Table 2.

Survey questions regarding EV biogenesis.

EV Biogenesis Survey Questions
Figure 1	Budding into the multivesicular body (MVB) as intraluminal vesicles (ILVs) and budding from the plasma membrane are the two major EV biogenesis pathways, with at least partially independent molecular machinery of biogenesis.
	It is possible that what we refer to as MVBs are actually physical extensions of the plasma membrane and not late endosomes.
	It is currently possible to distinguish, using protein, lipid, or other markers, an “exosome” (former ILV in the MVB) from a “microvesicle” (from the plasma membrane) after the respective vesicle has left the cell.
	Size can be used to separate EVs by biogenesis pathway.
	EVs from the endosomal system are smaller, on average, than EVs that bud from the plasma membrane.
	We know the basic size distribution of EVs from biofluids and cell culture.
Figure 2	Excluding apoptotic bodies and other “macrovesicles”, the average diameter of most EV populations is:	Significantly smaller than 100 nm
		Roughly in the 100–150 nm range
		Significantly larger than 150 nm
Figure 3	Asymmetric distribution of lipids (inner, outer leaflet) is the same in EVs as in the cell membrane of origin and remains stable over time.
	The inner and outer sides of the EV membrane are revealed by inner and outer domains of proteins in the expected orientation relative to the cell. That is, the cellular membrane topology is maintained by EVs.
Figure 4	The weight of the evidence supports preferential packaging of certain miRNAs or other RNA cargo into specific subsets of EVs.
	The RNA Cargo of larger EVs correlates with cellular expression, but that of small EVs does not.
Figure 5	It is possible to create cells or organisms that do not produce EVs.
	EV biogenesis is essential for life, as evidenced by lethality of TSG101 knockouts and knockouts of multiple biogenesis-linked proteins.
Figure 6	Lipid-raft domains (endosome-like domains, rich in cholesterol, etc.) play a role in EV biogenesis; without them, many EVs would not form.
	nSMase2 is not involved in biogenesis of all EV subtypes in all cells, hence discrepant results of nSMase2 blocking.
	Energetic requirements of EV biogenesis are largely unknown.