Biology subjects

Gillet, B.

Publications and source records attributed to Gillet, B..

At least 19 recordsLinked to original sources

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

Liver FGF21 mediates sex-specific adaptation to juvenile protein malnutrition

Dietary protein sufficiency during childhood is essential for healthy growth and tissue development. Chronic protein deficiency leads to stunting, height-for-age more than two standard deviations below the median, and affects 149 million children under five (https://www.who.int/publications/i/item/9789240073791). Linear growth is governed by the somatotropic axis, wherein pituitary growth hormone (GH) stimulates hepatic insulin-like growth factor 1 (IGF-1) to promote bone elongation1. Although boys are consistently more stunted than girls across populations (ranging from 18% to 45%)2,3, mechanisms underlying this apparent resilience remain unclear. Rodent studies suggest that the protein-to-carbohydrate ratio modulates life-history traits such as metabolism, lifespan, and reproduction, with evidence of sex-dependent sensitivity4,5. Whether such dimorphism reflects coordinated developmental adaptations in response to protein scarcity, particularly in females, has not been fully explored. Here, we show that juvenile dietary protein restriction causes sexually dimorphic adaptations with pronounced stunting in males and delayed reproductive maturation in females. Using liver-specific deletion, we identify fibroblast growth factor 21 (FGF21) as a sex-specific hepatic regulator of female somatic growth and reproductive development, revealing a dimorphic endocrine adaptation to early-life protein scarcity. Our findings position FGF21 as a critical physiological checkpoint coordinating growth and reproductive timing in response to nutritional stress.

physiology↗

bioRxiv2025

Drosophila larval gut transcriptome reveals a microbe-mediated intestinal tissue growth via Ecdysone during adaptive growth

Organ and systemic growth must remain coordinated during development, even under nutritional stress. In Drosophila larvae, the intestinal microbiota contributes to this coordination by promoting growth and maturation under chronic undernutrition. Using gnotobiotic models, we show that association with Lactiplantibacillus plantarum (Lp) selectively enhances midgut growth relatively to other organs, providing an adaptive mechanism that buffers the impact of dietary restriction. Transcriptomic profiling of larval midguts revealed a strong Ecdysone signaling signature upon Lp association. Functional analyses showed that local conversion of Ecdysone to its active form, 20-hydroxyecdysone, by the cytochrome P450 enzyme Shade, together with enterocyte Ecd receptor activity, is required for Lp-dependent intestinal and systemic growth. Pharmacological activation of Ecd signaling partially mimicked the bacterial effect, confirming its sufficiency to drive adaptive midgut expansion. Our results uncover an unexpected role of intestinal Ecd signaling in microbiota-driven developmental plasticity, revealing how commensal bacteria modulate local steroid signaling to fine-tune organismal growth and maturation.

cell biology↗

bioRxiv2025

LAM/TREM2+ macrophages release extracellular vesicles and extracellular lipid droplets which modulate the phenotype of recipient macrophages and homeostasis of skeletal muscle cells

The polarization of tissue-resident macrophages is influenced by a variety of signals from the immune system and the local tissue environment, including nutrition. Although it is known that the quality and quantity of ingested lipids have a significant effect on the lipid composition of extracellular vesicles and their fate, it is unknown how the nutritional environment modifies the release and the function of macrophage-derived EVs. In this study, we used a combination of palmitate and oleate (1:2, FFA) to generate lipid-associated TREM2-expressing macrophages (LAM/TREM2+) in vitro. Using various electron microscopy techniques (TEM, SEM, CryoEM) and fluorophores, we found that FFA overload not only induces lipid storage in LAM/TREM2+ macrophages, but also alters their morphology and reduces the diversity and the number of the lipid-derived structures they release. In addition, LAM/TREM2+ macrophages accumulated lipid droplets (LDs) below the plasma membrane and we discovered for the first time that they export and disseminate full LDs into their environment, in addition to extracellular vesicles, by using a cellular pathway associated to CD81. The use of 14C-palmitate confirmed the presence of 14C-triacylglycerols in the large extracellular vesicle pellet. LAM/TREM2+ macrophage-derived EVs induced TREM2 and Il-10 expression in recipient M0 macrophages. These data provide potential insights into how dietary factors and metabolic perturbations can shape the functions of macrophage-derived EVs in the context of metabolic diseases such as diabetes and obesity. In addition, LAM/TREM2+ macrophage-derived EVs modulated insulin-sensitivity, mitochondrial oxidative capacity, lipid profiles and the expressions of genes encoding extracellular matrix components in recipient skeletal muscle cells. Although previously postulated but never demonstrated, these data also highlight the LAM/TREM2+ macrophage-derived EVs as important players in SkM tissue renewal and metabolic homeostasis.

cell biology↗

bioRxiv2024

Targeting a shared neoepitope derived from non-canonical translation of c-MYC oncogene in cancer cells

Cancer cells rely on alternative modes of translation for protein synthesis, promoting internal ribosome entry site (IRES)-dependent translation of mRNA encoding pro-oncogenic factors. Furthermore, ribosomes translate mRNA with lower fidelity in tumor cells. We proposed that these translational modifications in cancer produce shared tumor-specific epitopes derived from IRES-containing oncogenes. To identify such neoepitopes, we developed an in silico-based method that we applied to c-MYC. We showed that the non-canonical translation of c-MYC mRNA in cancer cells, involving a (+1) ribosomal frameshift, generates a shared neoepitope which induces high-avidity T cells able to kill tumor cells in vitro and in vivo while sparing normal cells. Our data provide preclinical rationale for developing immunotherapies targeting this c-MYC-derived neoepitope and validate a new type of shared translation-associated neoantigens.

immunology↗

bioRxiv2024

AtVPS13M1 is involved in lipid remodeling in low phosphate and is located at the mitochondria surface in plants

VPS13 are conserved lipid transporters with multiple subcellular localizations playing key roles in many fundamental cellular processes. While the localization and function of VPS13 have been extensively investigated in yeast and animals, little is known about their counterparts in plants, particularly regarding their role in stress response. In this study, we characterized AtVPS13M1, one of the four VPS13 paralogs of the flowering plant Arabidopsis thaliana. We show that AtVPS13M1 binds and transports glycerolipids with a low specificity in vitro. AtVPS13M1 interferes with phospholipids degradation in response to phosphate starvation, a nutrient stress that triggers a massive remodeling of membrane lipids. AtVPS13M1 is mainly expressed in young dividing and vascular tissues. Finally, we show that AtVPS13M1 is mainly located at the surface of mitochondria in leaves. Overall, our work highlights the conserved role in lipid transport of VPS13 in plants, reveals their importance in nutrient stress response and opens important perspectives for the understanding of lipid remodeling mechanisms and for the characterization of this protein family in plants.

cell biology↗

bioRxiv2023

Optimization for High-Throughput BiFC screening

The Cell-PCA screen, since its inception, has provided an efficient method for analyzing cellular interactomes and has been used in various biological studies involving proteins like MYC, PER2, and ERK. With rapid advancements in biotechnology, including tools for protein function investigation, the Cell-PCA screen remains relevant. However, despite its successful application in recent studies, there are areas for optimization to ensure its continued relevance in the face of evolving technological advancements.

bioengineering↗

bioRxiv2023

Detection and identification of transposable element transcripts using Long Read RNA-seq in Drosophila germline tissues

Transposable elements (TEs) are repeated DNA sequences potentially able to move throughout the genome. In addition to their inherent mutagenic effects, TEs can disrupt nearby genes by donating their intrinsic regulatory sequences, for instance, promoting the ectopic expression of a cellular gene. TE transcription is therefore not only necessary for TE transposition per se but can also be associated with TE-gene fusion transcripts, and in some cases, be the product of pervasive transcription. Hence, correctly determining the transcription state of a TE copy is essential to apprehend the impact of the TE in the host genome. Methods to identify and quantify TE transcription have mostly relied on short RNA-seq reads to estimate TE expression at the family level while using specific algorithms to discriminate copy-specific transcription. However, assigning short reads to their correct genomic location, and genomic feature is not trivial. Here we retrieved full-length cDNA (TeloPrime, Lexogen) of Drosophila melanogaster gonads and sequenced them using Oxford Nanopore Technologies. We show that long-read RNA-seq can be used to identify and quantify transcribed TEs at the copy level. In particular, TE insertions overlapping annotated genes are better estimated using long reads than short reads. Nevertheless, long TE transcripts (> 4.5 kb) are not well captured. Most expressed TE insertions correspond to copies that have lost their ability to transpose, and within a family, only a few copies are indeed expressed. Long-read sequencing also allowed the identification of spliced transcripts for around 107 TE copies. Overall, this first comparison of TEs between testes and ovaries uncovers differences in their transcriptional landscape, at the subclass and insertion level.

bioinformatics↗

bioRxiv2023

Dual-topology of collagen XV and tenascin C acts in concert to guide and shape developing motor axons

During development, motor axons are guided towards their muscle target by various extrinsic cues including extracellular matrix (ECM) proteins those identities remain poorly documented. Using single-cell RNA-sequencing of differentiating slow muscle progenitors (SMP) in zebrafish, we charaterized the SMP as a major source of ECM proteins that were computationally predicted to form a basement membrane-like structure tailored for motor axon guidance. Multiple in vivo and in vitro approaches further revealed that motor axon shape and growth relies on the timely expression of the attractive cue Collagen XV-B (ColXV-B) that locally provides motor axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Bioprinted micropatterns mimicking their unique topology provide compelling evidence that it represents a sufficient condition to elicit directional motor axon growth. Our study provides the first evidence that ECM topology and stiffness critically influence motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury.

developmental biology↗

bioRxiv2023

Coordination of host and endosymbiont gene expression governs endosymbiont growth and elimination in the cereal weevil Sitophilus spp.

BackgroundInsects living in nutritionally poor environments often establish long-term relationships with intracellular bacteria that supplement their diets and improve their adaptive and invasive powers. Even though these symbiotic associations have been extensively studied on physiological, ecological and evolutionary levels, few studies have focused on the molecular dialogue between host and endosymbionts to identify genes and pathways involved in endosymbiosis control and dynamics throughout host development. ResultsWe simultaneously analyzed host and endosymbiont gene expression during the life cycle of the cereal weevil Sitophilus oryzae, from larval stages to adults, with a particular emphasis on emerging adults where the endosymbiont Sodalis pierantonius experiences a contrasted growth-climax-elimination dynamics. We unraveled a constant arms race in which different biological functions are intertwined and coregulated across both partners. These include immunity, metabolism, metal control, apoptosis, and bacterial stress response. ConclusionsThe study of these tightly regulated functions, which are at the center of symbiotic regulations, provides evidence on how hosts and bacteria finely tune their gene expression and respond to different physiological challenges constrained by insect development in a nutritionally limited ecological niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/535335v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@800121org.highwire.dtl.DTLVardef@1ca2c25org.highwire.dtl.DTLVardef@183d56org.highwire.dtl.DTLVardef@163074f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Larval microbiota primes the Drosophila adult gustatory response

The survival of animals depends, among other things, on their ability to identify threats in their surrounding environment. Senses such as olfaction, vision and taste play an essential role in sampling their living environment, including microorganisms, some of which are potentially pathogenic. This study focuses on the mechanisms of detection of bacteria by the Drosophila gustatory system. We demonstrate that the peptidoglycan (PGN) that forms the cell wall of bacteria triggers an immediate feeding aversive response when detected by the gustatory system of adult flies. Although we identify ppk23+ and Gr66a+ gustatory neurons as necessary to transduce fly response to PGN, we demonstrate that they play very different roles in the process. Time-controlled functional inactivation and in vivo calcium imaging demonstrate that while ppk23+ neurons are required in the adult flies to directly transduce PGN signal, Gr66a+ neurons must be functional in larvae to allow future adults to become PGN sensitive. Furthermore, the ability of adult flies to respond to bacterial PGN is lost when they hatch from larvae reared under axenic conditions. Recolonization of axenic larvae, but not adults, with a single bacterial species, Lactobacillus brevis, is sufficient to restore the ability of adults to respond to PGN. Our data demonstrate that the genetic and environmental characteristics of the larvae are essential to make the future adults competent to respond to certain sensory stimuli such as PGN.

animal behavior and cognition↗

bioRxiv2022

A live cell protein complementation assay for ORFeome-wide probing of human HOX interactomes

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies developing tools that allow capturing transient and low affinity protein-protein interactions (PPIs) in live conditions. Here we present an experimental strategy, Cell-PCA (Cell Protein Complementation Assay), which is based on BiFC (Bimolecular Fluorescence Complementation) and high throughput sequencing for ORFeome-wide analyses of different interactomes in the same live cell context. The specificity and sensitivity of Cell-PCA was established by using a wild type and a single amino-acid mutated HOXA9 protein, and the approach was subsequently applied for seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 for promoting its proliferative activity in a cancer cell context. Taken together, our work demonstrates that Cell-PCA is pertinent for revealing and, importantly, comparing interactomes between different or highly related bait proteins in the same cell context.

molecular biology↗

bioRxiv2022

Chronological aging impacts abundance, function and microRNA content of extracellular vesicles produced by human epidermal keratinocytes

The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin.

cell biology↗

bioRxiv2022

New insights from short and long reads sequencing to explore cytochrome b variants of Plasmopara viticola populations collected in vineyard and related to resistance to complex III inhibitors

Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease for grapevine. To preserve plants from the disease, complex III inhibitors are among the widely used fungicides that specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In French vineyard, P. viticola developed resistance against a first category of these fungicides, the Quinone outside inhibitors, by exhibiting a single amino acid substitution G143A in its cytb mitochondrial sequence. Their usage was restricted and another kind of fungicides, Quinone inside inhibitors, targeting the same gene and highly effective against oomycetes, were used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating resistant single-sporangia strains of P. viticola to these fungicides, we characterized new variants in cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with classical tools, pyrosequencing and RT-PCR, we then benchmarked both short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with the prospect to detect and assess the proportion of resistant variants of P. viticola at a natural population scale. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 show a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, long reads were able to identify variants, including SNPs, with confidence and detect a small proportion of P. viticola showing several variants along the same cytb sequence. Altogether, NGS appear promising methods to evaluate pathogen resistance towards fungicides related to cytb modifications at a population scale in the field. This approach could be rapidly a robust decision-support management tool for vineyard in future.

evolutionary biology↗

bioRxiv2022

Antimicrobial peptide secretion protects endosymbionts from bacteriome autoimmunity in insects

BackgroundMany insects house symbiotic intracellular bacteria (endosymbionts) that provide them with essential nutrients, thus promoting usage of nutrient-poor habitats. Endosymbiont seclusion within host specialized cells, called bacteriocytes, often organized in a dedicated organ, the bacteriome, is crucial in protecting them from host immune defenses while avoiding chronic host immune activation. Previous evidence obtained in the cereal weevil Sitophilus oryzae has shown that bacteriome immunity is activated against invading pathogens, suggesting endosymbionts might be targeted and impacted by immune effectors during an immune challenge. To pinpoint any molecular determinants associated with such challenges, we conducted a dual transcriptomic analysis of S. oryzaes bacteriome subjected to immunogenic peptidoglycan fragments. ResultsWe show that upon immune challenge the bacteriome actively participates in the innate immune response via an induction of antimicrobial peptides (AMPs). Surprisingly, endosymbionts do not undergo any transcriptomic changes, indicating that this potential threat goes unnoticed. Immunohistochemistry showed that TCT-induced AMPs are located outside the bacteriome, excluding direct contact with the endosymbionts. ConclusionsThis work demonstrates that endosymbiont protection during an immune challenge is mainly achieved by efficient confinement within bacteriomes, which provides physical separation between host systemic response and endosymbionts.

immunology↗

bioRxiv2021

Intestinal GCN2 controls Drosophila systemic growth upon AA imbalance in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNAoperons

Symbiotic bacteria interact with their host through symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically-defined diets, we found that association with Lp improves the growth of larvae fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its hosts growth through a molecular dialog that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the GCN2 kinase in Drosophilas enterocytes. Our data indicate Lps r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on our findings, we propose a novel beneficial molecular dialog between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.

microbiology↗

bioRxiv2021

Distinct gene expression dynamics in developing and regenerating limbs

Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned. HIGHLIGHTSO_LISingle-limb data on transcriptional dynamics of leg development and regeneration C_LIO_LIDeveloping embryonic legs show stereotypic transcriptional profiles C_LIO_LIRegenerating leg transcriptomes show a high degree on individual variation C_LIO_LIRegenerating leg transcriptomes are influenced by adult physiology, especially molting C_LIO_LIRegenerating leg transcriptomes reveal distinct phases of leg regeneration C_LIO_LILeg development and regeneration use overlapping sets of genes in different temporal patterns C_LI

developmental biology↗

bioRxiv2021

The genome sequence of the cereal pest Sitophilus oryzae: an unprecedented transposable element content

BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions. ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis. ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes.

genomics↗