Biology subjects

Schaeffer, M.

Publications and source records attributed to Schaeffer, M..

8 recordsLinked to original sources

bioRxiv2026

Diversification of ligand preference in conserved Brassicaceae LORE immune receptors matches proteobacterial 3-hydroxy fatty acid profiles

Pattern recognition receptors are often conserved across plant lineages, yet how ligand sensing diversifies during evolution remains poorly understood. The receptor kinase LORE mediates Arabidopsis thaliana immune responses to bacterial medium-chain 3-hydroxy fatty acids (mc-3-OH-FAs), but its distribution and functional variation across Brassicaceae remain largely unexplored. We combined phylogenetic analysis, species-wide immune profiling, ligand-binding assays, heterologous functional testing, and A. thaliana lore-1 complementation to define LORE diversification across Brassicaceae. LORE orthologs with conserved 3-OH-C10:0 binding are present in all major Brassicaceae lineages, including the basal lineage. Functional complementation confirmed mc-3-OH-FA-sensing capacity in orthologs spanning all four lineages. By contrast, chain-length preference profiles among mc-3-OH-FAs varied between species and orthologs, ranging from narrow 3-OH-C10:0-dominated profiles to broader profiles with comparable sensitivity to 3-OH-C10:0 and 3-OH-C12:0. 3-OH-FA profiling of a diverse collection of plant-associated bacteria revealed that high levels of mc-3-OH-FAs are prevalent in Proteobacteria, and that the range of mc-3-OH-FA chain lengths produced corresponds to the chain-length preferences of Brassicaceae LORE receptors. Our results establish LORE as a Brassicaceae-restricted pattern recognition receptor with conserved mc-3-OH-FA sensing but diversified chain-length preferences, and provide an evolutionary framework for studying how ligand preference can diversify within a biologically relevant ligand spectrum while core receptor function is maintained.

plant biology↗

bioRxiv2024

Chromatin folding motifs define the basic grammar of TAD architecture in single alleles

Chromatin folding exhibits extensive cell-to-cell variability, yet whether this variability is dominated by continuously varying conformations or by recurrent organizational patterns remains unclear. Here, we combined high-resolution chromatin tracing with a novel machine learning algorithm to show that chromatin organization can be compactly represented by a limited repertoire of recurrent folding motifs. Using polymer simulations and cohesin depletion experiments, we further show that these motifs arise primarily from loop extrusion. Analysis of distinct tissues reveals that a shared motif repertoire is sufficient to describe diverse structural ensembles, with biological differences arising primarily through changes in motif occupancy and combinatorial usage. More generally, rather than generating entirely new conformations, transcriptional and disease-associated changes, including those occurring during disease onset, act primarily by redistributing motif occupancy within this common repertoire. In particular, gene activation is associated with a shift toward decompacted states and reduced contact frequencies, revealing a quantitative link between transcription and chromatin organization at the single-allele level. Together, these findings reveal that single-allele chromatin variability is structured by a constrained repertoire of recurrent folding motifs whose usage is modulated by regulatory context, providing a new framework to interpret how chromatin organization relates to gene activity.

genomics↗

bioRxiv2023

pyHiM, a new open-source, multi-platform software package for spatial genomics based on multiplexed DNA-FISH imaging

The three-dimensional (3D) nuclear organization of chromatin in eukaryotes plays a crucial role in gene regulation, DNA replication, and DNA damage repair. While genome-wide ensemble methods have enhanced our understanding of chromatin organization, they lack the ability to capture single-cell heterogeneity and preserve spatial information. To overcome these limitations, a new family of imaging-based methods has emerged, giving rise to the field of spatial genomics. In this study, we present pyHiM, an open-source and modular software toolbox specifically designed for the robust, automatic analysis of sequential spatial genomics data. pyHiM enables the reconstruction of chromatin traces in individual cells from raw, multicolor images, offering novel, robust and validated algorithms, extensive documentation, and tutorials. Its user-friendly graphical interface and command-line interface allow for easy installation and execution on various hardware platforms. The software employs a modular architecture, allowing independent execution of analysis steps and customization according to sample specificity and computing resources. pyHiM supports preprocessing, spot detection, mask detection, and trace generation, generating human-readable reports and intermediate results for data validation and further analysis. Moreover, it offers additional features for data formatting, result display, and post-processing. pyHiMs scalability and parallelization capabilities enable the analysis of large, complex datasets in a reasonable time frame. Overall, pyHiM aims to facilitate the democratization and standardization of spatial genomics analysis, foster collaborative developments, and promote the growth of a user community to drive discoveries in the field of chromatin organization.

genomics↗

bioRxiv2022

Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we used multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc repressed genes. Finally, non-Hox Pc targets are proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not involve clustering of multiple distant Pc genes.

genetics↗

bioRxiv2021

LORE homomerization is required for 3-OH-C10:0 induced immune signaling

O_LIThe S-domain-type receptor-like kinase (SD-RLK) LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) from Arabidopsis thaliana is a pattern recognition receptor that senses medium-chain 3-hydroxy fatty acids, such as 3-hydroxydecanoic acid (3-OH-C10:0), to activate pattern-triggered immunity. Here, we show that LORE homomerization is required to activate 3-OH-C10:0-induced immune signaling. C_LIO_LIFluorescence lifetime imaging in Nicotiana benthamiana demonstrated that AtLORE homomerizes via the extracellular and transmembrane domains. Co-expression of AtLORE truncations lacking the intracellular domain exerts a dominant negative effect on AtLORE signaling in both N. benthamiana and A. thaliana, highlighting that homomerization is essential for signaling. C_LIO_LIScreening for 3-OH-C10:0-induced reactive oxygen species production revealed natural variation within the Arabidopsis genus. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3-OH-C10:0, although both possess a putative LORE orthologue. Both LORE orthologues have defective extracellular domains that bind 3-OH-C10:0 to a similar level but lack the ability to homomerize. Thus, ligand binding is independent of LORE homomerization. Analysis of AtLORE and AlyrLORE chimera suggests that the loss of AlyrLORE homomerization is caused by several amino acid polymorphisms across the extracellular domain. C_LIO_LIOur findings shed light on the activation mechanism of LORE and the loss of 3-OH-C10:0 perception within the Arabidopsis genus. C_LI

plant biology↗

bioRxiv2021

Low cost, medium throughput depletion-binding assay for screening S-domain-receptor ligand interactions using in planta protein expression

BackgroundPlant cell-surface receptors sense various ligands to regulate physiological processes. Matching ligand-receptor pairs requires evidence of their direct interaction and is often a bottleneck in functional receptor studies. The S-domain-type (SD) pattern recognition receptor LORE senses medium chain-3-hydroxy fatty acids (mc-3-OH-FAs) such as 3-hydroxy decanoic acid (3-OH-C10:0) via its extracellular domain (ECD) They are perceived as signals of danger from Gram-negative bacteria and activate immune responses in Arabidopsis thaliana. LORE is found in low levels in planta and is poorly expressed in heterologous systems. Furthermore, chemical modifications of the mc-3-OH-FA ligand affect its biological activity. Taken together, this makes LORE-mc-3-OH-FA binding studies particularly challenging. ResultsTo investigate the LORE-mc-3-OH-FA interaction, we have developed a sensitive assay system based on protein expression in planta. The ECDs of LORE and other proteins of interest were transiently expressed as soluble, apoplastic mCherry fusion proteins in Nicotiana benthamiana and collected in apoplastic washing fluids. Protein-ligand complexes and unbound ligand were separated according to their molecular weight. In a two-step procedure, we first investigated whether the ECD-mCherry fusion protein depletes 3-OH-C10:0 from the low molecular weight fraction (step depletion). Subsequently, protein-bound 3-OH-C10:0 retained in the high molecular weight fraction in the depletion step is released and detected (step binding). Both the unbound and the released 3-OH-C10:0 ligand are detected by a sensitive bioassay using LORE loss- and gain-of-function Arabidopsis plants. Using the depletion-binding assay, we show that the ECD of AtLORE and its ortholog from Capsella rubella, CrubLORE, bind 3-OH-C10:0. The ECD of AtSD1-23, the closest paralog of AtLORE, and mCherry did not bind 3-OH-C10:0 and are suitable negative controls. ConclusionThe depletion-binding assay is a simple method for reliably detecting interactions between plant-expressed SD-type receptor ectodomains and mc-3-OH-FAs. It does not require special equipment or expensive consumables and is suitable for medium throughput screening. The assay is very flexible and can be easily adapted to investigate ligand interactions of other extracellular receptor domains.

plant biology↗

bioRxiv2019

Maternal hypothyroidism in mice influences glucose metabolism in adult offspring

During pregnancy, maternal metabolic diseases and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offsprings remains poorly understood. Here, we show that when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation and increased insulin sensitivity. However, following high fat feeding, these offsprings became profoundly hyperinsulinemic, insulin-resistant and glucose-intolerant compared to controls from euthyroid mothers. Suggesting presence of epigenetic changes, altered glucose metabolism was maintained in a second generation of animals. As such, gestational hypothyroidism induces long-term and persistent alterations in endocrine pancreas function, which may have important implications for type 2 diabetes prevention in affected individuals.\n\nSIGNIFICANCEDiabetes and hypothyroidism are two major public health issues, affecting [~] 9 and 2 % of the population worldwide, respectively. As master metabolic gatekeepers, the thyroid hormones play an essential role in metabolism and fetal development. However, gestation increases demand on the thyroid axis in the mother, leading to hypothyroidism in 0.5 % of pregnancies. Maternal hypothyroidism is associated with deficits in fetal growth that may lead to long-term alterations in metabolism in the offspring. We therefore sought to investigate the effects of gestational hypothyroidism on glucose metabolism in adult offspring and their descendants, and how this may predispose to diabetes development.

physiology↗

bioRxiv2018

Integrin beta1 optimizes diabetogenic T cell migration and function in the pancreas

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas during autoimmune diabetes remains unclear. Using intra-vital 2-photon microscopy in a mouse model of diabetes, we found that CXCR3 chemokine downregulated CD8+ T cell motility specifically within islets, promoting effector cell confinement to their target sites. In contrast, T cell velocity and directionality in the exocrine tissue were enhanced along blood vessels and extra-cellular matrix fibers. This guided migration implicated integrin-dependent interactions, since integrin blockade impaired exocrine T cell motility. In addition, integrin {beta}1 blockade decreased CD4+ T cell effector phenotype specifically in the pancreas. Thus, we unveil an important role for integrins in the pancreas during autoimmune diabetes that may have important implications for the design of new therapies.

immunology↗