Biology subjects

Leforestier, A.

Publications and source records attributed to Leforestier, A..

7 recordsLinked to original sources

bioRxiv2026

Uncoupling mycomembrane biogenesis from mycolic acid synthesis reveals a distinct role for mycoloyltransferases in mycobacterial cell division.

Bacteria of the order Mycobacteriales, including the genera Mycobacterium and Corynebacterium, possess a unique outer membrane, termed the mycomembrane, which is structurally and chemically distinct from the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. A defining feature of the mycomembrane is its enrichment in mycolic acids, long-chain -branched, {beta}-hydroxylated fatty acids that occur as trehalose monomycolate (TMM), trehalose dimycolate (TDM), or are esterified to arabinogalactan, an unusual polymer that is itself covalently linked to peptidoglycan (PG). Mycoloyltransferases are Mycobacteriales-specific enzymes described to catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to various cell envelope acceptors, including trehalose and arabinogalactan. In several species, including Mycobacterium tuberculosis, these proteins are essential for viability; however, their occurrence as multiple paralogs with partially redundant functions has hindered the precise assignment of their cellular roles. Previously, we showed that Corynebacterium glutamicum remains viable in the absence of mycolic acids, and thus without a mycomembrane, following deletion of pks, the gene required for mycolic acid biosynthesis. Building on this finding, we systematically deleted all genes encoding mycoloyltransferases in C. glutamicum to further disclose their collective function in the cell. The resulting {Delta}myts mutant lacked arabinogalactan-bound mycolates and TDM, yet continued to synthesize TMM. Despite the high abundance of this major glycolipid, the {Delta}myts strain failed to assemble a mycomembrane and displayed pronounced cell aggregation. Unexpectedly, deletion of mycoloyltransferases also caused very severe defects in cell division and morphogenesis that are not observed in a {Delta}pks strain unable to synthesize mycolic acids. Together, these results demonstrate that mycoloyltransferases are essential for mycomembrane assembly but dispensable for TMM biosynthesis, and reveal an unexpected role for these enzymes in cell division that is independent of their canonical mycolic acid transfer activity. SIGNIFICANCEHow the mycomembrane is assembled and anchored to the cell wall remains a central question in Mycobacteriales, where this outer membrane is necessary for envelope integrity and intrinsic antibiotic resistance. By genetically separating mycolic acid synthesis from their incorporation into the outer membrane, we identify arabinogalactan-linked mycolates as the critical determinant for initiating membrane assembly. Unexpectedly, we also uncover a role for mycoloyltransferases beyond their canonical function in lipid metabolism, revealing a functional link with cell division. These findings point to a critical role of Mycoloyltransferases in the coordination between outer membrane biogenesis and bacterial cytokinesis.

microbiology↗

bioRxiv2025

Optimization of adhesion for high throughput cryo-electron tomography of vitreous sections

Cellular cryo electron tomography explores tissue and cells in their unstained flash-frozen native state, revealing in situ the structure of macromolecules together with their local environment and interactions with partners, also known as molecular sociology. To obtain thin samples, cryo-FIB milling is nowadays the most popular method, with impressive successes. The alternative, cryo-ultramicrotomy, is often overlooked on account of poorly reproducible attachment of cryo-sections to their support, resulting in extremely low throughput. We optimized the workflow, focusing on section adhesion and their support grids. We thus increased vitreous sections cryo electron tomography throughput to equal that of thin film, with typically several tens to hundreds of cryo-tomograms per sample. This open the way to new advances in cellular cryo electron tomography, as the method is devoid of beam damage, can provide large surfaces and serial sections of any type of sample from cells to tissues. In addition, section thickness can be tuned down to 30-50 nm, which may be an advantage for imaging small molecular complexes, such as DNA and nucleosomes.

biophysics↗

bioRxiv2024

Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

Toxoplasma gondii, the causative agent of toxoplasmosis, infects cells and replicates inside via the secretion of factors stored in specialized organelles (rhoptries, micronemes, dense granules) and the capture of host materials. The genesis of the secretory organelles and the processes of secretion and endocytosis depend on vesicular trafficking events whose molecular bases remain poorly known. Notably, there is no characterization of the BAR (Bin/Amphiphysin/Rvs) domain-containing proteins expressed by T. gondii and other apicomplexans, although such proteins are known to play critical roles in vesicular trafficking in other eukaryotes. Here, by combining structural analyses with in vitro assays and cellular observations, we have characterized TgREMIND (REgulators of Membrane Interacting Domains), involved in the genesis of rhoptries and dense granules, and TgBAR2 found at the parasite cortex. We establish that TgREMIND comprises an F-BAR domain that can bind curved neutral membranes with no strict phosphoinositide requirement and exert a membrane remodeling activity. Next, we establish that TgREMIND contains a new structural domain called REMIND, which negatively regulates the membrane-binding capacities of the F-BAR domain. In parallel, we report that TgBAR2 contains a BAR domain with an extremely basic membrane-binding interface able to deform anionic membranes into very narrow tubules. Our data show that T. gondii codes for two atypical BAR domain-containing proteins with very contrasting membrane-binding properties, allowing them to function in two distinct regions of the parasite trafficking system.

biochemistry↗

bioRxiv2022

Cryo-electron tomography and deep learning-based denoising reveal native chromatin landscapes of interphase nuclei

The chromatin machinery, with DNA-histone assembly and further folding of nucleosome chains, influences DNA availability for functional interactions necessary to the regulation of transcription, DNA replication and repair. Despite models based on in vitro studies, the nucleosome chain geometry within the crowded cell nucleus remains elusive. Using cryo-electron tomography of thin vitreous sections, we directly observed the path of nucleosomal and linker DNA in situ in intact flash-frozen organism - Drosophila embryo. We quantified linker length and curvature characterizing an irregular zig-zag chromatin folding motif, with a low degree of linker bending. Additionally, nucleosome conformational variability with non-canonical structures and non-octameric particles were observed as individual objects, without structure averaging, highlighting the high structural heterogeneity of native chromatin.

cell biology↗

bioRxiv2021

A complex CTCF binding code defines TAD boundary structure and function

Topologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair1-10. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundaries11-20. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary21-24. Here, we report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals that this clustering of CTCF binding does not only occur among peaks but also frequently within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites additively contribute to TAD separation. This clustering of CTCF binding may counter against the dynamic DNA-binding kinetics of CTCF25-27, which urges a re-evaluation of current models for the blocking of loop extrusion21-23. Our work thus reveals an unanticipatedly complex code of CTCF binding at TAD boundaries that expands the regulatory potential for TAD structure and function and can help to explain how distant non-coding structural variation influences gene regulation, DNA replication, recombination and repair5,28-34.

genetics↗

bioRxiv2020

Local structure of DNA toroids reveals curvature-dependent intermolecular forces

In viruses and cells, DNA is closely packed and tightly curved thanks to polyvalent cations inducing an effective attraction between its negatively charged filaments. Our understanding of this effective attraction remains very incomplete, partly because experimental data is limited to bulk measurements on large samples of mostly uncurved DNA helices. Here we use cryo electron microscopy to shed light on the interaction between highly curved helices. We find that the spacing between DNA helices in spermine-induced DNA toroidal condensates depends on their location within the torus, consistent with a mathematical model based on the competition between electrostatic interactions and the bending rigidity of DNA. We use our model to infer the characteristics of the interaction potential, and find that its equilibrium spacing strongly depends on the curvature of the filaments. In addition, the interaction is much softer than previously reported in bulk samples using different salt conditions. Beyond viruses and cells, our characterization of the interactions governing DNA-based dense structures could help develop robust designs in DNA nanotechnologies.

biophysics↗

bioRxiv2018

Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron miocroscopy of vitreous sections

In Eukaryotes, DNA is wound around the histone core octamer to form the basic chromatin unit, the nucleosome. Atomic resolution structures have been obtained from crystallography and single particle cryo-electron microscopy of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryo-electron microscopy and tomography of vitreous sections we analyse the conformation of native nucleosomes, both in vitro, using purified particles solubilised at physiologically relevant concentrations (25-50 %), and in situ, within interphase nuclei. We visualise individual nucleosomes at a level of detail that allows us to analyse the conformation of the DNA wrapped around, and measure the distance between the DNA gyres. We evidence a variety of conformations. In interphase nuclei open nucleosomes predominate, with an average inter-gyre distance larger than that of the canonical particle. In concentrated solutions, we evidence a salt-dependant transition, with high salt compact conformations resembling the canonical nucleosome, and open low salt ones, closer to nuclear nucleosomes. Although further particle characterisation and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ.

biophysics↗