Biology subjects

Belle, M.

Publications and source records attributed to Belle, M..

8 recordsLinked to original sources

bioRxiv2025

Genomic insights reveal community structure and phylogenetic associations of endohyphal bacteria and viruses in fungal endophytes

BackgroundEndohyphal microbiomes, comprising bacteria and viruses, significantly influence fungal phenotypes, host fitness, and ecological interactions. Endohyphal bacterial symbionts are known to affect fungal pathogenicity, secondary metabolite production, and adaptability, yet many aspects of their diversity and interactions remain uncertain. In contrast, endohyphal viruses, despite their widespread presence, are poorly understood in terms of their diversity, ecological roles, and evolutionary relationships with fungal hosts. These gaps highlight the need for integrative studies to explore the composition, diversity, host associations, and functional roles of both bacterial and viral communities in fungi. This study aimed to (1) characterize the diversity of endohyphal bacterial and viral communities within selected fungal endophytes using genomic and transcriptomic approaches, and (2) test for host specialization through phylogenetic signals and core microbial taxa. ResultsEndohyphal microbial communities from 19 fungal isolates from eight fungal orders (Amphisphaeriales, Botryosphaeriales, Diaporthales, Glomerellales, Mucorales, Pleosporales, Sordariales, and Xylariales) obtained from Fagus grandifolia leaves were characterized. Bacterial communities exhibited high diversity and significant phylogenetic signals, with core taxa such as Bacillales, Burkholderiales, Enterobacterales, Hyphomicrobiales, and Pseudomonadales, shared across all fungal samples. Specific bacterial taxa displayed potential host specialization, including Moraxellales, Sphingomonadales, and Streptosporangiaceae for Amphisphaeriales fungal samples; Enterobacterales (e.g., Enterobacteraceae), Hyphomicrobiales (e.g., Rhizobiaceae), and Micrococcales for Glomerellales; and Cytophagales for Diaporthales. Viral communities were less diverse, with Bamfordvirae and Heunggongvirae (double-stranded DNA viruses) identified as core taxa in metagenomic data, while metatranscriptomic data revealed no core taxa. Surprisingly, only a few reads of double-stranded RNA mycoviruses were detected. ConclusionsThe findings suggest a level of host specialization in bacterial communities and a more complex, limited association for viral communities, with dsDNA dominating the endohyphal virome. This study enhances our understanding of fungal-microbe interactions and highlights the ecological and evolutionary dynamics of endohyphal microbiota. Future efforts to expand reference databases and investigate the functional roles of these microbial taxa will further elucidate their contributions to fungal biology, impacts on their plant hosts, and ecosystem processes.

ecology↗

bioRxiv2024

Live-HPF CLEM using the HPM Live μ: Finding back all needles in every haystack. The last frontier in CLEM

Correlative light and electron microscopy is a scientific method that encompasses several technologies and workflows. One of the pioneering workflows consisted in vitrifying by high pressure freezing a sample, shortly after live observation1. Despite its extraordinary potential, it did not turn into a routine technique for practical reasons. We redesigned the entire tool set, from the sample carrier to the high-pressure freezing machine, to standardize and democratise the technique. In our manuscript, we present all the technological developments that lead to a routine workflow for live to HPF CLEM. We demonstrate our ability to track rapidly moving endosomes live and retrieve them at the electron microscopy level, in three dimensions, with high confidence.

cell biology↗

bioRxiv2024

Vitrification by high pressure freezing of a wide variety of sample using the HPM Live μ

This study explores the efficacy and reliability of high-pressure freezing (HPF) as a sample preparation technique for electron microscopy (EM) analysis across a diverse range of biological samples. Utilizing the HPM Live {micro} technology, based on the historical hydraulic HPM010 from BalTec, we demonstrate the reliability of our industrial equipment to achieve the critical parameters necessary for vitrification. By directly measuring physical values within the HPF chamber, we ensure the proper functioning of the equipment, contributing to the techniques reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, C. elegans, zebrafish, and A. thaliana root and seedlings were processed for EM analysis following HPF. The ultrastructure of each sample type was rigorously examined, revealing homogeneous preservation and minimal ice nucleation artifacts. Challenges such as plant leaf vitrification were addressed, highlighting the importance of methodological adaptation. Overall, our findings underscore the robustness and versatility of our HPM Live {micro} in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies.

cell biology↗

bioRxiv2023

Subcellular second messenger networks drive distinct repellent-induced axon behaviors

AO_SCPLOWBSTRACTC_SCPLOWSecond messengers, including cAMP, cGMP and Ca2+ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca2+ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalks, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo.

developmental biology↗

bioRxiv2022

The filipodia-like protrusions of adjacent somatic cells shape the developmental potential of mouse oocytes

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells which surround it. Part of this communication is through filopodialike protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component Myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes, and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

cell biology↗

bioRxiv2021

The sodium leak channel NALCN encodes the major background sodium ion conductance in murine anterior pituitary cells

The pituitary gland produces and secretes a variety of hormones that are essential to life, such as for the regulation of growth and development, metabolism, reproduction, and the stress response. This is achieved through an intricate signalling interplay between the brain and peripheral feedback signals that shapes pituitary cell excitability by regulating ion channel properties of these cells. In addition, endocrine anterior pituitary cells fire action potentials spontaneously to regulate intracellular calcium ([Ca2+]i) level, an essential signalling conduit for hormonal secretion. To this end, pituitary cells have to critically regulate their resting membrane potential (RMP) close to firing threshold, but the molecular identity of the ionic mechanisms involved remains largely unknown. Here, we revealed that the sodium leak channel NALCN, known to modulate neuronal excitability elsewhere in the brain, acts to regulate excitability in the mouse anterior endocrine pituitary cells. Using viral transduction combined with powerful electrophysiology methods and calcium imaging, we show that NALCN forms the major Na+ leak conductance in these cells, appropriately tuning cellular RMP for sustaining spontaneous firing activity. Genetic interruption of NALCN channel activity drastically hyperpolarised the cells, suppressing firing and ([Ca2+]i) oscillations. Remarkably, we uncover that NALCN conductance formed a very small fraction of the total cell conductance, but yet had a profound impact on pituitary cell excitability. Our results also provide a possible mechanism through which hypothalamic and hormone feedback signals can powerfully affect pituitary activity to influence hormonal function.

physiology↗

bioRxiv2020

HPM Live μ for full CLEM workflow

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

biophysics↗

bioRxiv2019

Cerebellar dopamine D2 receptors regulate preference for social novelty

The cerebellum, a primary center involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. The regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions. However, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown due to the lack of precise characterization of cerebellar dopaminoceptive neurons. By combining cell type-specific transcriptomic and histological analyses, 3D imaging and electrophysiology we demonstrate that cerebellar dopamine D2 receptors (D2R) in mice are preferentially expressed in Purkinje cells (PCs). While activation of D2R regulate synaptic efficacy onto PCs, their deletion or overexpression in PCs bidirectionally controls preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel D2Rs roles in PC function and causally link cerebellar D2R levels of expression to social behaviors.

neuroscience↗