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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170003	1/1	Mus musculus	mIMCD3	(d)(U)C	Nager AR	2017	44%
Study summary Full title An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling. All authors Nager AR, Goldstein JS, Herranz-Pérez V, Portran D, Ye F, Garcia-Verdugo JM, Nachury MV Journal Cell Abstract Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. H (show more...)Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts. (hide) EV-METRIC 44% (84th 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 GPCR signaling in BBS mutant Focus vesicles Ciliary Ectosome 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 Adj. k-factor 253.9 (pelleting) / 99.86 (washing) Protein markers EV: CD81/ Arl13B/ BiotinylatedGPCRs non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells mIMCD3 EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type TLS-55 Wash: speed (g) 100000 Wash: adjusted k-factor 99.86 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81, Arl13B Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Immune-EM EM protein Biotinylated GPCRs Image type Close-up, Wide-field Report size (nm) 70-120

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EV-TRACK ID	EV170003
species	Mus musculus
sample type	Cell culture
cell type	mIMCD3
condition	GPCR signaling in BBS mutant
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	44