Ectosome biogenesis and release processes observed by using live-cell dynamic imaging in mammalian glial cells

Background: Ectosomes are recognized as shedding from the plasma membranes into the extracellular environment. Recent research has demonstrated that ectosomes are surrounded by phospholipid membranes containing lipid rafts and caveolae. Some ectosomes contain cytokines in the lumen and have high levels of phosphatidylserine exposed to the outer membrane. Intracellular vesicles share both characters with ectosomes. Why the plasma membrane-derived ectosomes have the same characteristics as intracellular vesicles remain largely unknown. Methods: Using live-cell dynamic imaging, we recorded the process of ectosome biogenesis and release in primary cultured neural cells. Results: Our results show two different ectosome release methods: slow-releasing and fast-releasing. In the slow-releasing, multiple ectosomes emerge almost simultaneously on the cell surface and are released by outward budding from the plasma membrane. In the fast releasing, ectosomes squeeze out of the membrane domain and pinch off from a cell's surface. Using ER-tracker for live-cell imaging, we directly observed the process that intracellular vesicles jump out of the plasma membrane for release. This type of ectosomes has a reverse array of membrane proteins and phospholipids compared to the plasma membrane. So ectosomes should be divided into two groups: plasma membrane-derived and intracellular membrane-derived ectosomes. Conclusions: Both slow releasing and fast releasing EVs imply mechanisms of human diseases and for diagnostics and drug delivery.