How are EVs formed?

Last Update: August 2016


Eukaryotic Cell Membrane Structure and Regulation

Biological membranes are comprised of lipids and proteins, with carbohydrates linked to some of these. Membranes tend to have a thickness of 6-10nm, equivalent to two phospholipid molecules, forming a lipid bilayer. Lipids are structurally diverse, generally hydrophobic, and have three general functions: to store energy, to act as 1st or 2nd messengers, and to form cellular membranes from polar lipids28.

Amphipathic lipids form the lipid bilayer, which has an asymmetric distribution of both proteins and phospholipids. This asymmetric distribution is maintained by multiple factors, including biophysical properties, retentive mechanisms trapping lipids in a specific leaflet, and transporters aiding lipid translocation28. Structural integral lipids in eukaryotic cells are glycerophospholipids, which include phosphatidylcholine (PC), phosphatidylethanolamine (PE), PS, phosphatidylinositol (PI) and phosphatidic acid (PA) (28). The relative quantities of these phospholipids appear to differ between lipoproteins, cell types, and EVs. PC, however, makes up the majority of membrane lipid in all cells and EVs tested. The inclusion of PE within a PC bilayer asserts a curvature stress upon the membrane due to its shape, which is thought to be utilized in budding, fission and fusion (29).

The cell plasma membrane is enriched with sphingolipids and sterols. Sphingolipids contain a hydrophobic ceramide backbone, with the predominant mammalian sphingolipids being sphingomyelin (SM) and glycosphingolipids. Sterols are nonpolar lipids and are predominantly in the form of cholesterol in mammalian cells (28). This high density of sphingolipids and sterols, in comparison to glycerolipids, makes the cell membrane resistant to curvature stress. The lipidome of EVs has shown them to be more concentrated in SM than their cells of derivation, though lipidome studies in EVs do appear to yield varying results based on their isolation technique.

The asymmetric distribution of lipids is also a contributor to curvature stress, with PC, SM and sterols on the outer leaflet, while PS and PE are predominantly on the inner leaflet of inert cells. This distribution also plays a functional role. When membrane dysregulation occurs, causing a loss of phospholipid asymmetry and PS externalization, phagocytotic signalling, and a catalyst for thrombin generation is mediated. Transporters involved in maintaining lipid asymmetry include flippases, floppases and scramblases. Flippases are responsible for the ‘flipping’ of PS and PE from the exterior surface to the interior surface, whilst floppases are responsible for the opposite. Scramblases are capable of bi-directional trans-bilayer transport and can be activated through Ca2+ currents. It is also thought that some scramblases are activated through apoptosis.

Although steps have been made to elucidate the mechanisms in the biogenesis of EVs, much remains to be explained. This is predominantly due to detection and isolation methodology currently requiring development for smaller EVs.

Apoptotic Vesicle Formation

Apoptotic vesicles can be formed via a number of cell death pathways. Cellular death is generally discussed dichotomously, in terms of apoptosis or necrosis, with apoptosis being an active, programmed process, whilst necrosis has been presumed as passive, accidental cell death as a result of environmental factors. Understanding of the process of cell death has become increasingly complicated, however, and is not a dichotomous divide as has previously been assumed.

Shown are the main pathways that lead to apoptosis, these include the extrinsic, intrinsic and perforin/granzyme pathway. Each pathway has its own initiator, caspase-8/9/10, in turn activating caspase-3 leading to cytomorphological changes and apoptotic body formation.

Apoptosis is induced by three pathways, the extrinsic, intrinsic, and perforin/granzyme pathway. Each pathway ends in the activation of caspase-3 leading to cytomorphological changes such as cytoskeletal reorganisation, chromatin and cytoplasmic condensation, nuclear fragmentation, and more. This then results in the cell blebbing, forming apoptotic vesicles (For further details on pathway associated proteins see reviews (30-33).

Contrary to the passive, accidental cell death it was perceived to be, in recent years necrosis has been shown to be caused by a number of regulated pathways, some of which overlap with apoptosis. Some of these regulated modes include PARP1 hyperactivation, mitochondrial complex 1, Cys/Glu antiporter, necrosome, NADPH oxidases, inflammasome (34).

Other forms of cell death also exist; oncosis is a form of cell death accompanied by cellular and organelle swelling, along with membrane blebbing and membrane permeability. Along with oncosis, pyroptosis is another form of cell death that has been characterised and is induced by infection32.

The crucial message is that large vesicles, resulting from regulated cell death, are not solely derived from apoptosis but rather from cell death as a whole. The difference between these vesicle characteristics is not something that has been investigated, and those vesicles that are in the circulation are likely to depend upon the circumstance of the individual cells releasing vesicles. This, therefore, could be important when characterising these types of vesicles in translational studies. Here, the different large vesicles resulting from cellular death are collectively referred to as AVs.

Exosome Formation

Exosomes are formed within multivesicular bodies (MVBs). MVBs, however, are prone to fusion with lysosomes, within the lysosomal pathway, causing degradation of MVBs and their contents. There appears to be a biochemical and morphological distinction between MVBs who will undergo degradation and those secreted from cells. The composition of exosomes from studies to date show them to be formed of proteins from the endosome, plasma membrane and cytosolic origin. This suggests that exosomes do not contain just random proteins, but infers that they represent a specific compartment of the cell10. Databases have since been created to compile the published data on the exosome proteome from different origins (35, 36). Exosome formation within the MVB has been extensively studied at a molecular level, particularly in yeast (37). The endosomal sorting complex required for transport (ESCRT) machinery is very important in the formation of exosomes within the MVB. The ESCRT proteins essentially allow invagination of the membrane in the opposite direction to the cytosol. ESCRT-0 is important for ubiquitin-dependent cargo clustering, with ESCRT-I (TSG101) and ESCRT-II inducing bud formation, with vesicle scission being driven by ESCRT-III. Accessory proteins also have roles to play, with VPS4 ATPase allowing dissociation and recycling of the ESCRT machinery itself. Further details of the role of each protein’s role in exosome biogenesis can be found in reviews (38-41). ESCRT independent mechanisms of exosome formation have also been proposed. Mammalian cells depleted of key ESCRT machinery components are still capable of producing MVBs (42). These involve tetraspanins, lipids and heat shock proteins.

The illustration depicts the simplified biogenesis of exosomes from endocytosis, to early endosome formation, multivesicular body formation, to exosome, as well as RLV release, and microvesicle release through budding directly from the plasma membrane.

Once formed, MVBs fuse with the plasma membrane allowing the release of their exosomal contents. The trafficking and fusion of the MVB with the plasma membrane is regulated by the RAB family of small GTPases proteins. The RAB family of proteins appear to have major intracellular 9

localizations according to their role. The RAB2B protein is localised to the endoplasmic reticulum and Golgi apparatus, while RAB5A, RAB7, RAB9A, RAB11, RAB27A, RAB27B and RAB35 are localised to the endosome depending on its stage within the pathway (43-47).

Microvesicle Formation

MVs are formed upon cellular activation, death and even at rest, with diameters considered to vary from 20nm to above 1000nm. The majority of vesicles, however, are in the bottom half of this range (48). MVs are the result of plasma membrane vesiculation and were originally identified on the basis of phosphatidylserine (PS) exposure, due to dysregulation of the plasma membrane. A wide range of stimuli can induce MV vesiculation.

An initial clue to platelet MV formation came from the rare haemorrhagic disorder, Scott syndrome49. This syndrome is associated with a genetic mutation in a scramblase, transmembrane protein 16F (TMEM16F), also known as anoctamin-6. As a result of this mutation, platelets exposed to Ca2+ ionophore are unable to expose PS to their outer-membrane and are also thought not to shed MVs (50, 51). The inability to expose PS to outer-membrane is also true of erythrocytes, T-cells and B-cells derived from individuals with Scott Syndrome. These cells, however, all appear to display normal PS exposure and lipid scrambling rates when given an apoptotic trigger (52, 53). It has since been confirmed that TMEM16F can be activated both via Ca2+ currents and apoptotic signals (53).

The exact mechanism of MV formation is unknown. The majority of agonists known to result in MV formation produce a Ca2+ current or flux, with many research groups inducing MV formation using Ca2+ ionophores such as A23187 or ionomycin (54-58). Ca2+ activated K+ channels also aid MV release, likely providing a decreased cell volume which allows the reduction in the surface area required for MV release; this has previously been observed in apoptotic cells (59, 60). Further clues into MV budding have come from the study of the ESCRT machinery. The ESCRT proteins play an important role in the shedding of membrane vesicles, as well as the formation of MVBs, in response to small wounds that stimulate early signalling pathways such as Ca2+ influxes (61, 62). MV morphology and mechanisms leading to budding may, however, differ between cell types, with evidence in some models showing budding regulation by ADP-ribosylation factor 6, phospholipase D and a variety of kinases39.

Summary of EV Formation

In each case of EV formation there appear to be multiple pathways leading to vesiculation, but the end result of these pathways on the morphological and biochemical characteristics of the vesicles is ill defined. Making a true distinction between subsets of EVs is an area requiring study, that translational studies may benefit from.

Due to the current limitations in the distinction between EV subsets when analysing physiological fluids, density and diameter appear to be the best option for currently of allowing distinction between apoptotic vesicles and small vesicles (MVs & exosomes). However, due to some MVs overlapping in diameter with exosomes, it is debatable how effective density and diameter differentiation is in this case, and more likely protein markers and relative abundance also need to be factored in. It is questionable whether it is even possible to differentiate between exosomes and MVs (once they have been released) within their overlapping diameter ranges, at which both are at their most abundant.

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Text modified from Welsh, Joshua (2016) Flow cytometer optimisation and standardisation for the study of extracellular vesicles as translational biomarkers University of Southampton Doctoral Thesis, 209pp.