Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).

/pdf/exosome-mediated-transfer-of-mrnas-and-micrornas-is-a-novel-9o52kfcb0e.pdf

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.

https://rupress.org/jcb/article-pdf/200/4/373/1262520/jcb_201211138.pdf

Extracellular vesicles: exosomes, microvesicles, and friends.

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

/pdf/isolation-and-characterization-of-exosomes-from-cell-culture-4k118gm22m.pdf

Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

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Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

/pdf/biological-properties-of-extracellular-vesicles-and-their-bef7axtqvb.pdf

Biological properties of extracellular vesicles and their physiological functions

Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.

Metabolism and intracellular transport of glycosphingolipids.

The uptake of ganglioside analogues by a per- manent mouse fibroblast cell line has been studied by radio- tracer techniques and ESR spectroscopy with 3H- and ni- troxide-labeled compounds. Analogues of GMl, GMz, and GM~ monosialogangliosides and of GDla and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the gang- lioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature Gangliosides are characteristic constituents of the outer surface of animal cells, in which they are located with their ceramide portion embedded in the lipid layer and their sia- looligosaccharide residue facing the extracellular environment. Gangliosides of mammalian cells have long been assumed to be involved in cell-cell recognition and cell-cell adhesion, as well as in cell contact inhibition of growth and possibly tu- morigenesis (for review see Hakomori (198 l)). In addition, cell surface gangliosides interact with hormones, lectins, vi- ruses, interferon, and bacterial toxins (for review see Fishman & Brady (1976)l. One approach to studying the role of gangliosides in these crucial cellular processes is to investigate the uptake of ex- ogenously added gangliosides. Thus, in the study of (a) the regulation of ganglioside metabolism and ganglioside-metab- olizing enzymes, (b) the intracellular transport of gangliosides and their metabolites, and (c) the functional effects of ex- ogenous gangliosides on cultured cells, the incorporation of a defined labeled ganglioside into the outer leaflet of the plasma membrane can be of considerable importance. A critical question in such studies is whether the exogenous ganglioside is truly inserted into the membrane, rather than being simply adsorbed to its surface or alternatively trapped within membrane vesicles. A related question is whether the incorporated ganglioside is uniformly distributed throughout the membrane, as opposed to being incorporated in undispersed patches. gangliotriaitol/-monosialolactitol; ~~-G~~~~/-G~~~~/ -G~~I~/-GD~~~~/-

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study.

The synthesis of gangliosides GM3, GM2, GM1, and GD1a solely lacking the fatty acid moiety, and thus called lysogangliosides in analogy to lysophospholipids, is described. Since a selective elimination of the fatty acid residue has not been achieved as yet, the gangliosides were first subjected to alkaline hydrolysis. By this procedure the fatty acyl as well as the acetyl groups of the sialic acid residue(s) were completely removed. The acetamido group of the N-acetylgalactosamine moiety of the gangliosides GM2, GM1, and GD1a was very little (congruent to 10%) hydrolyzed. In a two-phase system composed of water and ether, the selective protection of the sphingoid amino group was accomplished with a hydrophobic protective group (9-fluorenylmethoxycarbonyl). Lysogangliosides were obtained after re-N-acetylation of the sialooligosaccharide amino group(s) followed by removal of the protecting group. The overall yield was about 30%. The structures of the lysogangliosides were confirmed by chemical analysis as well as negative ion FAB mass spectrometry and 1H NMR spectroscopy. By simple re-N-acylation of lysogangliosides with any labeled fatty acid, labeled gangliosides are now obtainable that are identical with their parent gangliosides except for their labeled fatty acid residue. This has been demonstrated by the synthesis of GM1 with a [1-13C]palmitic acid moiety in its ceramide portion. If desired, double-labeled gangliosides may be obtained by use of labeled acetic anhydride in the synthesis of the lysogangliosides.

Guenter Schwarzmann

Papers

Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.

Metabolism and intracellular transport of glycosphingolipids.

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study.

Synthesis of lysogangliosides.

Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion.