Progress and gaps of extracellular vesicle-mediated intercellular cargo transfer in the central nervous system

A fundamentally novel function proposed for extracellular vesicles (EVs) is to transfer bioactive molecules in intercellular signaling. In this minireview, we discuss recent progress on EV-mediated cargo transfer in the central nervous system (CNS) and major gaps in previous studies. We also suggest a set of experiments necessary for bridging the gaps and establishing the physiological roles of EV-mediated cargo transfer. &nbsp;</p

SNARE Zippering Is Suppressed by a Conformational Constraint that Is Removed by v-SNARE Splitting

Intracellular vesicle fusion is catalyzed by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-anchored v-SNAREs pair with target membrane-associated t-SNAREs to form trans-SNARE complexes, releasing free energy to drive membrane fusion. However, trans-SNARE complexes are unable to assemble efficiently unless activated by Sec1/Munc18 (SM) proteins. Here, we demonstrate that SNAREs become fully active when the v-SNARE is split into two fragments, eliminating the requirement of SM protein activation. Mechanistically, v-SNARE splitting accelerates the zippering of trans-SNARE complexes, mimicking the stimulatory function of SM proteins. Thus, SNAREs possess the full potential to drive efficient membrane fusion but are suppressed by a conformational constraint. This constraint is removed by SM protein activation or v-SNARE splitting. We suggest that ancestral SNAREs originally evolved to be fully active in the absence of SM proteins. Later, a conformational constraint coevolved with SM proteins to achieve the vesicle fusion specificity demanded by complex endomembrane systems. &nbsp;</p

Cyanine lipids promote shedding of extracellular vesicles from cell membranes

Extracellular vesicles (EV) have garnered significant attention in the fields of drug delivery, imaging, and immunotherapy. There is a need in methods to enhance release of EVs from cells. We found that at high labeling concentrations (100µM), indocarbocyanine lipids DiD and DiR that are commonly used for labeling cells, nanoparticles and EVs, promoted shedding of cell membrane tetraspanins with concomitant release of EVs in the medium. To further investigate this phenomenon, we screened a library of lipids and liposomal formulations for the release of membrane marker CD63 from THP-1 cells, and membrane red nanolantern (RNL) from 4T1 cells. We found a strong dependency of the EV release on lipid structure. In general, lipids that had a cyanine headgroup were more efficient than PEGylated phospholipids, neutral and cationic liposomes, with some lipids enhancing the release of CD63 up to 4-fold, and of RNL up to 8-fold, over vehicle treated control. A side-by-side comparison of cyanine lipid derivatives and corresponding precursor lipids confirmed that the cyanine headgroup significantly promoted shedding of RNL. Mutation of an exosome biogenesis regulator UNC13D did not hinder the release. Lipid-released EV could be modified with anti-interleukin 13 receptor alpha 2 antibody and targeted to glioma cells, suggesting potential utility in drug delivery. Furthermore, the impact of extraneously added lipids on cell membrane integrity should be carefully considered in cell labeling and drug delivery applications

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion. : Membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, Rathore et al. demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE.</p

AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of the AP2 adaptor complex, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34). In this study, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi network and the endosome. However, AAGAB is not involved in the formation of other adaptor complexes, including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the &gamma; and &sigma; subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis, and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Taken together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases. &nbsp;</p

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.</p

Research on Strain Measurements of Core Positions for the Chinese Space Station

The Chinese space station is designed to carry out manned spaceflight, space science research, and so on. In serious applications, it is a common operation to inject gas into the hull, which can produce strain of the bulkhead. Accurate measurement of strain for the bulkhead is one of the key tasks in evaluating the health condition of the space station. This is the first work to perform strain detection for the Chinese space station bulkhead by using optical fiber Bragg grating. In the period of measurements, the resistance strain gauge is used as the strain standard. The measurement error of the fiber optical sensor in the circumferential direction is very small, being less than 4.52 &mu;&epsilon;. However, the error in the axial direction is very large with the highest value of 28.93 &mu;&epsilon;. Because the measurement error of bare fiber in the axial direction is very small, the transverse effect of the substrate of the fiber optical sensor likely plays a role. The comparison of the theoretical and experimental results of the transverse effect coefficients shows that they are fairly consistent, with values of 0.0271 and 0.0287, respectively. After the transverse effect is compensated, the strain deviation in the axial detection is smaller than 2.04 &mu;&epsilon;. It is of great significance to carry out real-time health assessment for the bulkhead of the space station