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Cai, Kai, Bryan S. Sibert, Anil Kumar, et al. "Cryo-Electron Microscopy of Extracellular Vesicles." Microscopy and Microanalysis 28, S1 (2022): 1302–3. http://dx.doi.org/10.1017/s1431927622005347.
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Rodrigues, Marcio L., Ernesto S. Nakayasu, Debora L. Oliveira, et al. "Extracellular Vesicles Produced by Cryptococcus neoformans Contain Protein Components Associated with Virulence." Eukaryotic Cell 7, no. 1 (2007): 58–67. http://dx.doi.org/10.1128/ec.00370-07.
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ABSTRACT Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans, suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal products to host effector cells and tissues.
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Zhen, Ke, Xiaojuan Wei, Zelun Zhi, et al. "Comparison of Different Isolation Methods for Plasma-Derived Extracellular Vesicles in Patients with Hyperlipidemia." Life 12, no. 11 (2022): 1942. http://dx.doi.org/10.3390/life12111942.
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Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.
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Wolf, Julie M., Javier Espadas-Moreno, Jose L. Luque-Garcia, and Arturo Casadevall. "Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall." Eukaryotic Cell 13, no. 12 (2014): 1484–93. http://dx.doi.org/10.1128/ec.00111-14.
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ABSTRACTCryptococcus neoformansproduces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.
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Yuana, Yuana, Roman I. Koning, Maxim E. Kuil, et al. "Cryo-electron microscopy of extracellular vesicles in fresh plasma." Journal of Extracellular Vesicles 2, no. 1 (2013): 21494. http://dx.doi.org/10.3402/jev.v2i0.21494.
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Emelyanov, Anton, Tatiana Shtam, Roman Kamyshinsky, et al. "Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid." PLOS ONE 15, no. 1 (2020): e0227949. http://dx.doi.org/10.1371/journal.pone.0227949.
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Fertig, Emanuel T., Mihaela Gherghiceanu, and Laurentiu M. Popescu. "Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography." Journal of Cellular and Molecular Medicine 18, no. 10 (2014): 1938–43. http://dx.doi.org/10.1111/jcmm.12436.
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Dorward, David W., and Claude F. Garon. "Use of antibody and lectin-colloidal gold conjugates to detect surface and extracellular glycoproteins produced in vivo by the Lyme disease spirochete Borrelia bergdorferi." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 3 (1990): 934–35. http://dx.doi.org/10.1017/s0424820100162235.
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Despite the often serious pathological ramifications of Lyme disease, Borrelia burgdorferi, the causative agent, is rarely demonstrated in nor isolated from naturally-infected mammalian hosts. This dilemma has led to suggestions that a limited number of spirochetes expel extracellular bioproducts that may contribute to the clinical symptoms. Currently, no mammalian toxins have been attributed to B. burgdorferi, however, the production of extracellular vesicles by this bacterium was recently described. In order to assay the possible presence of such extracellular material in infected hosts, we developed a sensitive electron microscopic assay to detect and characterize B. burgdorferi products in biological samples.Polyclonal rabbit serum was raised against purified B. burgdorferi vesicles and against an electrophoretically-purified 83 kilodalton (kDa) vesicle-associated protein. After purification of IgG from the sera, anti-vesicle F(ab’)2 fragments were produced and used to “activate” parlodion-coated grids. Such grids were incubated with cultured spirochetes, or with fluids and tissues from infected and non-infected mammals and ticks. Captured antigens were then labeled with anti-83 kDa IgG and Protein A-colloidal gold conjugates, and/or lectin-colloidal gold conjugates, then examined and characterized by electron microscopy.
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De Vallée, Amelie, Jean-William Dupuy, Christine Moriscot, et al. "Extracellular Vesicles of the Plant Pathogen Botrytis cinerea." Journal of Fungi 9, no. 4 (2023): 495. http://dx.doi.org/10.3390/jof9040495.
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Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.
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Nicola, André Moraes, Susana Frases, and Arturo Casadevall. "Lipophilic Dye Staining of Cryptococcus neoformans Extracellular Vesicles and Capsule." Eukaryotic Cell 8, no. 9 (2009): 1373–80. http://dx.doi.org/10.1128/ec.00044-09.
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ABSTRACT Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNA-selective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components.
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Gerwing, Mirjam, Vanessa Kocman, Miriam Stölting, et al. "Tracking of Tumor Cell–Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis." Molecular Imaging and Biology 22, no. 6 (2020): 1501–10. http://dx.doi.org/10.1007/s11307-020-01521-9.
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Abstract Purpose Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis. Procedures Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum. Results In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV. Conclusions In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.
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Yastremsky, Evgeny, Luiza Garaeva, Elena Putevich, et al. "Abstract P-16: Cryo-Electron Microscopy Study of Vesicles from Various Species." International Journal of Biomedicine 11, Suppl_1 (2021): S18. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p16.
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Background: Plant-derived extracellular vesicles (PEVs) are studied as a natural carrier of functional biomolecules and as a potential system of targeted delivery of therapeutic agents. One of the urgent tasks in this direction is the selection of the carrier with optimal physicochemical parameters and morphology from a variety of plant sources. To date, vesicles from only a few plants were visualized using cryo-electron microscopy (cryo-EM). Here we investigated the morphology and physical parameters of extracellular vesicles from plant sources not previously studied utilizing this method. Methods: PEVs derived by ultracentrifugation from juice and cultural medium of 11 plants and mushrooms were studied using methods of cryo-EM. Samples were plunge frozen in liquid ethane with Vitrobot Mark IV and examined under cryogenic transmission electron microscope Titan Krios 60-300 (ThermoFisher Scientific, USA) in low dose mode using EPU software. Results: Most of the observed particles in each sample were classified as extracellular vesicles due to the presence of the lipid bilayer. Morphology and size characteristics of PEVs were determined and compared with each other. A variety of morphological configurations of PEVs were found: with single and multiple membranes, with different conformations and integrity state. Most of the isolated PEVs were single, round-shaped, and in a size range from 30 to 150 nm. Conclusion: Cryo-EM allowed us to obtain high-quality images of PEVs isolated from 11 plants and mushrooms (blueberry, chanterelle, cowberry, fly agaric, garlic, redcurrant, chlamydomonas, cucumber, shadberry, viburnum, gooseberry) which have been characterized by their size and morphology. From the data obtained, the most promising sources of vesicles were proposed. The approbation of the selected vesicles as effective delivery systems requires further research.
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Guyton, J. R., and K. F. Klemp. "Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques." Journal of Histochemistry & Cytochemistry 36, no. 10 (1988): 1319–28. http://dx.doi.org/10.1177/36.10.2458408.
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Electron microscopy of atherosclerotic arterial tissue commonly fails to distinguish lipid vesicles from droplets, especially when these are found in the extracellular space. The distinction is important, because vesicular or membranous lipid is composed of phospholipid and unesterified cholesterol, whereas neutral lipid in droplet form implies the presence of cholesteryl ester in atherosclerosis. A new procedure with sequential tannic acid and p-phenylenediamine treatments of osmicated tissue (TA-PDA) allows reliable ultrastructural discrimination of lipid vesicles and droplets. The multilamellar character of many vesicles is revealed. Extracellular droplets are found to possess many surface pits associated with membranous blebs. Pitting of droplets is especially evident after the use of an alternative tissue processing technique, the osmium-thiocarbohydrazide-osmium (OTO) sequence applied en bloc. The two complementary techniques will prove useful for electron microscopic studies of atherosclerotic and other lipid-rich tissues.
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Iša, Pavel, Arianna Pérez-Delgado, Iván R. Quevedo, Susana López, and Carlos F. Arias. "Rotaviruses Associate with Distinct Types of Extracellular Vesicles." Viruses 12, no. 7 (2020): 763. http://dx.doi.org/10.3390/v12070763.
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Rotaviruses are the leading cause of viral gastroenteritis among children under five years of age. Rotavirus cell entry has been extensively studied; however, rotavirus cell release is still poorly understood. Specifically, the mechanism by which rotaviruses leave the cell before cell lysis is not known. Previous works have found rotavirus proteins and viral particles associated with extracellular vesicles secreted by cells. These vesicles have been shown to contain markers of exosomes; however, in a recent work they presented characteristics more typical of microparticles, and they were associated with an increase in the infectivity of the virus. In this work, we purified different types of vesicles from rotavirus-infected cells. We analyzed the association of virus with these vesicles and their possible role in promotion of rotavirus infection. We confirmed a non-lytic rotavirus release from the two cell lines tested, and observed a notable stimulation of vesicle secretion following rotavirus infection. A fraction of the secreted viral particles present in the cell supernatant was protected from protease treatment, possibly through its association with membranous vesicles; the more pronounced association of the virus was with fractions corresponding to cell membrane generated microvesicles. Using electron microscopy, we found different size vesicles with particles resembling rotaviruses associated from both- the outside and the inside. The viral particles inside the vesicles were refractory to neutralization with a potent rotavirus neutralizing monoclonal antibody, and were able to infect cells even without trypsin activation. The association of rotavirus particles with extracellular vesicles suggests these might have a role in virus spread.
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Cavallaro, Sara, Petra Hååg, Kristina Viktorsson, et al. "Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis." Nanoscale Advances 3, no. 11 (2021): 3053–63. http://dx.doi.org/10.1039/d0na00948b.
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Nanoscale extracellular vesicles (EVs) have emerged as a valuable source of disease biomarkers. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy.
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Xun, Chengfeng, Lu Wang, Hailin Yang, et al. "Origin and Characterization of Extracellular Vesicles Present in the Spider Venom of Ornithoctonus hainana." Toxins 13, no. 8 (2021): 579. http://dx.doi.org/10.3390/toxins13080579.
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Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in the venom of snakes and parasitic wasps. Although vesicle secretion in spider venom glands has been observed, these secretory vesicles’ origin and biological properties are unknown. In this study, the origin of the EVs from Ornithoctonus hainana venom was observed using transmission electron microscopy (TEM). The Ornithoctonus hainana venom extracellular vesicles (HN-EVs) were isolated and purified by density gradient centrifugation. HN-EVs possess classic membranous vesicles with a size distribution ranging from 50 to 150 nm and express the arthropod EV marker Tsp29Fb. The LC-MS/MS analysis identified a total of 150 proteins, which were divided into three groups according to their potential function: conservative vesicle transport-related proteins, virulence-related proteins, and other proteins of unknown function. Functionally, HN-EVs have hyaluronidase activity and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) by affecting the cytoskeleton and cell cycle. Overall, this study investigates the biological characteristics of HN-EVs for the first time and sheds new light on the envenomation process of spider venom.
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Lin, Tzou-Yien, Tsong-Min Chang, and Huey-Chun Huang. "Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Attenuate Mast Cell Activation." Antioxidants 11, no. 11 (2022): 2279. http://dx.doi.org/10.3390/antiox11112279.
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The therapeutic potential of extracellular vesicles isolated from stem cells have been reported in several clinical diseases. Preclinical studies have demonstrated the beneficial effects of extracellular vesicles in the treatment of heart, kidney, liver, brain, and skin injuries. To address the putative therapeutic effects and mechanisms of extracellular vesicles derived from human umbilical cord mesenchymal stem cells on allergic activation in mast cells, we isolated extracellular vesicles from human umbilical cord-derived mesenchymal stem cells (UCMSCs) by tangential-flow filtration methods. The characteristics and identification of UCMSC-derived extracellular vesicles were examined via nanoparticle tracking analysis, transmission electron microscopy and protein marker analysis. Cytokines and tryptase in the cultured supernatant of KU812 cells were analyzed using an ELISA kit. Proteins in the MAPK and STAT5 signaling pathways were detected by Western blotting. This study showed that different doses of UCMSC-derived extracellular vesicles abolish IgE-stimulated KU812 cell activation and reduce the level of NF-κB, which subsequently leads to cell degranulation and the release of IL-1β, TNF-α and IL-6. Additionally, UCMSC-derived extracellular vesicles treatment blunted the IgE-induced signaling proteins p-P38, p-JNK and p-STAT5. Our results revealed a mechanism for anti-inflammation in which extracellular vesicles can affect the activation of mast cells and thus function in allergy regulation.
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Małys, Małgorzata Sabina, Christof Aigner, Martin Schulz Stefan, Schachner Helga, Jackson Rees Andrew, and Kain Renate. "Isolation of Small Extracellular Vesicles from Human Sera." International Journal of Molecular Sciences 22, no. 9 (2021): 4653. https://doi.org/10.3390/ijms22094653.
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Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exospin ™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.
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Albanese, Joseph, Sarkis Meterissian, Maria Kontogiannea, et al. "Biologically Active Fas Antigen and Its Cognate Ligand Are Expressed on Plasma Membrane-Derived Extracellular Vesicles." Blood 91, no. 10 (1998): 3862–74. http://dx.doi.org/10.1182/blood.v91.10.3862.
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Abstract Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.
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Albanese, Joseph, Sarkis Meterissian, Maria Kontogiannea, et al. "Biologically Active Fas Antigen and Its Cognate Ligand Are Expressed on Plasma Membrane-Derived Extracellular Vesicles." Blood 91, no. 10 (1998): 3862–74. http://dx.doi.org/10.1182/blood.v91.10.3862.3862_3862_3874.
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Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.
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Piffoux, Max, Nabeel Ahmad, Jaysen Nelayah, et al. "Monitoring the dynamics of cell-derived extracellular vesicles at the nanoscale by liquid-cell transmission electron microscopy." Nanoscale 10, no. 3 (2018): 1234–44. http://dx.doi.org/10.1039/c7nr07576f.
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Cordeiro, Luiz, Hsiu-Lien Herbie Lin, Anaïs Vitorino Carvalho, Isabelle Grasseau, Rustem Uzbekov, and Elisabeth Blesbois. "First insights on seminal extracellular vesicles in chickens of contrasted fertility." Reproduction 161, no. 5 (2021): 489–98. http://dx.doi.org/10.1530/rep-20-0462.
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Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.
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Tupitsyna, Anastasiya V., Alina E. Grigorieva, Svetlana E. Soboleva, et al. "Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix)." International Journal of Molecular Sciences 24, no. 16 (2023): 12907. http://dx.doi.org/10.3390/ijms241612907.
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Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30–100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.
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Kononova, I. V., and S. N. Mamaeva. "Comparison of the number of nano-sized particles in blood plasma and on the erythrocytes surface using scanning electron microscopy in a cervical cancer patient." YAKUT MEDICAL JOURNAL 84, no. 4 (2023): 134–36. https://doi.org/10.25789/ymj.2023.84.31.
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To explain the more productive isolation of HPV DNA from the blood component compared to plasma in cervical cancer patients, using scanning electron microscopy images of venous blood were studied. It was revealed that there are more nanosized bioparticles on the erythrocytes surface than in plasma. It has been suggested that among them there may be tumor extracellular vesicles carrying HPV DNA. To confirm that the erythrocyte fraction of blood is a more productive biological sample for isolating HPV DNA, continued studies are needed. Keywords: human papillomavirus, screening, extracellular vesicles.
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Filali, Samira, Nesrine Darragi-Raies, Layth Ben-Trad, et al. "Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions." International Journal of Molecular Sciences 23, no. 21 (2022): 13201. http://dx.doi.org/10.3390/ijms232113201.
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The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This “gel-in” vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a “gel-out” situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.
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Höög, Johanna L., and Jan Lötvall. "Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy." Journal of Extracellular Vesicles 4, no. 1 (2015): 28680. http://dx.doi.org/10.3402/jev.v4.28680.
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Nanou, Afroditi, Mateus Crespo, Penny Flohr, Johann De Bono, and Leon Terstappen. "Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles." Cancers 10, no. 11 (2018): 416. http://dx.doi.org/10.3390/cancers10110416.
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To explore morphological features of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs), we developed a protocol for scanning electron microscopy (SEM) of CTCs and tdEVs. CTCs and tdEVs were isolated by immunomagnetic enrichment based on their Epithelial Cell Adhesion Molecule (EpCAM) expression or by physical separation through 5 μm microsieves from 7.5 mL of blood from Castration-Resistant Prostate Cancer (CRPC) patients. Protocols were optimized using blood samples of healthy donors spiked with PC3 and LNCaP cell lines. CTCs and tdEVs were identified among the enriched cells by fluorescence microscopy. The positions of DNA+, CK+, CD45− CTCs and DNA−, CK+, CD45− tdEVs on the CellSearch cartridges and microsieves were recorded. After gradual dehydration and chemical drying, the regions of interest were imaged by SEM. CellSearch CTCs retained their morphology revealing various shapes, some of which were clearly associated with CTCs undergoing apoptosis. The ferrofluid was clearly distinguishable, shielding major portions of all isolated objects. CTCs and leukocytes on microsieves were clearly visible, but revealed physical damage attributed to the physical forces that cells exhibit while entering one or multiple pores. tdEVs could not be identified on the microsieves as they passed through the pores. Insights on the underlying mechanism of each isolation technique could be obtained. Complete detailed morphological characteristics of CTCs are, however, masked by both techniques.
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Shtam, Tatiana, Anton Emelyanov, Roman Kamyshinsky, et al. "Abstract P-25: Cryo-Electron Microscopy of Extracellular Vesicles from Cerebrospinal Fluid." International Journal of Biomedicine 9, Suppl_1 (2019): S27—S28. http://dx.doi.org/10.21103/ijbm.9.suppl_1.p25.
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Catanese, S., J. Burlaud-Gaillard, H. Blasco, et al. "Rapid identification of extracellular vesicles in basal tears using transmission electron microscopy." Journal Français d'Ophtalmologie 48, no. 4 (2025): 104446. https://doi.org/10.1016/j.jfo.2025.104446.
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Pérez-Doñate, Virginia, Facundo Pérez-Giménez, Lucas Del Castillo Agudo, Juan Alberto Castillo-Garit, Mar Soria-Merino, and Eulogio Valentín Gómez. "Isolation and characterization of extracellular vesicles in Candida albicans." Nereis. Interdisciplinary Ibero-American Journal of Methods, Modelling and Simulation., no. 12 (July 16, 2020): 99–108. http://dx.doi.org/10.46583/nereis_2020.12.611.
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 Background: The occurrence of systemic infections due to C. albicans has increased especially in critically ill patients. In fungal infections, secretory mechanisms are key events for disease establishment. Recent findings demonstrate that fungal organisms release many molecular components to the extracellular space in extracellular vesicles.Aims: We develop a method to obtain exosomes from yeast cultures of the Candida albicans.Methods: Yeast strains used in this work were C. albicans SC5314, C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258). Yeasts were grown at 37.º in liquid YPD medium. The cell cultures were centrifuged and the supernatant filtered through sterile nitrocellulose. Filtrates were concentrated and centrifuged using an ultracentrifuge. The sediment was analyzed by electron microscopy of transmission.Results: The transmission of electron microscopy and nanoparticle tracking analysis confirmed the presence of extracellular vesicles (exosomes) of sizes between 100 and 200 nm and the absence of cellular contaminants. This was ratified by the characterization of proteins performed through the western blot technique, where the absence of cell contamination in the preparations was assessed.Conclusions: The method proves to be highly effective due to the homogeneity and purity of the obtained microvesicles. The protocol developed in this paper proves to be effective for obtaining exosomes of other Candida species, which will allow future studies to determine its protein composition and the role that these vesicles can play.
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Eymieux, Sébastien, Rustem Uzbekov, Yves Rouillé, et al. "Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress." Cells 10, no. 8 (2021): 2047. http://dx.doi.org/10.3390/cells10082047.
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The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.
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Sesaki, H., and S. Ogihara. "Protrusion of cell surface coupled with single exocytotic events of secretion of the slime in Physarum plasmodia." Journal of Cell Science 110, no. 7 (1997): 809–18. http://dx.doi.org/10.1242/jcs.110.7.809.
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Exocytosis has been proposed to participate in the formation of pseudopods. Using video-enhanced microscopy, we directly visualized exocytosis of single vesicles in living Physarum plasmodia migrating on a substrate. Vesicles containing slime, the plasmodial extracellular matrix, of approximately 3.5 microm in diameter, shrank at the cell periphery at the average rate of approximately 1 microm/second, and became invisible. Immediately after exocytotic events, the neighboring cell surface extended to form a protrusion. The rate of extension was approximately 1 microm/second. The protrusion showed lamella-like morphology, and contained actin microfilaments. Electron microscopy suggested that the organization of microfilaments in such protrusions may be a random meshwork rather than straight bundles. These morphologies suggest that protruded regions are pseudopods. Importantly, only the slime-containing vesicle preferentially invaded the hyaline layer that consists of dense actin microfilaments while the other vesicular organelles remained in the granuloplasm. Quantitative analysis demonstrated a linear relationship in terms of their surface area, between individual protrusions and single slime-containing vesicles. It is, therefore, likely that most of the plasma membrane of the protrusion was supplied by fusion of the slime-containing vesicle during exocytosis.
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Furioso Ferreira, Rafaela, Morteza H. Ghaffari, Fabrizio Ceciliani, et al. "Untargeted lipidomics reveals unique lipid signatures of extracellular vesicles from porcine colostrum and milk." PLOS ONE 20, no. 2 (2025): e0313683. https://doi.org/10.1371/journal.pone.0313683.
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Extracellular vesicles (EV) are membranous vesicles considered as significant players in cell-to-cell communication. Milk provides adequate nutrition, transfers immunity, and promotes neonatal development, and milk EV are suggested to play a crucial role in these processes. Milk samples were obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet (ω-6:ω-3 = 13:1) or a test diet enriched in ω-3 (ω-6:ω-3 = 4:1). EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and assessment of EV markers via Western blotting. The lipidome was determined following a liquid chromatography–quadrupole time-of-flight mass spectrometry approach. Here, we show that different stages of lactation (colostrum vs mature milk) have a distinct extracellular vesicle lipidomic profile. The distinct lipid content can be further explored to understand and regulate milk EV functionalities and primordial for enabling their diagnostic and therapeutic potential.
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Qu, Pengxiang, Yuelei Zhao, Rong Wang, et al. "Extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice." Reproduction, Fertility and Development 31, no. 2 (2019): 324. http://dx.doi.org/10.1071/rd18203.
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Embryo transfer (ET) is an important procedure for assisted reproduction. However, the relatively lower success rate of ET hampers its application potential. In this study we aimed to elucidate the effects of extracellular vesicles derived from donor oviduct fluid (EDOF) on embryo development after ET. Extracellular vesicles from the oviduct were isolated and purified using ultracentrifugation and identified using transmission electron microscopy, NanoSight, bicinchoninic acid (BCA) protein assay and western blotting. The results revealed that extracellular vesicles were present in donor oviduct fluid in higher concentrations (P<0.05) and contained more proteins (P<0.05) than extracellular vesicles derived from recipient oviduct fluid (EROF). EDOF or EROF were supplemented in an ET medium (ETM) and the results showed that EDOF significantly improved birth rate via resisting apoptosis and promoting differentiation. In conclusion, our study indicated that there are differences in EDOF and EROF and that supplementing EDOF to ETM can improve the efficiency of ET; improved ET efficiency promotes the use of gene editing and benefits assisted reproductive technology and animal welfare.
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Rodrigues, Marcio L., Leonardo Nimrichter, Debora L. Oliveira, Joshua D. Nosanchuk, and Arturo Casadevall. "Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules?" Lipid Insights 2 (January 2008): LPI.S1000. http://dx.doi.org/10.4137/lpi.s1000.
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Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological significance. Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as ‘virulence bags’, with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to the well-described mammalian exosomes.
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Božič, Darja, Matej Hočevar, Marko Jeran, et al. "Ultrastructure and stability of cellular nanoparticles isolated from Phaeodactylum tricornutum and Dunaliella tertiolecta conditioned media." Open Research Europe 2 (October 20, 2022): 121. http://dx.doi.org/10.12688/openreseurope.14896.1.
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Background: Cells in general secrete nanoparticles (NPs) which are believed to mediate intercellular communication. Recently, great efforts have been made to utilize them as delivery vectors. We aimed to harvest and identify NPs from liquid cultures of two marine microalgae Dunaliella tertiolecta and Phaeodactyum tricornutum. Methods: NPs were isolated from the culture conditioned media by differential ultracentrifugation by the protocol used for the isolation of extracellular vesicles. Microalgae and isolated NPs were examined by scanning electron microscopy (SEM) while isolated NPs were examined also by cryogenic transmission electron microscopy (cryo-TEM). The Triton X-100 detergent and temperature sensitivity of NPs was assessed by dynamic light scattering (DLS) through monitoring the intensity of the scattered light (I) and the distribution of hydrodynamic radii of NPs (Rh). Results: Two mechanisms of formation of NPs with average Rh 200 nm were observed in the D. tertiolecta culture: a disintegration of tubular protrusions, and cell decay. A part of the imaged D. tertiolecta NPs were membrane-enclosed vesicles, but the isolates also contained electron-dense NPs and nanofilaments. P. tricornutum NPs in the culture and in the isolate were homogeneous in size and shape. Their average Rh was 104 nm. The addition of surfactant to isolates resulted in a change in Rh distribution and a decrease of I in samples from both species, indicating decay of a part of NPs. Changes in the width of the I(Rh) peaks were observed at temperatures above 45 °C. Conclusions: A part of NPs found in isolates from microalgae D. tertiolecta and P. tricornutum were membrane-enclosed vesicles. However, the isolates obtained by a standard protocol for extracellular vesicle isolation by ultracentrifugation contained also a significant amount of other similar-sized nanoparticles. The isolates were partly susceptible to the addition of detergent and to temperature up to 80 degrees.
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Wang, Zuopei, Menghan He, Han He, et al. "Nonenveloped Avian Reoviruses Released with Small Extracellular Vesicles Are Highly Infectious." Viruses 15, no. 7 (2023): 1610. http://dx.doi.org/10.3390/v15071610.
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Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.
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Usui, Kentaro, Haruki Yamamoto, Takao Oi, Mitsutaka Taniguchi, Hitoshi Mori, and Yuichi Fujita. "Extracellular Vesicle-Mediated Secretion of Protochlorophyllide in the Cyanobacterium Leptolyngbya boryana." Plants 11, no. 7 (2022): 910. http://dx.doi.org/10.3390/plants11070910.
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Protochlorophyllide (Pchlide) reduction in the late stage of chlorophyll a (Chl) biosynthesis is catalyzed by two enzymes: light-dependent Pchlide oxidoreductase (LPOR) and dark-operative Pchlide oxidoreductase (DPOR). The differential operation of LPOR and DPOR enables a stable supply of Chl in response to changes in light conditions and environmental oxygen levels. When a DPOR-deficient mutant (YFC2) of the cyanobacterium Leptolyngbya boryana is grown heterotrophically in the dark, Pchlide accumulates in the cells and is secreted into the culture medium. In this study, we demonstrated the extracellular vesicle-mediated secretion of Pchlide. Pchlide fractions were isolated from the culture medium using sucrose density gradient centrifugation. Mass spectrometry analysis revealed that the Pchlide fractions contained porin isoforms, TolC, and FG-GAP repeat-containing protein, which are localized in the outer membrane. Transmission electron microscopy revealed extracellular vesicle-like structures in the vicinity of YFC2 cells and the Pchlide fractions. These findings suggested that the Pchlide secretion is mediated by extracellular vesicles in dark-grown YFC2 cells.
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Miroshnikova, Valentina V., Kseniya V. Dracheva, Roman A. Kamyshinsky, et al. "Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus." PLOS ONE 18, no. 2 (2023): e0279652. http://dx.doi.org/10.1371/journal.pone.0279652.
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Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.
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Sohal, Ikjot Singh, Sydney N. Shaw, and Andrea L. Kasinski. "Abstract 322: Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer." Cancer Research 83, no. 7_Supplement (2023): 322. http://dx.doi.org/10.1158/1538-7445.am2023-322.
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Abstract Extracellular vesicles (EVs) act as key mediators of cell-to-cell communication in several pathophysiological processes including cancer. EVs inherit a cocktail of molecular cues (mRNAs, miRNAs, ncRNAs, proteins, metabolites, etc.) from cancer cells that mediate tumor microenvironment changes to facilitate cancer progression and metastasis. The heterogenous EV populations and their content dynamically change during cancer, which is why, there has been great progress in utilizing them as prognostic and diagnostic biomarkers. However, harnessing EVs for therapeutic applications requires greater understanding of the fundamental EV biology such as their origin, intracellular trafficking, cargo loading, and export mechanisms. Here, we utilized a genetically-encoded electron dense tag, APEX2 (an engineered peroxidase derived from soybean ascorbate peroxidase) and electron microscopy to elucidate that EVs, in the form of multivesicular bodies, can also originate at the trans-Golgi interface. We also identified a novel subpopulation of EVs, which expressed a classic trans-Golgi network protein (referred as TGNP) on their surface. While EVs of late endosomal origin are well known, and there are reports of mitochondrial origin as well, a trans-Golgi EV biogenesis pathway has not been reported before. Intriguingly, we determined that certain cells hijack the trans-Golgi pathway for EV biogenesis, which we hypothesize contributes to differential cancer cell EV cargo and phenotypic consequences. Initially, using confocal microscopy in non-small cell lung cancer (NSCLC) cells, we identified that some of the tetraspanins historically thought to be involved in late endosomal trafficking were instead enriched in the trans-Golgi compartments. We found that this colocalization pattern was conserved in several other NSCLC cell lines. To further elucidate this phenomenon at the trans-Golgi interface, we tagged TGNP to APEX2. After verifying the expression and localization of TGNP-APEX2 by immunostaining, we performed electron microscopy. TGNP-APEX2 was found to be present on the membrane of intraluminal vesicles inside multivesicular bodies. Interestingly, the multivesicular bodies containing TGNP-APEX2 vesicles were not of late endosome origin. Furthermore, TGNP was also found to be present in EVs of all NSCLC cell lines tested. Inhibiting Golgi trafficking using small molecule inhibitors (Brefeldin A) significantly reduced EV secretion and levels of trans-Golgi-enriched tetraspanins in the EV lysates. The data elucidates that EVs can also originate from trans-Golgi interface and identified novel subpopulation that expresses TGNP. The broader questions regarding the functional relevance of trans-Golgi-mediated EV biogenesis, the type of cargo exported, and mediators of loading and export in the context of cancer are currently under investigation. Citation Format: Ikjot Singh Sohal, Sydney N. Shaw, Andrea L. Kasinski. Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 322.
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Pulikkathodi, Anil, Indu Sarangadharan, Chiao-Yun Lo, Po-Hsuan Chen, Chih-Chen Chen, and Yu-Lin Wang. "Miniaturized Biomedical Sensors for Enumeration of Extracellular Vesicles." International Journal of Molecular Sciences 19, no. 8 (2018): 2213. http://dx.doi.org/10.3390/ijms19082213.
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In this research, we have realized a rapid extracellular vesicle (EV) quantification methodology using a high field modulated AlGaN/GaN high electron mobility (HEMT) biosensor. The unique sensing structure facilitated the detection of the sub-cellular components in physiological salt environment without requiring extensive sample pre-treatments. The high field operation of GaN HEMT biosensor provides high sensitivity and wide dynamic range of detection of EVs (107–1010 EVs/mL). An antibody specific to the known surface marker on the EV was used to capture them for quantification using an HEMT biosensor. Fluorescence microscopy images confirm the successful capture of EVs from the test solution. The present method can detect EVs in high ionic strength solution, with a short sample incubation period of 5 min, and does not require labels or additional reagents or wash/block steps. This methodology has the potential to be used in clinical applications for rapid EV quantification from blood or serum for the development of diagnostic and prognostic tools.
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Georgieva, Milena, Bela Vasileva, Penyo Ivanov, et al. "Cosmetic Potential of Haberlea rhodopensis Extracts and Extracellular Vesicles in Human Fibroblast Cells." Cosmetics 12, no. 3 (2025): 90. https://doi.org/10.3390/cosmetics12030090.
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Skin ageing is a complex biological process influenced by cellular senescence, oxidative stress, and extracellular matrix degradation. Emerging evidence suggests that plant-derived bioactive compounds and extracellular vesicles (EVs) play a crucial role in modulating cellular homeostasis, promoting tissue regeneration, and counteracting age-related morphological and functional changes. This study investigates the impact of Haberlea rhodopensis in vitro culture extracts, native and enriched with EVs, on key cellular processes, including morphology, mitochondrial dynamics, lysosomal activity, gene expression, and genotoxicity in human dermal fibroblasts. The extracellular vesicles were identified in terms of shape, size, and morphology using dynamic light scattering, negative staining and observation under a transmission electron microscope. A comprehensive in vitro analysis was conducted utilizing light microscopy to assess cellular morphology and lysosomal mass, fluorescence microscopy for actin cytoskeletal organization, mitochondrial integrity, and nuclear morphology, and gene expression profiling for markers associated with collagen synthesis (COL1A1, COL3A1), senescence (CDKN1A), and oxidative stress response (NFE2L2). Additionally, cell cycle progression was evaluated, and genotoxicity was assessed using the neutral comet assay. Haberlea rhodopensis in vitro culture extracts and EVs were found to preserve fibroblast morphology, enhance mitochondrial mass, and upregulate collagen-related gene expression. These effects were concentration-dependent. The extracts exhibited biocompatibility with minimal genotoxic effects, indicating their potential as safe bioactive agents for skin rejuvenation. The findings suggest that Haberlea rhodopensis in vitro culture extracts and their enrichment with extracellular vesicles hold promise for cosmetic and dermatological applications, particularly in enhancing collagen production, preserving cellular integrity, and mitigating age-related alterations in skin fibroblasts. Further studies are warranted to elucidate the underlying molecular mechanisms and optimize formulation strategies for clinical translation.
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Nizamudeen, Zubair Ahmed, Rachael Xerri, Christopher Parmenter, et al. "Low-Power Sonication Can Alter Extracellular Vesicle Size and Properties." Cells 10, no. 9 (2021): 2413. http://dx.doi.org/10.3390/cells10092413.
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Low-power sonication is widely used to disaggregate extracellular vesicles (EVs) after isolation, however, the effects of sonication on EV samples beyond dispersion are unclear. The present study analysed the characteristics of EVs collected from mesenchymal stem cells (MSCs) after sonication, using a combination of transmission electron microscopy, direct stochastic optical reconstruction microscopy, and flow cytometry techniques. Results showed that beyond the intended disaggregation effect, sonication using the lowest power setting available was enough to alter the size distribution, membrane integrity, and uptake of EVs in cultured cells. These results point to the need for a more systematic analysis of sonication procedures to improve reproducibility in EV-based cellular experiments.
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Kamińska, Kinga, Kasun Godakumara, Bianka Świderska, et al. "Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles." Theriogenology 205 (July 15, 2023): 79–86. https://doi.org/10.1016/j.theriogenology.2023.04.010.
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Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.
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Małys, Małgorzata S., Christof Aigner, Stefan M. Schulz, Helga Schachner, Andrew J. Rees, and Renate Kain. "Isolation of Small Extracellular Vesicles from Human Sera." International Journal of Molecular Sciences 22, no. 9 (2021): 4653. http://dx.doi.org/10.3390/ijms22094653.
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Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.
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Taylor, Sara K., Sahar Houshdaran, Joshua F. Robinson та ін. "Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα". Development 147, № 17 (2020): dev187013. http://dx.doi.org/10.1242/dev.187013.
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ABSTRACTThe placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000 g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000 g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000 g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.
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Quinelato, Valquiria, Carlos Fernando Mourão, Thalita Alves Barreto Santos, et al. "Protocols for Extraction of miRNA from Extracellular Vesicles of Lyophilized Human Saliva Samples." International Journal of Molecular Sciences 26, no. 7 (2025): 2891. https://doi.org/10.3390/ijms26072891.
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Extracellular vesicles (EVs) are emerging as crucial biomarkers in molecular diagnostics, providing early detection of disease progression. Although ultracentrifugation remains the gold standard for vesicle isolation from biofluids, it has limitations in scalability and accessibility. This study presents lyophilization as an innovative method for preserving EVs and isolating microRNAs from saliva, utilizing its proven ability to maintain biological activity and prevent unwanted chemical reactions. We assessed five different sample preparation protocols combined with a dual-purification strategy. Structural and molecular integrity analyses revealed that lyophilized samples retained essential EV characteristics, including CD63/CD9 membrane localization. QELS analysis and electron microscopy confirmed distinct vesicle populations in both ultracentrifuged (30–50 nm and 320–360 nm) and lyophilized samples (50–70 nm and 360–380 nm). Importantly, lyophilized samples exhibited higher total RNA concentrations (p < 0.0001) while preserving key microRNA signatures (miR-16, miR-21, miR-33a, and miR-146b) with high fidelity. The efficacy of lyophilization is linked to its ability to systematically reduce solvent content through sublimation while maintaining vesicle integrity and molecular cargo. This method offers a practical, scalable alternative for EV isolation with significant implications for biomarker-based diagnostics.
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Cole, Eric S., Thomas H. Giddings, Courtney Ozzello, et al. "Membrane Dynamics at the Nuclear Exchange Junction during Early Mating (One to Four Hours) in the Ciliate Tetrahymena thermophila." Eukaryotic Cell 14, no. 2 (2014): 116–27. http://dx.doi.org/10.1128/ec.00164-14.
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ABSTRACT Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila . Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.
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Ben-Trad, Layth, Constantin Ionut Matei, Mirela Maria Sava, et al. "Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances." International Journal of Molecular Sciences 23, no. 19 (2022): 11998. http://dx.doi.org/10.3390/ijms231911998.
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The quality of the lubricant between cartilaginous joint surfaces impacts the joint’s mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.
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Noble, Jade M., LaDeidra Monét Roberts, Netta Vidavsky, et al. "Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles." Journal of Structural Biology 210, no. 1 (2020): 107474. http://dx.doi.org/10.1016/j.jsb.2020.107474.
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