these novel probes, we identified a hitherto unknown pathway, in which calcium (Ca2+) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca2+-sensing capabilities into the splitFAST technology, we introduced PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), an unprecedented class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.

cell biology↗

bioRxiv2023

Humans progressively feel agency over events triggered before their actions

Artificial Intelligence (AI) has become increasingly efficient in anticipating our behavior. Will this impact, in the near future, how much we feel control over events generated with AI-assistance? This would in turn influence our decisions, actions, mental health and sense of responsibility. In everyday life, our sense of agency over events occurring at various delays after our actions has adapted to accommodate these delays. Here, we investigate whether our sense of agency can also adapt to an unusual situation where a consequence precedes an action. We used an online game in which players aimed to beat the computer at finding and clicking on a target to trigger an animation, while, in fact, an algorithm triggered the animation before the players click. The animation was not randomly controlled by the algorithm, but rather based on the history of the players past movements and on the beginning of their current movement. We used correlational, machine learning decoding, and modelling approaches to capture how players compute their reported sense of agency over the animation. We found evidence that, in less than an hour, players implicitly learned that the timing of the animation was related to their own actions, and adapted their sense of agency accordingly. Such findings may help us to anticipate how humans will integrate AI-assistance to guide their behavior.

neuroscience↗

bioRxiv2023

A tripartite chemogenetic fluorescent reporter for imaging ternary protein interactions

Most cellular processes are carried out by multiprotein assemblies. Although various molecular tools exist to visualize binary protein interactions in live cells, the visualization of multiprotein complexes remains a challenge. Here, we report the engineering of a complementation-based approach allowing one to visualize the interaction of three proteins through effective proximity-induced complementation of three fragments of pFAST, a chemogenetic fluorescent reporter that binds and stabilizes the fluorescent state of fluorogenic chromophores (so-called fluorogens). This tripartite-split-pFAST allowed the observation of dynamic ternary protein complexes in the cytosol, at the plasma membrane, in the nucleus and at the junction of multiple organelles, opening great prospects to study the role and function of multiprotein complexes in live cells and in various biologically relevant contexts.

cell biology↗

bioRxiv2023

Development of a tool combining multiplex PCR and high-throughput sequencing to study the polymorphism of eight Leptosphaeria maculans avirulence genes and its application to field surveys in France

Leptosphaeria maculans is a fungal pathogen causing stem canker of oilseed rape (Brassica napus). The disease is mainly controlled by the deployment of varieties with major resistance genes (Rlm). Rlm genes can rapidly become ineffective following the selection of virulent isolates of the fungus, i.e. with deletions or mutations in the corresponding avirulence genes (AvrLm). Reasoned and durable management of Rlm genes relies on the detection and monitoring of virulent isolates in field populations. Based on previous knowledge of AvrLm gene polymorphism, we developed a tool combining multiplex PCR and Illumina sequencing to characterise allelic variants for eight AvrLm genes in field L. maculans populations. We tested the method on DNA pools of 71 characterised L. maculans isolates and of leaf spots from 32 L. maculans isolates. After multiplex-PCR and sequencing with MiSeq technology, reads were mapped on an in-house AvrLm sequence database. Data were filtered using thresholds defined from control samples included in each run. Proportions of each allelic variant per gene, including deletions, perfectly correlated with expected ones. The method was then applied to around 1300 symptoms (42 pools of mainly 32 leaf spots) from nine B. napus fields. The proportions of virulent isolates estimated by sequencing leaf spot pools perfectly correlated with those estimated by pathotyping. In addition, the proportions of allelic variants determined at the national scale also correlated with those previously determined following individual sequencing of AvrLm genes in a representative collection of isolates. Finally, the method also allowed us to detect still undescribed and rare allelic variants. Despite the diversity of mechanisms generating virulent isolates and the gene-dependant diversity of AvrLm gene polymorphism, the method proved suitable for large-scale and regular monitoring of L. maculans populations, which will make it possible to choose effective Rlm genes and to detect resistance breakdowns at early stages.

molecular biology↗