Willem Stoorvogel

Extracellular vesicles: Exosomes, microvesicles, and friends

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.

Biological properties of extracellular vesicles and their physiological functions.

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.

Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes

Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50–60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.

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

Exosomes are 60–100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin α4, CD45, moesin, tubulin (α and β), actin, Giα2, and a multitude of other proteins. An α4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergent-resistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.

The biogenesis and functions of exosomes.

Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. They correspond to the internal vesicles of an endosomal compartment, the multivesicular body and are released upon exocytic fusion of this organelle with the plasma membrane. Intracellularly, they are formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. The unique composition of exosomes may confer specific functions on them upon secretion. Although their physiological role in vivo is far from being unraveled, it is apparent that they function in a multitude of processes, including intercellular communication during the immune response. Exosomes may have evolved early in the evolution of multicellular organisms and also seem to be important for tissue developmental processes.

Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.

Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used array analysis to establish the presence of microRNAs and mRNA in cell-derived vesicles from many sources. Here, we used an unbiased approach by deep sequencing of small RNA released by immune cells. We found a large variety of small non-coding RNA species representing pervasive transcripts or RNA cleavage products overlapping with protein coding regions, repeat sequences or structural RNAs. Many of these RNAs were enriched relative to cellular RNA, indicating that cells destine specific RNAs for extracellular release. Among the most abundant small RNAs in shuttle RNA were sequences derived from vault RNA, Y-RNA and specific tRNAs. Many of the highly abundant small non-coding transcripts in shuttle RNA are evolutionary well-conserved and have previously been associated to gene regulatory functions. These findings allude to a wider range of biological effects that could be mediated by shuttle RNA than previously expected. Moreover, the data present leads for unraveling how cells modify the function of other cells via transfer of specific non-coding RNA species.

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.

Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.

We provide a protocol for a high-resolution flow cytometry–based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of ∼100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2–4 h by an experienced flow cytometer operator.

Activated T-cells recruit exosomes secreted by dendritic cells via LFA-1

Dendritic cells (DCs) are known to secrete exosomes that transfer membrane proteins, like major histocompatibility complex class II, to other DCs. Intercellular transfer of membrane proteins is also observed during cognate interactions between DCs and CD4(+) T cells. The acquired proteins are functional and play a role in regulation of immune responses. How membrane protein transfer is achieved and regulated is unclear. Here we show that T cells can recruit major histocompatibility complex class II-containing DC exosomes secreted in the extracellular milieu during cognate DC-T-cell interactions. Recruitment of these exosomes required T-cell activation and was dependent on leukocyte function-associated antigen-1 (LFA-1) rather than on T-cell receptor specificity. Indeed, inducing a high-affinity state of LFA-1 on resting T cells was sufficient to provoke exosome binding. These results imply that DC exosomes secreted in the extracellular milieu during cognate T-cell-DC interactions are targeted to T cells activated in that microenvironment.