Biology subjects

van Niel, G.

Publications and source records attributed to van Niel, G..

9 recordsLinked to original sources

bioRxiv2026

Cutting-edge microfluidic technology allows detection and sorting of single fluorescent extracellular vesicles

Extracellular vesicles (EV) are cell-secreted, nanosized biological particles that are involved in most physiological and pathophysiological processes. Their composition and properties reflect the pathophysiological state of the producing cell, tissue, and organ. Released into biological fluids, they may be used as a potential source of non-invasive biomarkers for diagnosis, prognosis or therapeutic follow-up in several diseases such as cancer. However, despite the development of single-EV analysis technologies, the sorting of EV subpopulations for downstream analysis remains a major challenge. Here, we describe a novel instrument that combines a highly sensitive optical detection system with an advanced lab-on-a-chip device. Using engineered fluorescent small extracellular vesicles (sEV), we demonstrate that the Hekat NanoSorter enables sensitive, scalable, and reproducible detection and sorting of individual sEVs for downstream analysis by cryo-EM. Furthermore, detailed characterization of the sorted EV populations, together with the detection of sEVs labeled with an in vitro pathophysiological marker, demonstrates the ability of the NanoSorter to monitor and enrich clinically relevant EV-associated biomarkers. These findings highlight the clinical potential of extracellular vesicle sorting for their use as biomarkers for therapeutic monitoring and treatment outcome assessment.

bioengineering↗

bioRxiv2025

Single recipient cell tracking of tellurium-labeled extracellular vesicle proteomes (TeLEV) identifies EV-driven immunomodulation

Extracellular vesicles (EVs) mediate tumor-immune cell communication by carrying protein cargo that can immediately modulate signaling and antigen presentation. Yet mapping the uptake of primary EV proteomes by human immune cells at single-cell resolution has been constrained by a lack of labeling strategies. We show here that TeLEV, a tellurium-based metabolic mass tagging approach that incorporates L-2-tellurienylalanine (TePhe) into EV proteomes, can produce a biologically rare monoisotopic signal, which is detectable by mass cytometry, imaging mass cytometry, and nanoscale SIMS, without perturbing EV morphology, yield, or proteome composition. We applied TeLEV to label primary malignant B-cell-derived EVs (MBC-EVs) from chronic lymphocytic leukemia (CLL) patients and could follow EV uptake by distinct cell populations of healthy donor peripheral blood mononuclear cells. MBC-EV uptake occurred predominantly in cells of myeloid lineages. In direct control experiments with matched secreted proteins, a machine learning approach identified CD123, CD127, and CD25 as key markers distinguishing primary MBC-EV recipients from matched secreted protein recipient cells. Nanoscale imaging enabled localization of EV-delivered proteins within heterochromatin, whereas Te-labeled secreted proteins accumulated in the cytoplasm of recipient cells. We then generated a pan-immune EV uptake atlas by tracing the uptake of primary and cell-line EVs from nine cell lines and six donors with chronic lymphocytic leukemia into 2,977,094 recipient cells across 43 cell types and subpopulations. We found that the uptake of MBC-EVs by myeloid recipients induced monocyte-derived dendritic-cell polarization characterized by the co-expression of the interleukin-receptor triad (IL-RT: CD123, CD127, CD25) identified above. Time-resolved EV uptake analysis showed a rapid, transient expression of CD123/CD127 followed by CD25, both tightly coupled to MBC-EV uptake by myeloid cells. The intensity of IL-RT expression correlated with that of PD-L1 and BCL-2. Using different STAT degraders to bidirectionally modify the EV-induced STAT5 signal, we observed that MBC-EV uptake and IL-RT, PD-L1, and BCL-2 expression increased with STAT3 degradation and decreased with STAT5 degradation. To investigate the functional consequences of the MBC-EV-induced changes, we showed that MBC-EVs in the presence of IL-2 induced a high-CD25 immune state with low cytotoxic and high B cell proliferation. Taken together, TeLEV represents a novel tool for single-cell tracking of EV proteomes, revealing STAT5-dependent immune remodeling of recipient cells.

cancer biology↗

bioRxiv2023

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad- spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIII{beta} as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions.

cell biology↗

bioRxiv2023

Atheroprone shear stress stimulates noxious endothelial extracellular vesicle uptake by MCAM and PECAM-1 cell adhesion molecules

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, high shear stress (HSS) has atheroprotective effects on the endothelium. Endothelial cell-derived extracellular vesicles have been shown to regulate inflammation, senescence and angiogenesis and therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between hemodynamic forces and extracellular vesicle release, the exact consequences of shear stress on the release and uptake of endothelial EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours. Large and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by TEM, Western blot analysis of EV markers, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labeled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in HUVEC conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Additionally, endothelial LSS-EVs appeared to have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins such as PECAM1, MCAM, which were involved in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be involved in elevating reactive oxygen species levels in recipient cells. These findings suggest that endothelial shear stress has a significant impact during EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

cell biology↗

bioRxiv2022

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact single EV-based analysis and biomarker profiling, we investigated whether under physiological conditions LPPs and EVs can associate by using cryo-electron tomography, label-free synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high resolution single particle flow cytometric analysis. Furthermore, we evaluated the impact on flow cytometric analysis in the absence or presence of different types of LPPs using in vitro spike-in experiments of purified tumor cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrated that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations can influence EV analysis in a quantitative and qualitative manner. Our findings demonstrate that the biological colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis.

cell biology↗

bioRxiv2022

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages

Aging is characterized by gradual immune dysfunction and increased risk for many diseases, including respiratory infections. Genomic instability is thought to play a central role in the aging process but the mechanisms that damage nuclear DNA in aging are insufficiently defined. Cells that migrate or reside within confined environments experience forces applied to their nucleus, leading to transient nuclear envelope (NE) ruptures. NE ruptures are associated with DNA damage, and Lamin A/C is required to limit these events. Here, we show that Lamin A/C protects lung alveolar macrophages from NE rupture and hallmarks of aging. Lamin A/C ablation in immune cells results in a selective depletion of lung alveolar macrophages (AM) and a heightened susceptibility to influenza infection. Lamin A/C-deficient AM that persist display constitutive nuclear envelope rupture marks, DNA damage and p53-dependent senescence. In wild-type mice, we found that AM migrate within constricted spaces in vivo, at heights that induce NE rupture and DNA damage. AM from aged wild-type mice and from Lamin A/C-deficient mice share an upregulated lysosomal signature with CD63 expression, and we find that CD63 is required to clear damaged DNA in macrophages. We propose that induction of genomic instability by NE disruption represents a mechanism of aging in alveolar macrophages.

immunology↗

bioRxiv2021

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin released by virtually all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs and proteins between different cells, tissues or organs. However, the mechanisms that regulate the selective loading of cytosolic proteins into these vesicles are still largely unknow. Here we describe a mechanism whereby proteins containing a pentapeptide sequence, biochemically related to the KFERQ-motif, are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, this mechanism is independent of the ESCRT machinery components TSG101 and VPS4b and dependent on HSC70, CD63, Alix, Syntenin-1, Rab31 and ceramides. The transcription factor and master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. Additionally, by tagging fluorescent proteins with KFERQ-like sequences we were able to follow inter-organ transfer of exosomes in zebrafish larvae. Our findings identify LAMP2A as a key component in exosome biogenesis while opening new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

cell biology↗

bioRxiv2019

Origin of bidirectionality of cerebrospinal fluid flow and impact on long-range transport between brain and spinal cord

The circulation of cerebrospinal fluid (CSF) plays pivotal roles for body axis formation and brain development. During embryogenesis, CSF is rich in particles and proteins and flows bidirectionally in the central canal. The origins of bidirectional flow and its impact on development are unknown. Experiments combined with modeling and simulations demonstrate that the bidirectionality of CSF flow is generated locally by caudally-polarized motile cilia confined to the ventral wall of the central canal. Such active bidirectional flow of the CSF accelerates the long-range transport of particles propagating rostrally and caudally. In addition, spontaneous muscle contractions increase local CSF flow and consequently enhance long-range transport of extracellular lipidic particles. Focal ablation of the channel connecting brain ventricles to the central canal reduces embryo length, indicating that long-range transport contributes to embryonic growth. Our study also demonstrates that at this early stage, motile cilia ensure the proper formation of the central canal.

biophysics↗

bioRxiv2018

Live tracking of inter-organ communication by endogenous exosomes in vivo

Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.\n\nHighlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin\n- In the YSL, syntenin regulates exosome release into the blood for their propagation\n- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells\n- Uptake is scavenger receptor and dynamin-dependent and provides trophic support\n\n\nBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

cell biology↗