Understanding extracellular vesicle diversity – current status

ABSTRACT

Introduction: Extracellular vesicles (EVs) represent an important mode of intercellular communication. There is now a growing awareness that predominant EV subtypes; exosomes from endosomal origin, and shed microvesicles from plasma membrane budding, can be further stratified into distinct subtypes, however specific approaches in their isolation and markers that allow them to be discriminated are lacking.

Areas covered: Knowledge about these distinct EV subpopulations is important including the regulation of composition, release, targeting/localization, uptake, and function. This review discusses the mechanisms of distinct EV biogenesis and release, defining select EV classes (and subpopulations), which will be crucial for development of EV-based functions and clinical applications. We review the dynamics of cargo sorting leading to the mechanisms of EV heterogeneity, their mechanisms of formation, intracellular trafficking pathways, and provide an uptake about biochemical/functional differences. With advances in purification strategies and proteomic-based quantitation, allows significant benefit in accurately describing differences in EV protein cargo composition and modification.

Expert commentary: The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of these distinct EV subpopulations.     

Dynamics of spread of Salmonella enterica in the systemic compartment

Traditional microbiological and immunological tools, combined with modern imaging, and molecular and mathematical approaches, have revealed the dispersive nature of Salmonella infections. Bacterial escape from infected cells, spread in the tissues and attempts to restrain this process by the host give rise to fascinating scenarios that underpin the pathogenesis of salmonelloses.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

When S1P meets ATP

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.     

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic β cell death

There is a growing evidence of the role of autophagy in pancreatic β cell homeostasis. During development of type 2 diabetes, β cells are required to supply the increased demand of insulin. In such a stage, β cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, β cell death and overt diabetes. In this study, we used insulin secretion-deficient β cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived β cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased β cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient β cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in β cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.     

Pancreatic stellate cell-potentiated insulin secretion from Min6 cells is independent of interleukin 6-mediated pathway

Pancreatic stellate cells (PSCs) secrete various factors, which can influence the β-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on β-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP₃ pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of β-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP₃ pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP₃ pathway.