Biology subjects

Paul-Gilloteaux, P.

Publications and source records attributed to Paul-Gilloteaux, P..

7 recordsLinked to original sources

bioRxiv2025

Tripartite ER-Mitochondria-Lipid Droplets contact sites control adipocyte metabolic flexibility

Obesity is a major risk factor for cardiometabolic diseases, with adipocyte dysfunction playing a central role. Understanding how lipid storage and mobilization are regulated--and disrupted--in adipocytes is key to addressing obesity-associated complications. The ER-anchored protein Seipin controls lipid droplet (LD) biogenesis and maintenance, and its loss disrupts ER-LD contact sites. In humans, Seipin deficiency causes generalized lipodystrophy, a severe form of adipocyte dysfunction. We previously showed that Seipin also localizes at ER-mitochondria contact sites (MAM), where it regulates calcium exchange and mitochondrial function. Here, we examined whether Seipin targeting to MAM and ER-LD sites overlaps functionally. We analyzed subcutaneous adipose tissue (AT) from inducible Seipin-knockout mice using transmission electron microscopy (TEM) and proximity ligation assays (PLA) to quantify membrane contact sites (MCS) involving the ER, LDs, and mitochondria. In control mice, feeding reduced MAMs while increasing ER-LD and mitochondria-LD contacts, whereas Seipin deficiency abolished this remodeling. Specifically, under lipid loading, MAMs located in proximity to LDs--tripartite contact sites known as MAM-LD--were increased in control but not in Seipin-deficient adipocytes. Fluorescence recovery after photobleaching assays revealed that Seipin depletion impairs triglyceride transfer to LDs, an effect rescued by the MAM-LD-reinforcing synthetic peptide Linker-ER-Mi. Importantly, this rescue was abolished by silencing the mitochondrial calcium uniporter, demonstrating that calcium exchange is critical for triglyceride storage in LDs. We further investigated how MAM-LD remodeling influences adipocyte metabolic flexibility. Using TEM and PLA, we monitored two MAM subtypes: those forming MAM-LD and those engaging cytosolic mitochondria (MAM-CM). During adipogenesis, MAM-LD frequency increased while MAM-CM decreased. Similarly, in mouse AT and 3T3-L1 adipocytes, lipid loading selectively promoted MAM-LD. Notably, this adaptive remodeling of membrane contact sites was blunted in the adipose tissue of diet-induced obese mice. Genetic disruption of MCS in 3T3-L1 adipocytes altered lipid flux, impaired lipolysis, and reduced insulin signaling. Together, our findings identify MAM-LD contacts as key regulators of adipocyte lipid handling and metabolic flexibility, whose disruption may underlie the metabolic inflexibility of obesity.

cell biology↗

bioRxiv2024

TYPE THREE SECRETION SYSTEM-INDUCED MECHANOPORATION OF VACUOLAR MEMBRANES

SO_SCPLOWUMMARYC_SCPLOWEndomembrane breaching is a crucial strategy employed by intracellular pathogens enclosed within vacuoles to access the nutrient-rich cytosol for intracellular replication. While bacteria use various mechanisms to compromise host membranes, the specific processes and factors involved are often unknown. Shigella flexneri, a major human pathogen, accesses the cytosol relying on the Type Three Secretion System (T3SS) and secreted effectors. Using in-cell correlative light and electron microscopy, we tracked the sequential steps of Shigella host cell entry. Moreover, we captured the T3SS, which projects a needle from the bacterial surface, in the process of puncturing holes in the vacuolar membrane. This initial puncture ensures disruption of the vacuolar membrane. Together this introduces the concept of mechanoporation via a bacterial secretion system as a crucial process for bacterial pathogen-induced membrane damage. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/613840v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1669028org.highwire.dtl.DTLVardef@8259cborg.highwire.dtl.DTLVardef@1d522caorg.highwire.dtl.DTLVardef@1df0f2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Label-free imaging of large samples: 3D rendering and morphological analysis within histological workflows using serial block face imaging

Serial block face imaging (SFBI) is a method used to generate 3-dimensional (3D) reconstruction of a sample via serial image acquisition. Several SBFI approaches have been proposed for large samples, differing in the ability to generate contrast as well as in the nature of the detected signal. We propose a new system that detects the endogenous autofluorescence signal of paraffin-embedded samples. The sample preparation is simplified compared to other approaches, and adapted to be integrated into a routine histological preparation. More specifically, it was designed to limit reagent toxicity and to be compatible with downstream histological processing. We show the usefulness of the technique with a wide range of tissues based on the intrinsic autofluorescence signal. Optimization of quality section recovery offers the possibility to develop correlative approaches and multimodal analysis between the 3D dataset with the 2-dimensional (2D) sections. In addition, contrast and resolution of block-face images allow us to successfully perform post processing analysis and morphology quantifications. Overall, our methodology offers a simple, cost effective and rapid approach to obtain quantitative data on a large sample with no specific staining.

bioengineering↗

bioRxiv2021

In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging

1.Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo-fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo-FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells.

cell biology↗

bioRxiv2020

Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and demonstrate that species-specific kinetics of Plasmodium invasion is shaped by the mosquito complement-like system. Author SummaryThe traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood. Here, we harnessed transgenic reporters to track invasion of Plasmodium parasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two anopheline species and demonstrated an unexpected role of mosquito complement-like system in regulation of parasite invasion.

immunology↗

bioRxiv2019

BIAFLOWS: A collaborative framework to benchmark bioimage analysis workflows

Automated image analysis has become key to extract quantitative information from scientific microscopy bioimages, but the methods involved are now often so refined that they can no longer be unambiguously described using written protocols. We introduce BIAFLOWS, a software tool with web services and a user interface specifically designed to document, interface, reproducibly deploy, and benchmark image analysis workflows. BIAFLOWS allows image analysis workflows to be compared fairly and shared in a reproducible manner, safeguarding research results and promoting the highest quality standards in bioimage analysis. A curated instance of BIAFLOWS is available online; it is currently populated with 34 workflows that can be triggered to process image datasets illustrating 15 common bioimage analysis problems organized in 9 major classes. As a complete case study, the open benchmarking of 7 nuclei segmentation workflows, including classical and deep learning techniques, was performed on this online instance. All the results presented can be reproduced online.

bioinformatics↗

bioRxiv2018

Development of a robust automated tool for the annotation of embryo morphokinetic parameters

Study QuestionIs it possible to automatically annotate human embryo development in time-lapse devices, with results comparable to manual annotation?\n\nSummary AnswerWe developed an automated tool for the annotation of embryo morphokinetic parameters having a high concordance with expert manual annotation in a large scale-study.\n\nWhat is Known AlreadyMorphokinetic parameters obtained with time-lapse devices are increasingly used for human embryo quality assessment. However, their annotation is timeconsuming and can be operator-dependent, highlighting the need of developing automated approaches.\n\nStudy Design, Size, DurationThis monocentric pilot study was conducted using 701 blastocysts originating from 584 couples undergoing IVF with embryo culture in a time-lapse device and on 4 mouse embryos.\n\nParticipants/Materials, Setting, MethodsAn automated annotation tool was developed based on grey level coefficient of variation and detection of the thickness of the zona pellucida. The timings of cellular events obtained with the automated tool were compared with those obtained manually by 2 expert embryologists. The same procedure was applied on 4 mouse preimplantation embryos obtained with a different device in a different setting.\n\nMain Results and the Role of ChanceAlthough some differences were found when embryos were considered individually, we found an overall excellent concordance between automated and manual annotation of human embryo morphokinetics from fertilization to expanded blastocyst stage (r2=0.94). Moreover, the automated annotation tool gave promising results across species (human, mice).\n\nLimitations, Reasons for CautionThese results should undergo multi-centric external evaluation in order to test the overall performance of the annotation tool.\n\nWider Implications of the FindingsOur system performs significantly better than the ones reported in the literature and on a bigger cohort, paving the way for high-throughput analysis of multicentric morphokinetic databases, providing new insights into the clinical value of morphokinetics as predictor of embryo quality and implantation.\n\nStudy Funding/Competing Interest(s)This study was partly funded by Finox Forward Grant 2016.\n\nTrial Registration NumberNA

developmental biology↗