Population Of Membrane Vesicles Research Articles

MicroRNA of extracellular vesicles as biomarkers and regulators of pathological and physiological processes

Extracellular vesicles (EV) are currently considered potential biomarkers of diseases, as well as “biological constructs” for targeted drug delivery. Extracellular vesicles represent a heterogeneous population of membrane vesicles formed by various cell populations, including immune cells. At present time, EV are divided into exosomes, microvesicles, and apoptotic bodies, depending on their size and formation manner. EV have been found in various human biological fluids. Therefore, the possibility of their usage as diagnostic biomarkers is under discussion. The vesicles have a diverse internal composition and express a wide repertoire of receptors on their surface, thus allowing them to participate in different intercellular communications by transferring to the cells various molecules, including genetic material. E.g., microRNAs transmitted via extracellular vesicles are evolutionarily conserved non-coding RNA molecules 18-25 nucleotides long. Their main function is to regulate gene expression at the post-transcriptional level. MicroRNAs are synthesized by different cell types. However, some microRNAs are found ubiquitously, whereas others are present only in certain types of tissues. MicroRNAs are found both inside, and outside the cells (extracellular or circulating microRNAs). The microRNAs are resistant to RNases and stable in the extracellular environment, due to their secretion as protein complexes, or as part of extracellular vesicles. The variable microRNA profile in extracellular vesicles depends on the physiological conditions and presence of various pathological disorders. Multiple studies show that microRNAs can determine the functional activity of extracellular vesicles, e.g., therapeutic usage of microRNAs carried by EV as well as diagnostic applications in various pathologies. This review considers distinct populations of extracellular vesicles and their main properties, describes the characteristics of intra- and extracellular (circulating) microRNAs, mechanisms of their biosynthesis, and techniques for detection and assessing contents of microRNAs. The review describes microRNAs as a component of exosomes and microvesicles formed by various cells, including cells of the immune system in the course of physiological and pathological processes, with respect to functions of these microRNAs as well as their diagnostic and therapeutic potential.

Increased Yield of Extracellular Vesicles after Cytochalasin B Treatment and Vortexing.

Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring extracellular vesicles and cytochalasin B-induced membrane vesicles (CIMVs) from mesenchymal stem cells (MSCs). In order to maintain accuracy in the comparative analysis, the same culture was used for the isolation of EVs and CIMVs: conditioned medium was used for EVs isolation and cells were harvested for CIMVs production. The pellets obtained after centrifugation 2300× g, 10,000× g and 100,000× g were analyzed using scanning electron microscopy analysis (SEM), flow cytometry, the bicinchoninic acid assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). We found that the use of cytochalasin B treatment and vortexing resulted in the production of a more homogeneous population of membrane vesicles with a median diameter greater than that of EVs. We found that EVs-like particles remained in the FBS, despite overnight ultracentrifugation, which introduced a significant inaccuracy in the calculation of the EVs yield. Therefore, we cultivated cells in a serum-free medium for the subsequent isolation of EVs. We observed that the number of CIMVs significantly exceeded the number of EVs after each step of centrifugation (2300× g, 10,000× g and 100,000× g) by up to 5, 9, and 20 times, respectively.

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

Extracellular vesicles-based drug delivery systems for cancer immunotherapy

Extracellular vesicles (EVs), a heterogeneous population of membrane vesicles, are secreted by almost all living cells and contain different proteins, lipids and nucleic acid depending on their sources. Recently, numerous reports indicated EVs could interact with diverse cells in tumor microenvironment and influence both physiological and pathological conditions. Given that EVs regulate tumor immune microenvironment, EVs-based strategies are explored for anti-tumor immunotherapy. In this review, about nine kinds of EVs derived from diverse cells are indicated. The biological functions in tumor immunity, strategies for anti-tumor immunotherapy and further clinical trials of these EVs will be introduced in detail.

Differential expression of CD42b and CD9 proteins in platelets and extracellular membrane vesicles during platelet-concentrate storage

Platelet concentrate is used to restore and maintain hemostasis in patients with a reduced number or activity of platelets. Platelet concentrate changes its properties when stored, which leads to a weakening of the therapeutic effect, as well as the occurrence of transfusion side effects. One of the processes that alter platelet concentrates during storage is the secretion of several types of membrane vesicles. Despite the fact that platelet-derived membrane vesicles affect homeostasis and transmit signals of intercellular communication, mechanisms of their formation, features of regulatory activity and molecular composition are still poorly understood. In this paper, the results of analysis of CD42b and CD9 membrane proteins expression in platelets and platelet-derived membrane vesicles during platelet-concentrate storage are presented. Populations of membrane vesicles of different sizes were isolated from the platelet concentrates and characterized. Aggregation and morphological alteration of platelets are observed during storage; the protein composition of platelets, as well as membrane vesicles, changes, and there is a significant increase in the levels of CD42b and CD9 proteins in fractions of membrane vesicles. The results obtained indicate that platelet concentrates contain different populations of membrane vesicles, the molecular composition of which varies during storage.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30–300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.

Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

Graded distribution of intracellular second messengers, such as Ca(2+) and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca(2+) have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca(2+) signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca(2+)-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Human Platelet Exosome Proteomics Leads to the Identification of WNT Positive Exosomes Which Impact Canonical WNT Signalling in Target Cells

Upon activation platelets release a myriad of soluble proteins as well as two distinct membrane vesicle populations- exosomes and microparticles (MPs) into the external milieu [1]. Although there have been many studies characterising platelet MPs and their increased circulating numbers in disease [2, 3], platelet exosomal vesicles have been characterised to date only by transmission electron microscopy (TEM) [4]. Here, we characterize the proteome of human platelet exosomes and reveal that a population of these vesicles carry active WNT glycoproteins on their surface that can modulate WNT signalling activity in both endothelial and monocytic cells.To establish the exosomal proteome of human platelets we compared platelet exosomes isolated from three healthy volunteers by (i) differential ultracentrifugation (DC) and (ii) ExoQuickTM (ExoQ) precipitation. Assessment of both exosomal populations was performed using nanoparticle tracking analysis (NTA), TEM, western blot and proteomics analysis (in triplicate) using a high performance Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer coupled with quantitative computational analysis using MaxQuant software. We concluded that DC was the superior isolation method for platelet exosomes, for example, our NTA revealed a 2-fold greater enrichment (6x108 /ml) of platelet exosomes following isolation by DC in comparison to ExoQ. Proteomic analysis of platelet exosomes identified 704 proteins across 18 MS runs, 119 of which were present in all 18 runs and represented the core human platelet exosomal proteome. Using western blotting and immunogold TEM, we confirmed the presence of numerous well-established exosomal membrane markers (CD63, CD9, and HSP70) in our proteome, as well as several platelet-specific proteins including GPIb, as previously described [4], and GPV.We also detected several of the 19 human WNT glycoproteins to be specifically secreted in the exosomal fraction upon platelet activation and verified their surface expression on platelet-derived exosomes using immunogold TEM. As we have previously demonstrated, platelets themselves can be modulated directly by WNT glycoproteins [5], hence here, we assessed the paracrine impact of these WNT-positive platelet exosomes on intracellular signalling events in monocytes and endothelial cells, both cell types closely linked to the pathogenesis of atherosclerosis. Using fluorescence microscopy, we found that these platelet exosomes were readily endocytosed by both endothelial (EAHY296) and monocytic (THP-1) cell lines where they specifically modulated beta-catenin nuclear expression and canonical WNT signalling.In conclusion our proteomic analysis has revealed that platelet exosomes are new players in the regulation of WNT signalling in both endothelial and monocytic cells.1. Coppinger, J.A., et al., Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood, 2004. 103(6): p. 2096-104.2. Knijff-Dutmer, E.A., et al., Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum, 2002. 46(6): p. 1498-503.3. van der Zee, P.M., et al., P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem, 2006. 52(4): p. 657-64.4. Heijnen, H.F., et al., Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood, 1999. 94(11): p. 3791-9.5. Steele, B.M., et al., Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A, 2009. 106(47): p. 19836-41. DisclosuresNo relevant conflicts of interest to declare.

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles

Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical EditorThe authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.

NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes

In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.

Compartmentalization of Lipid Biosynthesis in Mycobacteria

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

ALIX-ing phospholipids with endosome biogenesis.

Endosomes, which comprise a diverse population of membrane vesicles and tubules, sort proteins and lipids to various cellular destinations. The organization and functions of these pleiomorphic cellular organelles have been extensively studied. Matsuo et al.1 now provide new exciting evidence on the role of lysobiphosphatidic acid (LBPA), a resident phospholipid of internal vesicles of the late endosome, in the control of membrane invagination and endosome biogenesis. In vivo, LBPA functions are controlled by the adaptor protein Alix and depend on pH gradients along the endosomal compartments.

Glucose Transporter Glut3 Is Targeted to Secretory Vesicles in Neurons and PC12 Cells

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

Regulation of membrane release in apoptosis.

Apoptosis is a fundamental process of cell regulation whereby cells execute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to play roles in the regulation of apoptosis, including the activation of phospholipase A2 (PLA2), usually measured as release of 3H-labelled arachidonic acid (AA) from prelabelled cells. The current study was aimed at examining the role of PLA2 in regulating apoptosis in response to several inducers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [3H]fatty acids and the released radioactivity was characterized. These studies indicated that the AA release assay did not reflect release of non-esterified fatty acid via activation of the PLA2 pathway. Rather, studies using TLC and electron microscopy showed that AA release reflected a previously unsuspected shedding of a heterogeneous population of membrane vesicles and fragments, probably as components of apoptotic bodies. Further studies demonstrated that this process is an integral part of apoptosis. Overexpression of Bcl-2 or the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethane prevented the characteristic morphological changes of cell death, and completely inhibited the release of membrane vesicles and fragments. On the other hand, release of membrane vesicles and fragments was caused by various inducers of apoptosis, as measured by release of either 3H-labelled AA or palmitic acid. Thus the present study demonstrates that the release of membrane lipids during apoptosis defines a new assay for apoptosis and has allowed the investigation of the mechanisms regulating formation of apoptotic bodies.

Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells.

Previous investigations in several systems have demonstrated that Rab3 family members redistribute to soluble fractions on fusion of secretory granules with target plasma membranes. Rab proteins are then recycled back onto mature secretory vesicles after reinternalization of the membrane. Although this cycle is well established for Rab3, far less is known about redistribution of other Rab proteins during vesicle fusion and recycling. In the gastric parietal cell, Rab11a is associated with H-K-ATPase-containing tubulovesicles, which fuse with the apical plasma membrane (secretory canaliculus) in response to agonists such as histamine. We have analyzed distribution of Rab11a and other tubulovesicle proteins in resting and histamine-stimulated rabbit parietal cells. Stimulation of isolated gastric glands in the presence of 100 microM histamine and 100 microM 3-isobutyl-1-methylxanthine did not cause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a, Rab25, syntaxin 3, and SCAMPs increased immunoreactivity in stimulus-associated vesicles prepared from rabbits treated with histamine compared with those from ranitidine-treated animals. The large GTPase dynamin was found in both vesicle preparations, but there was no change in amount of immunoreactivity. Immunofluorescence staining of resting and histamine-stimulated primary cultures of parietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate that Rab11a does not cycle off the membrane during the process of tubulovesicle fusion with the secretory canaliculus. Thus Rab11a may remain associated with recycling apical membrane vesicle populations.

KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro

We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2 beta binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2 alpha binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2 beta. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.

Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier

Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that gamma-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5'-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Na+/K(+)-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K(+)-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.

Characterization of a voltage-dependent Ca2+-selective channel from wheat roots

A new mechanism for calcium flux in wheat (Triticum aestivum L.) root cells has been characterized. Membrane vesicles were enriched in plasma membrane using aqueous-polymer two-phase partitioning and incorporated into artificial lipid bilayers, allowing characterization of single channels under voltage-clamp conditions. Membrane marker activities showed 74% and 83% purity in plasma membrane when expressed in terms of membrane area and activity, respectively. Since membrane vesicles obtained by aqueous-polymer two-phase partitioning yield a population of membrane vesicles of regular orientation, and vesicle fusion into planar lipid bilayers occurs in a defined manner, the orientation of the channel upon vesicle incorporation could be determined. Thus ionic activities and potentials could be controlled appropriately on what we propose to be the cytosolic (trans) and extracellular (cis) faces of the channel. The unitary conductance in symmetrical 1 mM CaCl2 was 27±0.4 (pS). The correlation between the theoretical and observed reversal potentials in asymmetrical conditions showed that the channel was highly selective for Ca2+ over Cl−. Experiments simulating physiological ionic conditions showed a PCa 2+/PK + of 17–26, decreasing in this range as the extracellular CaCl2 concentration increased from 0.1 to 1 mM. The channel was also permeable to the essential nutrient ions, Mg2+ and Mn2+. The open probability of the channel was strongly dependent on the membrane potential. Inactivation with time was observed at more negative membrane potentials, and was immediately reversed as soon as the membrane potential was decreased. At membrane potentials more negative than -130mV, the channel remained mainly in the closed state, suggesting that in vivo the channel would remain largely closed and would open only upon membrane depolarization. The channel was blocked by micromolar concentrations of extracellular verapamil and trivalent cations, Al3+ being the most effective of those tested. Exposure of the cytosolic and extracellular sides of the channel to inositol 1,4,5-trisphosphate had no effect on the channel activity. We suggest a plasma-membrane origin for the channel as shown by biochemical and electrophysiological evidence, and discuss possible physiological roles of this channel, both in Ca2+ uptake into roots and in signal transduction.