The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform

Cees W. M. Haest - One of the best experts on this subject based on the ideXlab platform.

  • Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte Membrane
    Biochimica et Biophysica Acta, 1998
    Co-Authors: Dietrun Kamp, Cees W. M. Haest
    Abstract:

    Abstract Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1,3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner Membrane Leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner Membrane Leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

  • Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte Membrane depends on Membrane potential.
    Biochemistry, 1997
    Co-Authors: Cees W. M. Haest, Adrienne Oslender, Dietrun Kamp
    Abstract:

    The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner Membrane Leaflet of human erythrocytes and vice versa depends on Membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on Membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer Membrane Leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The Membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer Leaflets. The distribution is shifted to the inner Leaflet by increasingly positive potentials and to the outer Leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.

  • Fast translocation of phosphatidylcholine to the outer Membrane Leaflet after its synthesis at the inner Membrane surface in human erythrocytes.
    Biochimica et biophysica acta, 1991
    Co-Authors: Corinna Andrick, Karin Bröring, Bernhard Deuticke, Cees W. M. Haest
    Abstract:

    The translocation rate of [14C]phosphatidylcholine to the outer Membrane Leaflet of human erythrocytes after its primary synthesis from lysophosphatidylcholine by acylation with 14C-labeled oleic or palmitic acid in the inner Leaflet has been measured by following the time-dependent increase of cleavability of 14C-labeled phospholipids by external phospholipase A2 (5 min, 37 degrees C). Immediately after a short acylation time period of 10 min about 20% of the newly synthesized [14C]phosphatidylcholine are already detectable in the outer Leaflet. After an incubation of 1 h at 37 degrees C following 10 min of acylation the fractions of labeled and native phosphatidylcholine accessible to the lipase are identical, which demonstrates that [14C]phosphatidylcholine has attained the same asymmetric distribution as its endogenous analogue. The calculated halftime of the outward translocation is about 20 min and its activation energy is low, 30 kJ/mol. Translocation is inhibited by a 5 min treatment with phenylglyoxal following acylation. A fast translocation is not observed for newly synthesized phosphatidylethanolamine. Results suggest a selective, protein-mediated outward translocation of newly synthesized phosphatidylcholine.

Arnd Pralle - One of the best experts on this subject based on the ideXlab platform.

  • Transient Effect of Calcium Influx on PIP2 Clusters in the Inner Plasma Membrane Leaflet of Intact Cells
    Biophysical Journal, 2016
    Co-Authors: Weixiang Jin, Arnd Pralle
    Abstract:

    Phospjatidylinositol 4,5-bisphosphated(PIP2) is known to interact with many cytoskeletal proteins electrostatically. When calcium ion channels open, local calcium levels are estimated up to 500uM. Experiments in model lipid bilayer have shown that such calcium level is sufficient to affect PIP2 in the inner Leaflet of plasma Membrane (PM).PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between lipid acyl-chains, stabilized by cholesterol, lead to transient submicroscopic nano-domains, and head-group interactions of charged lipids with divalent ions may cluster lipids. In addition, Membrane cytoskeleton may impose further structure on Membrane. Here we study influence of calcium on formation of PIP2 clusters in intact cells. We study changes of domains over time and upon calcium channel activation by analyzing diffusion of GFP-tagged inner-Leaflet Membrane proteins. Using bimFCS, developed by us, we measure diffusion on multiple length scales simultaneously to derive information about domains. To study formation of PIP2 clusters in the PM, we use GFP-PHPLCdelta to directly mark PIP2. We observe that opening TRPV1 channels leads to a transient rise in calcium as imaged using GCaMP5G, increases interaction strength between GFP-PHPLCdelta and PIP2 domains, which decreases to base lines within 1-2 minutes. Using ionophores, we determine that the increasing interaction strength is due to calcium-PIP2 interaction. By Vinculin depletion or actin filaments (de-)polymerization, we find that both anchor proteins and actin filaments play important roles in PIP2 clusters, and the formation of larger PIP2 clusters can potentially further activate Plasma Membrane Ca2+ ATPase. These results suggest that in intact cells, calcium ions are able to induce changes of PIP2 clusters that are also regulated by cytoskeleton structure, changes of PIP2 clusters can further affect other signaling events.

  • transient effect of calcium influx on pip2 clusters and cholesterol stabilized nano domains in the inner plasma Membrane Leaflet of intact cells
    Biophysical Journal, 2014
    Co-Authors: Weixiang Jin, Arnd Pralle
    Abstract:

    When calcium ion channels open, local calcium levels are estimated to reach up to 500uM, which may be sufficient to affect negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), in the inner Leaflet of the plasma Membrane (PM). The PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between the acyl-chains of the lipids, stabilized by Cholesterol, lead to transient submicroscopic nano-domains, and lipid head-group interactions of charged lipids with divalent ions may cluster lipids.

Frances J Sharom - One of the best experts on this subject based on the ideXlab platform.

  • Flipping and flopping--lipids on the move.
    IUBMB life, 2011
    Co-Authors: Frances J Sharom
    Abstract:

    The rapid movement of polar lipids from one Membrane Leaflet to the other is facilitated by lipid flippases or translocases. Although their activity was first observed over 30 years ago, the structures, physiological roles, and molecular mechanisms of this group of proteins remain enigmatic. Lipid flippases maintain Membrane lipid asymmetry, and in eukaryotes they are also intimately involved in Membrane budding and vesicle trafficking. The ATP-dependent flippases are members of well-characterized protein families, whose other members transport nonlipid substrates across cell Membranes. The P(4)-type ATPases carry out the inward translocation of phospholipids, and various ABC transporters are involved in outward lipid movement. The ATP-independent flippases move lipid substrates in both directions between Membrane Leaflets. With only a few exceptions, the molecular identity of these proteins is still unknown, despite their involvement in key biosynthetic pathways in both bacteria and eukaryotes. This review provides an overview of the different classes of flippases, and summarizes recent progress in their identification and functional characterization. The possible mechanisms of action of lipid flippases are discussed, and future directions explored.

  • proximity of bound hoechst 33342 to the atpase catalytic sites places the drug binding site of p glycoprotein within the cytoplasmic Membrane Leaflet
    Biochemistry, 2002
    Co-Authors: Qin Qu, Frances J Sharom
    Abstract:

    : The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic Membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the Membrane surface, within the cytoplasmic Leaflet of the Membrane, in both resting-state and transition-state P-glycoprotein.

Ilpo Vattulainen - One of the best experts on this subject based on the ideXlab platform.

  • The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2.
    Communications biology, 2020
    Co-Authors: Cyril Legrand, Roberto Saleppico, Jana Sticht, Fabio Lolicato, Hans-michael Müller, Sabine Wegehingel, Eleni Dimou, Julia P. Steringer, Helge Ewers, Ilpo Vattulainen
    Abstract:

    FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma Membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma Membrane Leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma Membrane Leaflet being required for efficient Membrane translocation of FGF2 to cell surfaces.

  • Interdigitation of long-chain sphingomyelin induces coupling of Membrane Leaflets in a cholesterol dependent manner.
    Biochimica et biophysica acta, 2015
    Co-Authors: Tomasz Róg, Adam Orłowski, Alicia Llorente, Tore Skotland, Tuulia Sylvänne, Dimple Kauhanen, Kim Ekroos, Kirsten Sandvig, Ilpo Vattulainen
    Abstract:

    It has been a long-standing question how the two Leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid Membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric Membranes the amide-linked chain of SM is observed to extend deep into the opposing Membrane Leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing Leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite Leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) Leaflet.

  • Atomistic Simulations of Functional Gold Nanoparticles Au144(Sr)60 Interacting with Membranes
    Biophysical Journal, 2013
    Co-Authors: Elena Heikkilä, Ilpo Vattulainen, Andrey A. Gurtovenko, Hector Martinez-seara, Hannu Häkkinen, Jaakko Akola
    Abstract:

    Gold nanoparticles (AuNps) are used in nanomedicine in, e.g., drug delivery and bio-imaging. However, it is regrettable that the understanding of nanoparticle properties in cellular surroundings is incompletely understood. Here, we have complemented our previous studies [1] by performing extensive atomistic molecular dynamics simulations of lipid Membranes interacting with charged gold nanoparticles. We have elucidated the action of these nanoparticles on Membranes characterized by lipid compositional asymmetry in the two Leaflets, thereby unraveling the interactions of AuNPs with both the extracellular and the cytosolic sides of plasma Membranes of eukaryotic cells. We have found that there is an appealing interplay between AuNps and the two Membrane Leaflets, where both the Membrane Leaflet composition and the charged nature of the nanoparticle (cationic vs. anionic) play a role. Here we discuss the resulting effects from both structural and dynamical points of view, and highlight the role of electrostatics in nanoparticle-Membrane interactions.Figure 1Visualization of (a) anionic AuNP with the extracellular and (b) cationic AuNP with the cytosolic Leaflet.View Large Image | View Hi-Res Image | Download PowerPoint Slide[1] E. Heikkila, et al. 2012. J. Phys. Chem. C 116: 9805-9815.

Weixiang Jin - One of the best experts on this subject based on the ideXlab platform.

  • Transient Effect of Calcium Influx on PIP2 Clusters in the Inner Plasma Membrane Leaflet of Intact Cells
    Biophysical Journal, 2016
    Co-Authors: Weixiang Jin, Arnd Pralle
    Abstract:

    Phospjatidylinositol 4,5-bisphosphated(PIP2) is known to interact with many cytoskeletal proteins electrostatically. When calcium ion channels open, local calcium levels are estimated up to 500uM. Experiments in model lipid bilayer have shown that such calcium level is sufficient to affect PIP2 in the inner Leaflet of plasma Membrane (PM).PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between lipid acyl-chains, stabilized by cholesterol, lead to transient submicroscopic nano-domains, and head-group interactions of charged lipids with divalent ions may cluster lipids. In addition, Membrane cytoskeleton may impose further structure on Membrane. Here we study influence of calcium on formation of PIP2 clusters in intact cells. We study changes of domains over time and upon calcium channel activation by analyzing diffusion of GFP-tagged inner-Leaflet Membrane proteins. Using bimFCS, developed by us, we measure diffusion on multiple length scales simultaneously to derive information about domains. To study formation of PIP2 clusters in the PM, we use GFP-PHPLCdelta to directly mark PIP2. We observe that opening TRPV1 channels leads to a transient rise in calcium as imaged using GCaMP5G, increases interaction strength between GFP-PHPLCdelta and PIP2 domains, which decreases to base lines within 1-2 minutes. Using ionophores, we determine that the increasing interaction strength is due to calcium-PIP2 interaction. By Vinculin depletion or actin filaments (de-)polymerization, we find that both anchor proteins and actin filaments play important roles in PIP2 clusters, and the formation of larger PIP2 clusters can potentially further activate Plasma Membrane Ca2+ ATPase. These results suggest that in intact cells, calcium ions are able to induce changes of PIP2 clusters that are also regulated by cytoskeleton structure, changes of PIP2 clusters can further affect other signaling events.

  • transient effect of calcium influx on pip2 clusters and cholesterol stabilized nano domains in the inner plasma Membrane Leaflet of intact cells
    Biophysical Journal, 2014
    Co-Authors: Weixiang Jin, Arnd Pralle
    Abstract:

    When calcium ion channels open, local calcium levels are estimated to reach up to 500uM, which may be sufficient to affect negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), in the inner Leaflet of the plasma Membrane (PM). The PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between the acyl-chains of the lipids, stabilized by Cholesterol, lead to transient submicroscopic nano-domains, and lipid head-group interactions of charged lipids with divalent ions may cluster lipids.