Biology subjects

Roelofs, M.

Publications and source records attributed to Roelofs, M..

3 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles from Manila clam (Ruditapes philippinarum): tailored isolation from hemolymph and insights into water-derived vesicles

Extracellular vesicles (EVs) are evolutionarily conserved mediators of intercellular communication released by cells into biological fluids and the extracellular environment. Despite their growing relevance in biomedical and veterinary research, knowledge on EVs in marine bivalves remains limited. The aim of this study was to optimize tailored protocols for EV isolation from the hemolymph of the Manila clam (Ruditapes philippinarum) based on density gradient ultracentrifugation (dgUC) or size exclusion chromatography (SEC). EV-enriched fractions were identified through nanoparticle tracking analysis, protein quantification, transmission electron microscopy, and cryo-electron microscopy. Both methods successfully isolated small EVs (<200 nm). While dgUC yielded higher-purity preparations, SEC provided a higher recovery rate and compatibility with downstream metabolomic analyses. Metabolomics performed on SEC fractions and on hemolymph, revealed that EV-enriched fractions possessed a distinct metabolic signature including enrichment in metabolites associated with nucleotide metabolism, glycolysis, redox regulation, and energy metabolism. Furthermore, we performed a pilot investigation into the presence of EVs released into conditioned water by Manila clams. Using tangential flow filtration and ultrafiltration, EVs were successfully concentrated from water samples and characterized by nanoparticle tracking analysis, CONAN assay, atomic force microscopy, and electron microscopy. Our findings demonstrate the feasibility of isolating EVs both from Manila clam hemolymph and from conditioned water, providing the first evidence of water-derived EV recovery in aquatic animals. Although further methodological refinement is needed to improve the purity of EVs isolated from water, and additional characterization studies are required to better define the molecular composition of clam-derived EVs, these results establish a foundation for future investigations into the role of EVs in bivalve biology and their potential application as minimally invasive biomarkers for aquaculture, environmental monitoring, and ecosystem health assessment.

systems biology↗

bioRxiv2022

Unraveling the intricate microtubule inner protein networks that reinforce mammalian sperm flagella

To find and fuse with the egg, mammalian sperm must complete an arduous voyage through the female reproductive tract. The sperm cells remarkable odyssey is powered by its flagellum, a microtubule-based molecular machine ornamented with accessory structures that stabilize the sperm tail in viscous media. Recently, cryo-electron tomography (cryo-ET) revealed that mammalian sperm flagella are further reinforced at the molecular scale with sperm-specific microtubule inner proteins (sperm-MIPs), but the identities of these sperm-MIPs are unknown. Here, we use cryo-electron microscopy to resolve structures of native bovine sperm doublet microtubules, thus identifying most sperm-MIPs. In the A-tubule, several copies of testis-specific Tektin-5 contribute to an extended protein network spanning nearly the entire microtubule lumen. Different copies of Tektin-5 adopt a range of conformations and organizations based on their local interactions with other MIPs. The B-tubule is in turn stabilized by sperm-MIPs that bind longitudinally along and laterally across protofilaments. We further resolve structures of endpiece singlet microtubules, revealing MIPs shared between singlets and doublets. Our structures shed light on the molecular diversity of cilia across different cell types of the vertebrate body and provide a structural framework for understanding the molecular underpinnings of male infertility.

cell biology↗

bioRxiv2021

In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

cell biology↗