Biology subjects

Daraspe, J.

Publications and source records attributed to Daraspe, J..

12 recordsLinked to original sources

bioRxiv2026

Cryo-ET and Sub-Volume Analysis of Fibrillar Pathology in the Neurodegenerative Human Brain

Structural studies of amyloid fibrils extracted from brain tissue have identified disease-specific fibril polymorphs. However, the mechanisms driving distinct polymorphs remain unclear because no method currently links the cellular context, composition and ultrastructure of individual aggregates to their constituent fibril polymorphs. Here, we present a workflow for in situ cryo-electron tomography (cryo-ET) to study amyloids within individual aggregates in the human neurodegenerative postmortem brain. Using chemically fixed tissue, we studied in situ tau fibrils in the hippocampus of an Alzheimer's disease (AD) donor and -synuclein fibrils within a glial nuclear inclusion in the cingulate gyrus of a multiple systems atrophy (MSA) donor. Subtomogram averaging and helical reconstruction of fibril subvolumes yielded low-resolution (~30-35A) density maps, which were compared to existing ex vivo structures. Preserved ultrastructure enabled detailed observations of pathology, including an alpha-synuclein fibril penetrating the nuclear envelope, providing mechanistic insight into intranuclear aggregate formation. Combined with existing high-resolution fibril structures lacking spatial context, cryo-ET offers a powerful approach to understanding amyloid polymorphism in neurodegenerative diseases, advancing anti-amyloid therapies and diagnostic tools.

neuroscience↗

bioRxiv2026

Atonosomes, compartments involved in membrane tension decrease

To ensure survival, cells need to buffer the effects of environmental stress on their plasma membrane, yet the structural mechanisms by which this is acutely achieved remain largely unknown. Here, we propose atonosomes as a unifying identity for a class of previously observed but enigmatic, tension-responsive, plasma membrane-derived compartments that arise across contexts of acute and chronic membrane tension loss. Leveraging unprecedented high resolution cryo-FIB-ET imaging in yeast, we show that atonosomes are complex, organelle-containing structures bounded by membranes and cell wall material, spanning hundreds of nanometers, and displaying a remarkable morphological diversity. Atonosomes form within seconds in response to reduced plasma membrane tension, and their emergence appears to require no dedicated molecular machinery, arising instead as a direct consequence of membrane biophysics. Upon formation, they recruit key membrane-associated proteins, including TORC2, Slm1, and septins. Under conditions of chronic disruption of PM homeostasis, atonosomes become constitutively present. Their stability and reversibility are further modulated by the cell wall, whose polymerization state influences atonosome dynamics. Structural conservation in fungi and ichthyosporea, demonstrates that atonosomes are a conserved stress-triggered response of cell-wall enclosed organisms. Together, these findings establish atonosomes as a novel compartment that mediates cellular responses to plasma membrane tension variation, coupling membrane remodeling and lipid homeostasis to preserve cellular integrity under stress.

cell biology↗

bioRxiv2026

Alternative exon splicing reveals hidden mitochondrial targeting of PLIN3

Perilipin 3 (PLIN3) is a lipid droplet-associated protein implicated in cellular lipid metabolism. We characterize a previously unrecognized alternative splicing event in human PLIN3. The generated transcript PLIN3B is detectable at the RNA level across human tissues and is modulated by antisense oligonucleotide-mediated splicing. Despite clear evidence for PLIN3B transcript expression, endogenous PLIN3B protein could not be detected using multiple complementary state-of-the-art detection strategies. When expressed exogenously, PLIN3B coding sequence encodes an unstable protein that localizes predominantly within mitochondria, in contrast to canonical PLIN3. Using super-resolution microscopy, correlative light-electron microscopy, and interaction proteomics, we show that PLIN3B accumulates intramitochondrially and engages protein quality control machinery. Mitochondrial targeting depends on residues shared by both isoforms, not altered by alternative splicing, indicating that mitochondrial targeting is normally repressed. Together, these results show that alternative exon splicing in PLIN3 reveals latent mitochondrial targeting without producing detectable endogenous protein, underscoring the need to distinguish transcript diversity from stable protein expression.

cell biology↗

bioRxiv2025

Cryo-electron tomography reveals lineage-specific replication features of Zika virus

Zika virus (ZIKV), a member of the Flavivirus genus, can cause severe neurodevelopmental disorders when infection occurs during pregnancy. Phylogenetic analyses classify ZIKV into two major lineages: African and Asian. Despite their genetic similarity, only the Asian lineage has been linked to congenital anomalies. ZIKV possesses a single-stranded RNA genome and replicates by remodelling the host endoplasmic reticulum (ER) to form membrane-bound replication complexes, which support viral RNA synthesis, virion assembly, and immune evasion. ZIKV also induces the formation of convoluted membranes (CMs), whose function remains unclear. Most ultrastructural studies of ZIKV replication have relied on transmission electron microscopy, which, despite its value, can alter membrane architecture due to sample preparation. To overcome these limitations, we employed cryo-focused ion beam milling combined with electron tomography to visualize ZIKV replication in a near-native state. By comparing infections with African and Asian ZIKV lineages, we uncovered lineage-specific ultrastructural features, despite similar replication dynamics and growth curves. In cells infected with the Asian strain, virions at different maturation stages were often co-packaged within the same compartment. In contrast, the African lineage ZIKV showed distinct compartmentalization of virions and vesicles. Furthermore, the ER-virion spacing was uniform in African-lineage-infected cells, while morphological heterogeneity was evident in the Asian lineage, including irregular viral sacs. Vesicle diameter was also significantly larger in cells infected with the Asian lineage. Together, these findings reveal both conserved and distinct replication structures between ZIKV lineages. The Asian lineage induces more extensive membrane remodelling and CM formation, potentially contributing to its association with congenital disease.

cell biology↗

bioRxiv2025

The Luminal Ring Protein C2CD3 Acts as a Radial In-to-Out Organizer of the Distal Centriole and Appendages

Centrioles are polarized microtubule-based structures with appendages at their distal end that are essential for cilia formation and function. The protein C2CD3 is critical for distal appendage assembly, with mutations linked to orofaciodigital syndrome and other ciliopathies. However, its precise molecular role in appendage recruitment remains unclear. Using Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, and in situ cryo-electron tomography (cryo-ET), we reveal that C2CD3 adopts a radially symmetric 9-fold organization within the centrioles distal lumen. We show that the C-terminal region of C2CD3 localizes close to a [~]100 nm luminal ring structure consisting of [~]27 nodes, while its N-terminal region localizes close to a hook-like structure that attaches to the A-microtubule as it extends from the centriole interior to exterior. This hook structure is adjacent to the DISCO complex (MNR/CEP90/OFD1), which marks future appendage sites. C2CD3 depletion disrupts not only the recruitment of the DISCO complex via direct interaction with MNR but also destabilizes the luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14, as well as the distal microtubule tip protein CEP162. This reveals an intricate "in-to-out" molecular hub connecting the centriolar lumen, distal microtubule cap, and appendages. Although C2CD3 loss results in shorter centrioles and appendage defects, key structural elements remain intact, permitting continued centriole duplication. We propose that C2CD3 forms the luminal ring structure and extends radially to the space between triplet microtubules, functioning as an architectural hub that scaffolds the distal end of the centriole, orchestrating its assembly and directing appendage formation.

cell biology↗

bioRxiv2025

Imaging of specialized plant cell walls by improved cryo-CLEM and cryo-electron tomography

Cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM) has become essential for preparing electron-transparent lamellae from cryo-plunged and high-pressure frozen specimens. However, targeting specific cellular features within large, complex organs remains challenging. Here we present a series of technical improvements significantly enhancing the efficiency and accessibility of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures that are revolutionizing the field. We were able to extend the cryo-FIBSEM session from 24 hours to 5 days without interruptions. In addition, we describe a modified silver-plated EasyLiftTM needle that eliminates the need of the copper or gold block between the original tungsten needle and the sample. Moreover, we describe a strategy that significantly reduces curtaining effects. Finally, we report a precise routine to target a lamella with a precision of approximately 1 {micro}m in X,Y and Z. Together, these modifications considerably reduce contamination risk and preparation time, making cryo-lift-out techniques more accessible for routine structural biology applications on any type of tissue. Here, we demonstrate the power of our technique by targeting several specific wall structures that are of crucial importance for root function in plants and that were previously inaccessible to cryo-electron tomography (cryo-ET). High-pressure freezing (HPF) of plant tissues presents unique challenges for cryo-electron microscopy sample preparation due to the overall sample size, the individual cells size, their rigid cell wall and finally, their large vacuoles, which contain large amounts of rather diluted water solutions compared to cytosol. The internal root structures targeted are the Casparian strip (CS), suberin lamellae (SL), as well as secondary wall of xylem vessels, requiring reaching a targeting precision of 5 m in a 3 mm long and 80-120 m thick root tip. Our technological improvements for the cryo-correlative light and electron microscopy (cryo-CLEM) workflow enabled successful, targeted cryo-ET in plant roots. We noticed that, despite ice formation in vacuoles and to some degree in the cytosol, the plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents and in a dehydrated state.

plant biology↗

bioRxiv2024

A versatile correlative light and electron microscopy protocol for human brain and other biological models

Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies, and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe the step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room-temperature and can be also adapted to other human and animal tissue samples. The procedure requires 8-days to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition, multi-modal image correlation, and is executable within standard EM laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions. Key pointsO_LIThe protocol for correlative light and electron microscopy (CLEM) is optimized for analyzing chemically fixed human brain tissues. It focuses on maintaining the integrity of ultrastructural features, thereby minimizing artifacts and structural alterations. C_LIO_LIExamining brain tissues at the ultrastructural level can provide an unprecedented amount of detail which may help advance our understanding of the mechanisms underlying neurodegenerative disorders. C_LI

neuroscience↗

bioRxiv2023

Uptake without inactivation of human adenovirus type 2 by Tetrahymena pyriformis ciliates

Human adenoviruses are ubiquitous contaminants of surface water. Indigenous protists may interact with adenoviruses and contribute to their removal from the water column, though the associated kinetics and mechanisms differ between protist species. In this work, we investigated the interaction of human adenovirus type 2 (HAdV2) with the ciliate Tetrahymena pyriformis. In co-incubation experiments in a freshwater matrix, T. pyriformis was found to efficiently remove HAdV2, with [≥] 4 log10 removal over 72 hours. Neither sorption onto the ciliate nor secreted compounds contributed to the observed loss of infectious HAdV2. Instead, internalization was shown to be the dominant removal mechanism, resulting in the presence of viral particles inside food vacuoles of T. pyriformis, as visualized by transmission electron microscopy. The fate of HAdV2 once ingested was scrutinized and no evidence of virus digestion was found over the course of 48 hours. This work shows that T. pyriformis can exert a dual role on microbial water quality: while they remove infectious adenovirus from the water column, they can also accumulate and potentially protect infectious viruses from exposure to environmental or engineered stressors. Environmental significanceHuman viruses are ubiquitous contaminants of surface water. The fate of human viruses, once discharged into the environment, is modulated by indigenous microorganisms in the surrounding water body. Among microorganisms, the role of protists in controlling virus persistence has been overlooked. Here, we investigate the interactions of the ciliate Tetrahymena pyriformis with human adenovirus type 2. We demonstrate that T. pyriformis can serve as a sink of adenovirus from the water column, but also act as a reservoir and potential protective barrier for infectious viruses against stressors. T. pyriformis ciliates are among the most abundant protists in surface waters. Understanding this dual role of protists is important for assessing and maintaining microbial water quality and infection risks.

microbiology↗

bioRxiv2023

Foraging on host synthesized metabolites enables the bacterial symbiont Snodgrassella alvi to colonize the honey bee gut

Dietary nutrients and microbial cross-feeding allow diverse bacteria to colonize the animal gut. Less is known about the role of host-derived nutrients in enabling gut bacterial colonization. We examined metabolic interactions within the evolutionary ancient symbiosis between the honey bee (Apis mellifera) and the core gut microbiota member Snodgrassella alvi. This Betaproteobacteria is incapable of metabolizing saccharides, yet colonizes the honey bee gut in the presence of only a sugar diet. Using comparative metabolomics, 13C tracers, and Nanoscale secondary ion mass spectrometry (NanoSIMS), we show in vivo that S. alvi grows on host-derived organic acids, including citrate, glycerate and 3-hydroxy-3-methylglutarate which are actively secreted by the host into the gut lumen. S. alvi additionally modulates tryptophan metabolism in the gut by converting kynurenine to anthranilate. These results suggest that S. alvi is adapted to a specific metabolic niche in the gut that depends on host-derived nutritional resources.

microbiology↗

bioRxiv2021

Extracellular membrane tubules involved in suberin deposition in plant cell walls

Suberin is a fundamental plant biopolymer, found in protective tissues, such as seed coats, exodermis and endodermis of roots, the outer layers of stems and roots with secondary growth, as well as in wound-induced tissues. Its presence allows organs to resist various environmental stresses, such as pathogen attack, drought or excessive salt concentrations. Suberin is a mostly aliphatic polyester of long-chain fatty acids and alcohols, often co-occurring with lignin-like polymers in the same cells. Most suberizing cells appear to deposit suberin in the form of lamellae just outside of the plasma membrane, below the primary cell wall. The monomeric precursors of suberin are thought to be glycerated fatty acids, synthesized at the endoplasmic reticulum. However, it has remained obscure how these monomers are transported outside of the cell, where they will be polymerized to form suberin lamellae. Here, we demonstrate that extracellular vesicular-tubular structures accumulate specifically in suberizing cells. By employing various, independent mutational and hormonal challenges, known to affect suberization in distinct ways, we demonstrate that their presence correlates perfectly with root suberization. Surprisingly, no endosomal compartment marker showed any conspicuous changes upon induction of suberization, suggesting that this compartment might not derive from endosomal multi-vesicular bodies, but possibly form directly from endoplasmic reticulum subdomains. Consistent with this, we could block formation of both, suberin deposition and vesicle accumulation by a pharmacogenetic manipulation affecting early steps in the secretory pathway. Whereas many previous reports have described extracellular vesicle occurrence in the context of biotic interactions, our results suggest a developmental role for extracellular vesicles in suberin formation. One Sentence SummarySuberin lamellae formation is associated with extracellular membrane tubules.

plant biology↗

bioRxiv2020

Hidden cell diversity in Placozoa: Ultrastructural Insights from Hoilungia hongkongensis

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly six major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species - Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

evolutionary biology↗

bioRxiv2019

ROS production by localized SCHENGEN receptor module drives lignification at subcellular precision

Production of reactive-oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and direct cellular targets of ROS. A well-understood ROS target in plants are lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here we establish a full kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicates that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

plant biology↗