Exosomes and Other Microvesicles
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Exosomes are microvesicles shed by a wide range of living cells.
Figure 1: Exosome biogenesis begins with formation of intraluminal vesicles (ILVs) through inward budding of endosomes (B) to form multivesicular bodies (MVBs).
Figure 2: The MVBs (C) fuse with the outer cell membrane to release their cargo of ILVs (now exosomes) to the extracellular environment (D).
Exosomes are one of many different sub-populations of microvesicles that can be isolated from biofluids such as blood, urine and cerebrospinal fluid (CSF) and from which high quality RNA and DNA can be extracted and purified for analysis. Exosomes are shed by cells under both normal and pathological conditions. Although the nomenclature and terminology of the field are still developing, there is a growing consensus that exosomes are characterized in their biogenesis by formation of intraluminal vesicles (ILVs) through the inward budding of endosomes to form multivesicular bodies (MVBs). These MVBs then fuse with the outer cell membrane to release their cargo of ILVs (now exosomes) to the extracellular environment. The endosome is first formed by inward budding of the cell membrane by endocytosis and leads to inversion of the lipid membrane, trapping some of the extracellular environment on the intraluminal side. Similarly, the second inward budding of the endosome membrane traps a volume of the cell's cytoplasm and results in a positive orientation of the ILVs lipid membrane. When the ILVs (now exosomes) are released to the extracellular environment, they have the same orientation as the cell membrane and have been shown to display many of the surface markers from their cell of origin. However, the sorting process of membrane proteins during ILV formation is an active process and thus, exosomal surface proteins are not a simple one-to-one representation of the surface markers from the cell of origin.
Figure 3: Other pathways also play a role in formation of microvesicles, including direct budding of the cellular membrane (A' -> B').
It is clear that exosomes (or at least "microvesicles of endocytic origin") make an important contribution to the overall population of circulating microvesicles. However, other pathways also play significant roles in the formation of microvesicles, including direct budding of the cellular membrane. Both processes (through MVBs or through direct budding) lead to an encapsulation of cytoplasm volume from the cell of origin, including any nucleic acid present in the cytoplasm. The exact mechanism of how nucleic acid is packaged into microvesicles is still being investigated, but there is evidence to suggest that this takes place both by simple inclusion of molecules in the engulfed cytoplasm and by active packaging of certain nucleic acid species, possibly as a means of cell-to-cell communication. When the microvesicles leave the cell of origin, some will enter the blood stream, the urine, cerebrospinal fluid, or other bodily fluids where they can be harvested, extracted and the nucleic acid contents isolated for analytical and molecular diagnostic applications.
For the purpose of molecular diagnostic applications, the optimal microvesicle subpopulation might vary depending on the indication and the biomarker in question. For more advanced applications, we have found it necessary to modify the microvesicle isolation and/or the nucleic acid extraction procedure to increase enrichment for individual biomarkers.
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