Biology subjects

Loconte, V.

Publications and source records attributed to Loconte, V..

6 recordsLinked to original sources

bioRxiv2025

Decoding acute neuroinflammatory states from the 3D architecture of in vitro microglia

Microglia, core brain immune defenders, rapidly polarize into pro- or anti-inflammatory states that shape neuronal survival during acute brain inflammation. Yet, how these inflammatory states are encoded at the native cellular level remains unclear. While microglial states associate with specific molecular and organelle markers, it is unknown whether their cellular architecture integrates robust metabolic and structural features. Here, we quantitatively decode inflammatory states from the 3D architecture of individual in vitro mouse microglia (BV-2). Using soft X-ray tomography on established BV-2 polarization, we identify coordinated intracellular organization distinguishing homeostatic, pro-inflammatory, and anti-inflammatory cells, including characteristic lipid droplet-endosome architectures. We further link lipid-endosome reorganization with mTORC1 pathway, by functionally implicating sestrin-2 in promoting anti-inflammation. Finally, we resolve the time-dependent remodeling of lipid droplet 3D profiles by their distinct lipidomic composition across inflammatory states. Overall, our work enables deciphering microglia states in disease-relevant models, with potential to ultimately understand brain immunity.

cell biology↗

bioRxiv2025

Classifying Biophysical Subpopulations of Insulin Secretory Granules using Quantitative Whole Cell Structure Analysis

Pancreatic {beta}-cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft x-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole {beta}-cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts towards mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary {beta}-cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

biophysics↗

bioRxiv2025

HP1B and H3K9me3 Regulate Olfactory Receptor Choice and Transcriptional Identity

Diverse epigenetic regulatory mechanisms ensure and regulate cellular diversity. Among others, the histone 3 lysine 9 me3 (H3K9me3) post translational modification participates in silencing lineage-inappropriate genes. H3K9me3 restricts access of transcription factors and other regulatory proteins to cell-fate controlled genes. In mice, olfactory sensory neurons (OSN) express one olfactory receptor (OR) gene out of 2,600 possibilities. This monoallelic and stochastic OR choice happens as OSNs differentiate and undergo dramatic changes in nuclear architecture. OR genes from different chromosomes converge into specialized nuclear bodies and chromatin compartments as H3K9me3 and chromatin binding proteins including heterochromatin protein 1 (HP1) are incorporated. In this work, we have uncovered an unexpected role for HP1{beta} in OR choice and neuronal identity that cannot be rescued by HP1 in vivo. With the use of a conditional knock-in mouse model that replaces HP1{beta} for HP1, we observe changes in H3K9me3 levels, DNA accessibility, and Hi-C contacts over OR gene clusters. These changes alter the expression patterns that partition the mouse olfactory epithelium into five OR expression zones, which results in a reduced OR repertoire leading to a loss of olfactory sensory neuron diversity. We propose that HP1{beta} modulates the competition of OR-promoters for enhancers to promote receptor diversity, by establishing repression gradients in a zonal fashion.

molecular biology↗

bioRxiv2025

Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic {beta}-cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G-protein coupled receptor 40 (GPR40) promote ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell altering overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.

biophysics↗

bioRxiv2024

Soft X-ray tomography reveals variations in B.subtilis biofilm structure upon tasA deletion

Bacterial biofilms are complex communities of cells within a self-produced extracellular matrix. They play crucial roles in healthcare, nutrition, agriculture and environmental research, yet an analysis of their elaborate 3D architecture remains challenging. Understanding mechanisms of biofilm formation, particularly the effects of chemical, physical, and genetic influences or modifications, is crucial but requires structural information at subcellular resolution to enable a community-level analysis of biofilms. In this work, we developed a "biofilm-in-capillary" growth method compatible with full-rotation soft X-ray tomography, providing high-resolution 3D imaging of bacterial cells and their surrounding extracellular matrix during biofilm formation, without drying or fixation steps. This approach offers 50 nm isotropic spatial resolution, rapid imaging time, and quantitative native analysis of biofilm structure. We demonstrate the potential of our method using Bacillus subtilis biofilms, detecting coherent alignment and chaining of wild-type cells while they are travelling towards the oxygen-rich capillary tip region. In stark contrast, the genetic knock-out {Delta}tasA shows a loss of cellular orientation, including changes in the extracellular matrix in volume and chemical density. Notably, we show that the addition of TasA protein to a culture of a {Delta}tasA strain restores the extracellular matrix density and leads to a compaction of cell assemblies, yet no chaining is observed as for the wildtype. Our approach to imaging biofilms is scalable and transferable, opening new avenues for examining biofilm structure and function across various species, including mixed biofilms, and observing 3D reorganization in response to genetic and environmental factors.

microbiology↗

bioRxiv2023

3-O-methyltolcapone and Its Lipophilic Analogues are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinsons disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation which is responsible of the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in-vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in-vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

biochemistry↗