161 research outputs found
The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles
Engineered inorganic nanoparticles are essential components in the
development of nanotechnologies. For applications in nanomedicine, particles
need to be functionalized to ensure a good dispersibility in biological fluids.
In many cases however, functionalization is not sufficient : the particles
become either coated by a corona of serum proteins or precipitate out of the
solvent. In the present paper, we show that by changing the coating of iron
oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small
carboxylated polymers (poly(acrylic acid)), the colloidal stability of the
dispersion is improved and the adsorption/internalization of iron towards
living mammalian cells is profoundly affected. Citrate-coated particles are
shown to destabilize in all fetal-calf-serum based physiological conditions
tested, whereas the polymer coated particles exhibit an outstanding
dispersibility as well as a structure devoid of protein corona. The
interactions between nanoparticles and human lymphoblastoid cells are
investigated by transmission electron microscopy and flow cytometry. Two types
of nanoparticle/cell interactions are underlined. Iron oxides are found either
adsorbed on the cellular membranes, or internalized into membrane-bound
endocytosis compartments. For the precipitating citrate-coated particles, the
kinetics of interactions reveal a massive and rapid adsorption of iron oxide on
the cell surfaces. The quantification of the partition between adsorbed and
internalized iron was performed from the cytometry data. The results highlight
the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.Comment: 21 pages, 11 figures, accepted at Biomaterials (2011
Interplay of packing and flip-flop in local bilayer deformation. How phosphatidylglycerol could rescue mitochondrial function in a cardiolipin-deficient yeast mutant
In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin head-groups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondria! activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondria! function. n a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin headgroups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function
Active Membrane Fluctuations Studied by Micropipet Aspiration
We present a detailed analysis of the micropipet experiments recently
reported in J-B. Manneville et al., Phys. Rev. Lett. 82, 4356--4359 (1999),
including a derivation of the expected behaviour of the membrane tension as a
function of the areal strain in the case of an active membrane, i.e.,
containing a nonequilibrium noise source. We give a general expression, which
takes into account the effect of active centers both directly on the membrane,
and on the embedding fluid dynamics, keeping track of the coupling between the
density of active centers and the membrane curvature. The data of the
micropipet experiments are well reproduced by the new expressions. In
particular, we show that a natural choice of the parameters quantifying the
strength of the active noise explains both the large amplitude of the observed
effects and its remarkable insensitivity to the active-center density in the
investigated range. [Submitted to Phys Rev E, 22 March 2001]Comment: 14 pages, 5 encapsulated Postscript figure
Field Theoretic Study of Bilayer Membrane Fusion III: Membranes with Leaves of Different Composition
We extend previous work on homogeneous bilayers to calculate the barriers to
fusion of planar bilayers which contain two different amphiphiles, a
lamellae-former and a hexagonal former, with different compositions of the
twoin each leaf. Self-consistent field theory is employed, and both standard
and alternative pathways are explored. We first calculate these barriers as the
amount of hexagonal former is increased equally in both leaves to levels
appropriate to the plasma membrane of human red blood cells. We follow these
barriers as the composition of hexagonal-formers is then increased in the cis
layer and decreased in the trans layer, again to an extent comparable to the
biological system. We find that, while the fusion pathway exhibits two barriers
in both the standard and alternative pathways, in both cases the magnitudes of
these barriers are comparable to one another, and small, on the order of 13 kT.
As a consequence, one expects that once the bilayers are brought sufficiently
close to one another to initiate the process, fusion should occur rapidly.Comment: 9 figure
Photo-induced proton gradients for the in vitro investigation of bacterial efflux pumps
We describe an original activity assay for membrane transport that uses the proton motive force-dependent efflux pump MexAB from Pseudomonas aeruginosa. This pump is co-reconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump
Distinct Regions of the Large Extracellular Domain of Tetraspanin CD9 Are Involved in the Control of Human Multinucleated Giant Cell Formation
Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role
Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein
Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens
Nanoelectropulse-driven membrane perturbation and small molecule permeabilization
BACKGROUND: Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization. RESULTS: In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X(7 )receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts. CONCLUSION: Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane
Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity
Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents
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