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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140051	1/1	Cryptococcus neoformans	NAY	(d)(U)C Filtration UF	Wolf JM	2014	43%
Study summary Full title Interaction of Cryptococcus neoformans extracellular vesicles with the cell wall All authors Wolf JM, Espadas-Moreno J, Luque-Garcia JL, Casadevall A Journal Eukaryot Cell Abstract Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including vir (show more...)Cryptococcus neoformans produces 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. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: Urease/ Phosphatase/ Laccase non-EV: Proteomics yes Show all info Study aim Biogenesis/Sorting Sample Species Cryptococcus neoformans Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Urease/ Phosphatase/ Laccase ELISA Antibody details provided? No Detected EV-associated proteins Urease/ Phosphatase/ Laccase Characterization: Particle analysis DLS EM EM-type transmission EM/ cryo EM/ scanning EM Image type Close-up, Wide-field Report size (nm) Not reported

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EV-TRACK ID	EV140051
species	Cryptococcus neoformans
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	43