Identification and characterization of cell-bound membrane vesicles.

In contrast to the released/circulating membrane vesicles (extracellular vesicles), cell-bound membrane vesicles are poorly identified and characterized. In this study, cell-bound membrane vesicles on human umbilical vein endothelial cells (HUVECs) and human hepatoma HepG-2 cells were investigated. We identified that cell-bound membrane vesicles are not co-localized with the major markers for extracellular vesicles (e.g. phosphatidylserine, CD63, CD107α, CD31, and DNA fragments for the three well-known types of extracellular vesicles) and for intracellular organelles with similar sizes (e.g. MitoTracker and LAMP1/LAMP3 for mitochondria and multivesicular bodies or lysosomes, respectively). The data imply that cell-bound membrane vesicles are neither the precursors of extracellular vesicles nor a false structure pushed up by an intracellular organelle but probably a novel unknown structure in the plasma membrane. Moreover, we revealed that cell-bound membrane vesicles are resistant to various detergents including but probably not limited to Triton X-100, SDS, and saponin. We further characterized that these unique vesicles are soluble in organic solvents (e.g. chloroform-methanol mixture and ethanol) which can be prevented by a lipid-stabilizing fixative (e.g. OsO4) and that they are co-localized with, but do not monopolize, the major markers (e.g. caveolin-1 and GM1) for lipid rafts (a nano-sized detergent-resistant domains in the plasma membrane). The data imply that cell-bound membrane vesicles contain the lipid component and lipid rafts. Involvement of other specific unknown components might explain the detergent resistance of cell-bound membrane vesicles. Further research will mainly depend on the establishment of an effective approach for isolation/purification of these vesicles from the plasma membrane.