scholarly journals Generating Giant Membrane Vesicles from Live Cells with Preserved Cellular Properties

Research ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Qiaoling Liu ◽  
Cheng Bi ◽  
Jiangling Li ◽  
Xuejiao Liu ◽  
Ruizi Peng ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Transport ◽  
Plasma Membranes ◽  
Biomedical Application ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Membrane Properties ◽  
Live Cells ◽  
Plasma Membrane Vesicles ◽  
High Yields

Biomimetic giant membrane vesicles, with size and lipid compositions comparable to cells, have been recognized as an attractive experimental alternative to living systems. Due to the similarity of their membrane structure to that of body cells, cell-derived giant plasma membrane vesicles have been used as a membrane model for studying lipid/protein behavior of plasma membranes. However, further application of biomimetic giant membrane vesicles has been hampered by the side-effects of chemical vesiculants and the utilization of osmotic buffer. We herein develop a facile strategy to derive giant membrane vesicles (GMVs) from mammalian cells in biofriendly medium with high yields. These GMVs preserve membrane properties and adaptability for surface modification and encapsulation of exogenous molecules, which would facilitate their potential biological applications. Moreover, by loading GMVs with therapeutic drugs, GMVs could be employed for drug transport to tumor cells, which represents another step forward in the biomedical application of giant membrane vesicles. This study highlights biocompatible GMVs with biomimicking membrane surface properties and adaptability as an ideal platform for drug delivery strategies with potential clinical applications.

Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
Author(s):  
Christie Cefaratti ◽  
Andrea Romani ◽  
Antonio Scarpa
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Channel Blockers ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles

2021 ◽  
Author(s):  
Nikolas K. Teiwes ◽  
Ingo Mey ◽  
Phila C. Baumann ◽  
Lena Strieker ◽  
Ulla Unkelbach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Two Phases Of Ferricyanide Reductase Activity In Ehrlich Cell Plasma Membranes

1992 ◽  
Vol 47 (11-12) ◽  
pp. 929-931 ◽  
Author(s):  
Antonio del Castillo-Olivares ◽  
Javier Márquez ◽  
Ignacio Núñez de Castro ◽  
Miguel Angel Medina
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Cell Plasma Membrane ◽  
Ferricyanide Reductase ◽  
Ehrlich Cell ◽  
Cell Plasma ◽  
Two Phases

Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases

cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes

1997 ◽  
Vol 273 (4) ◽  
pp. G842-G848 ◽  
Author(s):  
Sunil Mukhopadhayay ◽  
M. Ananthanarayanan ◽  
Bruno Stieger ◽  
Peter J. Meier ◽  
Frederick J. Suchy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Plasma Membrane Vesicles ◽  
Short Term ◽  
Kinase A

Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

scholarly journals Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi

1995 ◽  
Vol 306 (1) ◽  
pp. 299-303 ◽  
Author(s):  
G Benaim ◽  
S N J Moreno ◽  
G Hutchinson ◽  
V Cervino ◽  
T Hermoso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
High Sensitivity ◽  
Bovine Brain ◽  
Calcium Pump ◽  
Plasma Membrane Vesicles ◽  
P Type

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

scholarly journals Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Peter J. A. van den BROEK ◽  
Angeline E. van GOMPEL ◽  
Marijke A. H. LUTTIK ◽  
Jack T. PRONK ◽  
Carla C. M. van LEEUWEN
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Transport Systems ◽  
Sugar Accumulation ◽  
Plasma Membrane Vesicles ◽  
Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

scholarly journals Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation

1998 ◽  
Vol 333 (3) ◽  
pp. 621-630 ◽  
Author(s):  
Frances J. SHAROM ◽  
Peihua LU ◽  
Ronghua LIU ◽  
Xiaohong YU
Keyword(s):  
Cyclic Peptides ◽  
Drug Transport ◽  
Membrane Vesicles ◽  
Drug Uptake ◽  
Multidrug Transporter ◽  
Chemotherapeutic Drugs ◽  
Plasma Membrane Vesicles ◽  
Hydrophobic Peptides ◽  
P Glycoprotein ◽  
Highly Correlated

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 µM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

scholarly journals The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

1991 ◽  
Vol 273 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Darnanville ◽  
R Bredoux ◽  
K J Clemetson ◽  
N Kieffer ◽  
N Bourdeau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Glycoprotein Ib ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

Bicarbonate-chloride exchange in gill plasma membranes of blue crab

1985 ◽  
Vol 249 (5) ◽  
pp. R544-R550 ◽  
Author(s):  
S. H. Lee ◽  
J. B. Pritchard
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
External Medium ◽  
Callinectes Sapidus ◽  
Exchange Process ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Plasma Membrane Vesicles ◽  
Low Salinity ◽  
Chloride Exchange

The uptake of chloride was studied in gill plasma membrane vesicles from low-salinity-adapted blue crabs (Callinectes sapidus). Cl- uptake was not Na+ dependent. However, when a HCO-3 gradient (in greater than out) was imposed across the membrane, a transient overshoot of about 2.5-fold was produced. Approximately 90% of the Cl- uptake reflected entry into the osmotically active intravesicular space. Cl- itself, nitrate, hydroxyl, and sulfite could substitute for HCO-3. The HCO-3/Cl- exchange process appeared to saturate at higher concentrations of either HCO-3 or Cl-. The apparent Km for Cl- was 15 mM. HCO-3-dependent Cl- uptake was significantly inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and thiocyanate but not by amiloride, furosemide, or ouabain. Alterations in membrane potential had no effect on Cl- uptake. Addition of Cl- or HCO-3 to the external medium also accelerated efflux of 36Cl- and H14CO-3 from preloaded vesicles. These results indicate that the uptake of Cl- by the crab gill plasma membrane is a carrier-mediated Na+-independent anion exchange process.

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