Biology subjects

Lefebvre, A. E. Y. T.

Publications and source records attributed to Lefebvre, A. E. Y. T..

6 recordsLinked to original sources

bioRxiv2026

Senescent cells induce vascular MHC II to recruit CD4+ T cells and drive inflammation in aging adipose tissue

Adipose tissue exhibits pronounced inflammation during aging, yet the mechanisms sustaining this chronic state are not well understood. By creating an atlas integrating histology, single-nucleus transcriptomics and flow cytometry across the murine lifespan, we find that age-associated inflammation is distinct from the obesity-like inflammatory profile observed at mid-life. Specifically, age-associated inflammation is characterized by a potent interferon-gamma (IFN{gamma}) response signature and the accumulation of T cells. We demonstrate that senescent cells act as an upstream trigger, indirectly initiating an IFN{gamma} response that upregulates vascular MHC II to promote extravasation of CD4+ T cells into the aging tissue. These recruited T cells then act as a critical source of IFN{gamma}, thereby perpetuating a positive feedback loop that maintains chronic immune infiltration and tissue inflammation. These results provide a multi-modal view of adipose aging and identify a mechanism that sustains its age-associated inflammation.

immunology↗

bioRxiv2025

Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

The increasing micro- and nanoplastic (MNP) pollution poses significant risks to human and animal health, yet the mechanisms of their accumulation and effects on absorptive tissues such as the gastrointestinal tract remain poorly understood. Addressing these knowledge gaps requires tractable models coupled to dynamic live cell imaging methods, to enable multi-parameter analysis at single cell resolution. Here we report a new method combining adult stem cell-derived small intestinal organoid cultures with multi-parameter live Fluorescence Lifetime Imaging Microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an apical-out topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) exhibiting different surface charges, and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles differently interacted with apical and basal membranes of the organoids and even showed a species-specific pattern of cellular uptake. Using a phasor-FLIM approach, we demonstrate better sensitivity of FLIM over conventional intensity-based microscopy. The fluorescence lifetime barcoding enabled distinguishing different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 days)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total energy budget and epithelial inflammation and found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and help tracing other types of fluorescent nanoparticles in live organoid and 3D ex vivo systems.

pharmacology and toxicology↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2023

The actin binding protein profilin 1 is critical for mitochondria function

Profilin 1 (PFN1) is an actin binding protein that is vital for the polymerization of monomeric actin into filaments. Here we screened knockout cells for novel functions of PFN1 and discovered that mitophagy, a type of selective autophagy that removes defective or damaged mitochondria from the cell, was significantly upregulated in the absence of PFN1. Despite successful autophagosome formation and fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells still accumulate damaged, dysfunctional mitochondria. Subsequent imaging and functional assays showed that loss of PFN1 significantly affects mitochondria morphology, dynamics, and respiration. Further experiments revealed that PFN1 is located to the mitochondria matrix and is likely regulating mitochondria function from within rather than through polymerizing actin at the mitochondria surface. Finally, PFN1 mutants associated with amyotrophic lateral sclerosis (ALS) fail to rescue PFN1 knockout mitochondrial phenotypes and form aggregates within mitochondria, further perturbing them. Together, these results suggest a novel function for PFN1 in regulating mitochondria and identify a potential pathogenic mechanism of ALS-linked PFN1 variants.

cell biology↗

bioRxiv2023

Microenvironmental Stiffness Induces Metabolic Reprogramming in Glioblastoma

The mechanical properties of solid tumors influence tumor cell phenotype and ability to invade into surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peri-tumoral brain tissues, but tumor stiffness is heterogenous where tumor edges are softer than the tumor core. Then, we developed three-dimensional scaffolds with {micro}-compressive moduli resembling either stiffer, tumor core or softer, peri-tumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.

cancer biology↗

bioRxiv2020

Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the expansion and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis.

cancer biology↗