Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

On the origin of membrane vesicles in Gram-negative bacteria

It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.     

Analysis of post-lysosomal compartments

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.     

Lipid fatty acid and protein pattern of equine prostasome-like vesicles

The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein pattern (SDS-PAGE electrophoresis) shows that the 30–50-kDa fraction is less abundant in stallion vesicles. In addition, fluidity (measured as fluorescence anisotropy of diphenylhexatriene) is higher in horse prostasome-like vesicles than in human prostasomes, albeit being much lower than that of most membranes. These findings may be connected to some species-related differences in reproductive physiology: the vaginal milieu of the mare is not acidic and the deposition of semen is intrauterine in the horse but vaginal in humans.     

Rolling properties of rGPIbalpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 microm using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility.     

Rapid,combinatorial analysis of membrane compartments in intact plants with a multicolor marker set

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill‐defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non‐toxic expression. We demonstrate the power of this multicolor ‘Wave’ marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live‐imaging and immunoelectron microscopy. Among other findings, our systematic co‐localization analysis revealed that a class of plant Rab1‐homologs has a much more extended localization than was previously assumed, and also localizes to trans‐Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.     

Tvp38, Tvp23, Tvp18 and Tvp15: novel membrane proteins in the Tlg2-containing Golgi/endosome compartments of Saccharomyces cerevisiae

Four previously uncharacterized proteins (Tvp38, Tvp23, Tvp18 and Tvp15) were found in Tlg2-containing membrane by proteomic analysis of immunoisolated Golgi subcompartments of Saccharomyces cerevisiae (Inadome et al., Mol. Cell. Biol., 25 (2005) 7696-7710). Immunofluorescence double staining of HA-tagged Tvp proteins and myc-tagged tSNAREs supported that these proteins mainly localize in the Tlg2-containing compartments. Conserved sequences of Tvp38, Tvp23 and Tvp18 are found in higher eukaryotes, but these homologues have not been characterized yet. All Tvp proteins were nonessential for growth under laboratory conditions. Immunoprecipitation of Tvp proteins indicated that Tvp23, Tvp18 and Tvp15 are in an interactive network with Yip1-family proteins, Yip4 and Yip5. They may collectively assist in the effective maintenance/function of the late Golgi/endosomal compartments. Disruptions of tvp15 and tvp23 showed synthetic aggravation with ypt6 or ric1 null mutation. Processing of carboxypeptidase Y and alkaline phosphatase in tvp disruptants occurred as in the wild type.     

Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Regulation of ROCKII by localization to membrane compartments and binding to DynaminI

ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context. Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1.     

Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles

Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.     

Investigation of the role of ubiquinone in rat liver subcellular compartments

The role of ubiquinone in the Golgi apparatus is still unknown, even if it might be considered as a lipid marker of the Golgi compartment because of its high content in these subcellular fractions. In vivo modulation of ubiquinone with ethanol and in vitro pentane extraction show that ubiquinone is not required either for NADH-ferricyanide reductase, acetaldehyde dehydrogenase activity, or Ca2+ and Mg2+ stimulated ATPases. Since ubiquinone does not seem to be involved in these enzymic activities in Golgi compartments, other possible functions are discussed, related to a role in membrane fluidity or as a barrier to the propagation of free radicals.     

Single point mutation in Bin/Amphiphysin/Rvs (BAR) sequence of endophilin impairs dimerization, membrane shaping, and Src homology 3 domain-mediated partnership

Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors.     

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Summary Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.Abbreviations ATP adenosine 5-triphosphate - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - CCD counter-current distribution - DTT dithiothreitol - EDTA ethylene-diamine-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - GSII 1,3--glucan synthase - HEPES N-[2-hydroxyethyl]-piperazine-N-[2-ethane sulfonic acid] - MES 2-(N-morpholino)ethane sulfonic acid - NBT p-nitro blue tetrazolium chloride - IDP inosine 5-diphosphate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - PPB potassium phosphate buffer - PM plasma membrane - PMSF phenylmethylsulfonyl fluoride - PVDF polyvinylidene difluoride - PVPP polyvinylpolypyrrolidone - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TTBS Tris-buffered saline with Tween 20 - Tris tris(hydroxymethyl) aminomethane     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

蜜蜂大脑的分区与功能

蜜蜂Apis mellifera L.是神经生物学研究的重要模式生物。尽管工蜂脑的体积不足1mm3,包含的神经元数量不到百万,但却拥有丰富的个体和社会行为,甚至还有学习、记忆、认知等高级行为。如此微小的大脑也是通过不同结构分区来实现其丰富复杂的行为。本文对蜜蜂大脑的精细解剖结构以及脑区功能研究进行了综述,为昆虫科学和神经生物学研究提供参考。     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.     

Lipid-protein surface films generated from membrane vesicles: selfassembly,composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm² film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm². Offprint requests to: H. Schindler     

A method to investigate protein association with intact sealed mycobacterial membrane vesicles

In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins.