The Role of Exosome and the ESCRT Pathway on Enveloped Virus Infection

The endosomal sorting complex required for transport (ESCRT) system consists of peripheral membrane protein complexes ESCRT-0, -I, -II, -III VPS4-VTA1, and ALIX homodimer. This system plays an important role in the degradation of non-essential or dangerous plasma membrane proteins, the biogenesis of lysosomes and yeast vacuoles, the budding of most enveloped viruses, and promoting membrane shedding of cytokinesis. Recent results show that exosomes and the ESCRT pathway play important roles in virus infection. This review mainly focuses on the roles of exosomes and the ESCRT pathway in virus assembly, budding, and infection of enveloped viruses. The elaboration of the mechanism of exosomes and the ESCRT pathway in some enveloped viruses provides important implications for the further study of the infection mechanism of other enveloped viruses.

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EV production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the PfVps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production.

The Redox Balance and Membrane Shedding in RBC Production, Maturation, and Senescence

Membrane shedding in the form of extracellular vesicles plays a key role in normal physiology and pathology. Partial disturbance of the membrane–cytoskeleton linkage and increased in the intracellular Ca content are considered to be mechanisms underlying the process, but it is questionable whether they constitute the primary initiating steps. Homeostasis of the redox system, which depends on the equilibrium between oxidants and antioxidants, is crucial for many cellular processes. Excess oxidative power results in oxidative stress, which affects many cellular components, including the membrane. Accumulating evidence suggests that oxidative stress indirectly affects membrane shedding most probably by affecting the membrane–cytoskeleton and the Ca content. In red blood cells (RBCs), changes in both the redox system and membrane shedding occur throughout their life—from birth—their production in the bone marrow, to death—aging in the peripheral blood and removal by macrophages in sites of the reticuloendothelial system. Both oxidative stress and membrane shedding are disturbed in diseases affecting the RBC, such as the hereditary and acquired hemolytic anemias (i.e., thalassemia, sickle cell anemia, and autoimmune hemolytic anemia). Herein, I review some data-based and hypothetical possibilities that await experimental confirmation regarding some aspects of the interaction between the redox system and membrane shedding and its role in the normal physiology and pathology of RBCs.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)–containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel–mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicles (EVs) production in parasitized red blood cells (pRBC), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery that is activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular bodies formation and membrane shedding, both reported mechanisms of EVs production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy. In accordance, disruption of the Pfvps60 gene led to a reduction in the number of the produced EVs in the KO strain when compared with the parental 3D7 strain. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EVs production.

Multi-Omics Data Integration in Extracellular Vesicle Biology—Utopia or Future Reality?

Extracellular vesicles (EVs) are membranous structures derived from the endosomal system or generated by plasma membrane shedding. Due to their composition of DNA, RNA, proteins, and lipids, EVs have garnered a lot of attention as an essential mechanism of cell-to-cell communication, with various implications in physiological and pathological processes. EVs are not only a highly heterogeneous population by means of size and biogenesis, but they are also a source of diverse, functionally rich biomolecules. Recent advances in high-throughput processing of biological samples have facilitated the development of databases comprised of characteristic genomic, transcriptomic, proteomic, metabolomic, and lipidomic profiles for EV cargo. Despite the in-depth approach used to map functional molecules in EV-mediated cellular cross-talk, few integrative methods have been applied to analyze the molecular interplay in these targeted delivery systems. New perspectives arise from the field of systems biology, where accounting for heterogeneity may lead to finding patterns in an apparently random pool of data. In this review, we map the biological and methodological causes of heterogeneity in EV multi-omics data and present current applications or possible statistical methods for integrating such data while keeping track of the current bottlenecks in the field.

MMP-9 Shedding Releases Truncated CD73 from Astrocytes

Previously we had reported that astrocytes physiologically express high levels of CD73 in their membrane, converting extracellular AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. Following an interaction with effector T cells (CD4+CD25- ), astrocytes lost most of their membrane expressed CD73, which rendered astrocytes’ immune suppressive function and accelerated neural inflammation such as EAE. Here, we investigated the mechanism leading to the loss of membrane CD73 in astrocytes. Our results revealed that there was no significant difference in Cd73 mRNA expressions between CD73high and CD73low astrocytes. Membrane shedding of CD73 by matrix metalloproteinase-9 (MMP-9) accounted for its membrane loss in astrocytes; meanwhile, C terminal truncated CD73 could be found in the medium of induced CD73low astrocytes. With an MMP-9 inhibitor in existence, the shedding of CD73 in wt-astrocytes, when interacted with CD73-/- effector CD4 cells, was almost completely blocked, and the production of pro-inflammatory cytokines, such as IL-17 and IFNγ, from interacted CD73-/- effectors, were significantly decreased. However, when a CD73 inhibitor was added together with MMP-9 inhibitor, decreased production of pro-inflammatory cytokines were completely restored. As conclusion, our findings suggested that under active inflammatory condition, MMP-9 releases CD73 from astrocytes. The block of CD73 shedding in astrocytes by the addition of MMP-9 inhibitor could significantly decrease the activation of interacted effector T cells.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia

Repairing plasma membrane damage is vital to eukaryotic cell survival. Membrane shedding is thought to be key to this repair process, but a detailed view of how the process occurs is still missing. Here we used electron cryotomography to image the ultrastructural details of plasma membrane wound healing. We found that filopodia-like protrusions are built at damage sites, accompanied by retraction of neighboring filopodia, and that these repurposed protrusions act as scaffolds for membrane shedding. This suggests a new role for filopodia as reservoirs of membrane and actin for plasma membrane damage repair. Damage-induced shedding was dependent on F-actin dynamics and Myo1a, as well as Vps4B, an important component of the ESCRT machinery. Thus we find that damage shedding is more complex than current models of simple vesiculation from flat membrane domains. Rather, we observe structural similarities between damage-mediated shedding and constitutive shedding from enterocytes that argue for conservation of a general membrane shedding mechanism.