Difffusion Dynamics of AChR Receptors on Live Muscle Cell Membrane

Sunday, February 28, 2016 426-Pos Board B206 In Silico Modeling of Biologically Complex Membranes Helgi I. Ingolfsson1, Manuel N. Melo1, Svetlana Baoukina2, Tsjerk A. Wassenaar1, Xavier Periole1, Alex H. de Vries1, D. Peter Tieleman2, Siewert J. Marrink1. 1 University of Groningen, Groningen, Netherlands, 2University of Calgary, Calgary, AB, Canada. The detailed lipid organization of cellular membranes remains elusive. A typical plasma membrane contains hundreds of different lipid species that are actively regulated by the cell. Currently over 40,000 biologically relevant lipids have been identified and specific organisms often synthesize thousands of different lipid types. This is far greater diversity than is needed to maintain bilayer barrier properties and to solvate membrane proteins. Why do organisms go through the costly process of creating and maintaining such a large diversity of lipids? What is the individual role of these lipids, and how do they interact and organize in the membrane plane? To start to address these questions we model biologically realistic membranes using coarse-grained Martini molecular dynamics simulations. We optimized and developed the Martini lipidome and systematically explored physiochemical properties of >100 different Martini lipid types. Bulk properties of each type (e.g. bilayer thickness, area per lipid, diffusion, order parameter and area compressibility) were analyzed and overall trends compared to experimental values. Biologically realistic idealized membrane compositions were constructed and simulated, such as in (Ingo´lfsson, et al. Lipid organization of the plasma membrane. JACS, 136:14554-14559, 2014). These large-scale simulations (~70 by 70 nm and multi microsecond long) are in terms of lipid composition by an order of magnitude the most complex simulations to date. They provide a high-resolution view of the lipid organization of biologically relevant membranes; revealing a complex global non-ideal lipid mixing of different species at different spatiotemporal scales. We analyze a variety of membrane physicochemical properties, including: lipid-lipid interactions, bilayer bulk material properties, domain formation and coupling between the bilayer leaflets, for a number of lipid mixtures and conditions. 427-Pos Board B207 Vitamin E Promotes the Inverse Hexagonal Phase via a Novel Mechanism: Implications for Antioxidant Role Paul E. Harper1, Andres T. Cavazos2, Jacob J. Kinnun2, Horia I. Petrache2, Stephen R. Wassall2. 1 Department of Physics and Astronomy, Calvin College, Grand Rapids, MI, USA, 2Department of Physics, Indiana University, Purdue University Indianapolis, Indianapolis, IN, USA. Vitamin E (alpha-tocopherol) protects polyunsaturated membrane phospholipids from oxidation. How it accomplishes this task at relatively low concentrations is an ongoing area of investigation. An interesting property of alphatocopherol is that it promotes the inverse hexagonal (HII) phase in PE membranes. It has been well established that other compounds, such as dodecane and similar purely hydrophobic compounds, promote the HII phase by relieving extensive stress. We argue that alpha-tocopherol promotes the HII phase by a novel mechanism, by instead relieving compressive stress. With this new understanding, we examine the hypothesis that alpha-tocopherol will preferentially partition close to polyunsaturated lipids to maximize its effectiveness as an antioxidant. 428-Pos Board B208 Anticancer Drug Colchicine Increases Disorder and Reduces Complexity in the Macrophage Membrane Arkady Bitler1,2, Ron Dover3, Yechiel Shai3. 1 Department of Physics & Institute of Nanotechnology, Bar Ilan University, Ramat Gan, Israel, 2Weizmann Institute of Science, Rehovot, Israel, 3 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel. Macrophages are part of the immune system and play critical role in the host defense and tissue homeostasis ingesting a dead tissue and fighting invading pathogens. Exploring interactions of anticancer therapies with macrophages as part of innate immunity is important both for biomedical researches and applications and can help to combine various approaches with immunotherapy. In this study we evaluated the influence of anticancer drug colchicine on the disorder and complexity of the macrophage membrane from atomic force microscope (AFM) images. Fixed and dried mouse RAW 264.7 macrophage membranes were imaged with AFM operating in Peak Force mode. The disorder of the membrane was characterized by entropy and complexity by fractal dimension. These parameters were calculated for a set of AFM images of untreated macrophages and macrophages pretreated with anticancer

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drug colchicine, using three different methods. Processing of AFM images and calculations were done with custom MATLAB code. We show that colchicine treatment yields entropy increase and therefore produces higher disorder of the macrophage membrane. Furthermore the membrane complexity is reduced demonstrating lower fractal dimension. In addition we studied also changes in the macrophage membrane disorder and complexity produced by microtubule stabilizing agent taxol, having opposite to the colchicine (inhibiting the microtubule polymerization) effect. These results demonstrate at the level of single cell that anticancer drug colchicine affects macrophage membrane structures producing more disordered state. Finally we discuss some possible consequences of this more disordered state on the macrophage activity. 429-Pos Board B209 Hydration Mediated G-Protein-Coupled Receptor Activation Udeep Chawla1, Suchithranga M.D.C. Perera1, Andrey V. Struts1,2, Michael C. Pitman1, Michael F. Brown1,3. 1 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, 2Laboratory of Biomolecular NMR, St. Petersburg State University, St. Petersburg, Russian Federation, 3Department of Physics, University of Arizona, Tucson, AZ, USA. G-protein-coupled receptors (GPCRs) comprise about 50% of the known drug targets and play crucial roles in numerous of physiological processes. Rhodopsin is a canonical GPCR, which upon photoactivation undergoes a series of conformational changes leading to a chemical equilibrium between closed inactive Meta-I and open active Meta-II states [1,2]. We hypothesize that activation of rhodopsin leads to an increase in hydrated protein volume in the active Meta-II state. Using UV-visible spectroscopy, we tested our hypothesis by evaluating how the Meta-I/Meta-II equilibrium and thermodynamic parameters are influenced by changing the membrane environment. We discovered a surprising bulk water influx (about 75 water molecules) during formation of the active protein. Moreover, our results with high molar mass osmolytes differ from previous osmotic stress studies with small osmolytes. We discovered that osmolytes of varying molar mass affect rhodopsin activation differently. Large osmolytes shift the equilibrium to the inactive Meta-I state, leading to efflux of water. By contrast, small osmolytes lead to an influx of water upon activation. We propose that small osmolytes affect rhodopsin activation similarly to the G-protein transducin, which stabilizes the active Meta-II state. Large osmolytes cannot gain access to the transducin binding site and exert osmotic stress on the protein. Our results are in agreement with molecular dynamics studies showing an influx of water during rhodopsin photoactivation [3]. Hence we propose that rapid high-fidelity signaling by rhodopsin involves cycling of water into and out of the protein core together with activation of transducin. Our studies give important insight in the role of water in activation of GPCRs like rhodopsin. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A. V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [3] N. Leioatts et al. (2013) Biochemistry 53, 376-385. 430-Pos Board B210 Difffusion Dynamics of AChR Receptors on Live Muscle Cell Membrane Wei He1, Hao Song1, Lin Geng2, H. Benjamin Peng2, Penger Tong1. 1 Department of Physics, HKUST, Hong Kong, China, 2Division of Life Science, HKUST, Hong Kong, China. The dynamic structure of a cell membrane allows it to become an effective platform for various biological functions, such as signal transduction, molecule transportation and endocytosis. We report here a single-molecular tracking experiment on a quantum-dot-labeled transmembrane protein, acetylcholine receptor (AChR), in cultured Xenopus muscle cells. We carried out a complete statistical analysis on a large set of AChR trajectories with more than 500 cells examined. Various drug treatments were used to perturb F-actin and scaffold proteins and examine their roles in regulating the motion of the AChRs. The diffusion dynamics of AChRs was characterized by three quantities: the mean-square displacement hDr2(t)i, the probability density function P(Dx) of instantaneous displacement Dx(t) and the probability distribution f(d) of instantaneous diffusion coefficient d. After a careful analysis, we conclude that (1) AChRs show a hindered motion by the surrounding membrane molecules at short time and become diffusive at long time. (2) The mobile AChRs have a broad distribution in diffusion coefficient d with a long exponential tail, which is universal and independent of different sample conditions. (3) The exponential distribution f(d) leads to an exponential distribution P(Dx). Our measurements of membrane diffusion based on a large number of single molecular trajectories provide a complete statistical description of dynamic heterogeneity on live cell membrane. By combining all the experimental results available, we propose a dynamic picket-fence model of membrane organization involving slow active remodeling of the underlying cortical actin network to

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explain the observed non-Gaussian statistics and dynamic heterogeneity. (Work was supported by the Research Grants Council of Hong Kong SAR.) 431-Pos Board B211 Cholesterol’s Aliphatic Side Chain Structure Modulates Membrane Properties Daniel Huster1, Thomas Meyer1, Jo¨rg Nikolaus2, Dong Jae Baek3, Ivan Haralampiev2, Robert Bittman4, Peter Mu¨ller2, Andreas Herrmann2, Holger A. Scheidt1. 1 Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany, 2Institute of Biology/Biophysics, Humboldt University Berlin, Berlin, Germany, 3College of Pharmacy, Mokpo National University, Mokpo, Korea, Republic of, 4Department of Chemistry and Biochemistry, Queens College of CUNY, Flushing, NY, USA. The influence of the length of the cholesterol alkyl chain on essential membrane properties such as lipid ordering (condensation), lateral diffusion, membrane permeability, and membrane domain formation was studied using a series of synthetic cholesterol derivatives without a side chain or with an iso-branched chain bearing 5 to 14 carbons in length. We found that cholesterol’s aliphatic side chain is crucial for all of the membrane properties investigated. First, we detected that the side chain is responsible for more than half of the phospholipid condensation in bilayers. Second, the length of the sterol side chain strongly determines membrane permeation, since a slightly longer or shorter side chain renders the bilayer significantly more permeable. Third, lateral lipid and sterol diffusion highly depends on the side chain length. Fourth, the length of the cholesterol side chain also determines the lateral organization of lipids in raft mixtures. While all tested sterols induce domain formation, the lipid distribution between the liquid disordered or raft phase is strongly dependent on side chain length. Fifth, the sterol side chain also influences the partitioning of a transmembrane peptide domain into liquid-ordered or liquid-disordered domains. Therefore, in addition to the flat tetracyclic ring system of cholesterol, the iso-branched side chain of cholesterol plays a key role in producing the relevant membrane properties of eukaryotic cells that result in an optimal interaction with phospholipids and presumably with membrane proteins. Sterols bearing an unbranched alkyl side chain of varying length also influence important properties of lipid membranes. However, the extent of this impact is lower compared with that measured for the respective branched iso-side chain sterols. Obviously, sterols having a branched iso-chains with two terminal methyl groups exhibit altered cholesterol-phospholipid-interactions compared to molecules with a straight unbranched chain. 432-Pos Board B212 Content of Plasmalogen Lipids Markedly Decreases in Barth Syndrome Tomohiro Kimura1, Atsuko Kimura1, Bob Berno2, Mindong Ren3, Michael Schlame3, Richard M. Epand1. 1 Biochemistry, McMaster University, Hamilton, ON, Canada, 2Chemistry, McMaster University, Hamilton, ON, Canada, 3Department of Cell Biology, NYU Langone Medical Center, New York, NY, USA. Barth Syndrome, with a major symptom of dilated cardiomyopathy, is caused by mutations in the enzyme tafazzin that catalyzes acylchain exchange between phospholipids and lysophospholipids. In a tafazzin-deficient heart, the acyl chain composition of cardiolipin, an essential component of cardiac lipids, becomes heterogeneous unlike in a normal heart with a single dominant species. In addition, cardiolipin decreases and monolysocardiolipin appears. In this work we have compared the lipid composition of hearts obtained from normal mice with that of hearts obtained from tafazzin knock-down mice. An extensive series of 31P NMR experiments using a cryoprobe on the biological materials with different solution conditions enabled us to determine detailed compositions of phospholipids in heart tissue. In addition to confirming that hearts obtained from tafazzin knock-down mice had less cardiolipin and detectable amounts of monolysocardiolipin, we found a decrease in plasmenylcholine. While the decrease in both cardiolipin and plasmenylcholine were large, both ~35%, the decrease in plasmenylcholine in terms of changes in the total lipid content was drastic, ~ 11% (32.4/21.5%); while the decrease in cardiolipin was only 2.3% (6.3/4.0%). In order to obtain an accurate and stable value for the amount of plasmalogen from highly resolved NMR resonances, it was required to have both a chelating agent EDTA and an antioxidant BHT present. Different tissues of the same mammalian species vary with regard to the relative amounts of the two principle forms of plasmalogen: plasmenylcholine and plasmenylethanolamine. Heart contains mostly plasmenylcholine, while lymphoblasts have principally plasmenylethanolamine. As a system directly related to the disease, we also studied human lymphoblasts derived from individuals with Barth Syndrome vs. normal controls. In this case we observed a marked decrease in the plasmenylethanolamine in mitochondria from individuals with Barth Syndrome.

433-Pos Board B213 Oxidation of Cholesterol Changes the Physical Properties of Lipid Membranes Waldemar Kulig1, Agnieszka Olzynska2, Piotr Jurkiewicz2, Anu M. Kantola3, Moutusi Manna1, Mohsen Pourmousa1, Mario Vazdar1,4, Lukasz Cwiklik2,5, Tomasz Rog1, George Khelashvili6, Daniel Harries7, Ville-Veikko Telkki3, Martin Hof2, Ilpo Vattulainen1,8, Pavel Jungwirth1,5. 1 Department of Physics, Tampere University of Technology, Tampere, Finland, 2J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3Department of Physics and Chemistry, University of Oulu, Oulu, Finland, 4Rudjer Boskovic Institute, Division of Organic Chemistry and Biochemistry, Zagreb, Croatia, 5Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 6Weill Cornell Medical College, New York, NY, USA, 7Institute of Chemistry and the Fritz Haber Research Center, The Hebrew University of Jerusalem, Jerusalem, Israel, 8MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. Oxysterols are oxidative derivatives of cholesterol playing a crucial role in many regulatory processes in the human body. They are produced naturally from cholesterol during an enzymatic side chain hydroxylation (catalysed by cytochrome P450) or non-enzymatic oxidation (due to direct exposure of cholesterol to reactive oxygen species). Using several experimental techniques, including dynamic light scattering, fluorescence spectroscopy, and NMR, together with atomistic molecular dynamics simulations and a quantitative description of molecular tilt, we characterized the behavior of a family of oxysterols in phospholipid membranes and compared the resulting data to that of cholesterol. We found that the two distinct groups of oxysterols, i.e., ring-oxidized sterols (mostly produced by free radicals) and tail-oxidized sterols (mostly produced enzymatically) behave differently in lipid membranes, influencing them in different ways. Unlike tail-oxidized sterols or cholesterol, ring-oxidized sterols can efficiently acquire tilted orientations in the bilayer, leading to a stronger disruption of the membrane structure. These changes in physical properties of lipid bilayers may have implications in membrane biochemistry: tail-oxidized sterols behave similarly to cholesterol in terms of membrane stiffening, indicating that their effect on biochemical processes in membranes is similar to that of cholesterol, while ring-oxidized sterols exhibit behavior distinct from cholesterol, implying potential disruptions in membrane functionality such as in signalling processes. 434-Pos Board B214 Clusters of Cholera Toxin B Subunit on the Outer Leaflet Stabilize Lipid Heterogeneity on the Inner Leaflet of B Cell Membranes Marcos F. Nunez, Sarah L. Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. Protein sorting based on liquid-ordered or liquid-disordered phase preference is readily observed in giant plasma membrane vesicles (GPMVs) at low temperatures but is not observed directly in the intact cells from which they are derived. Here we utilize two-color super-resolution microscopy to quantify the sorting of two minimal inner leaflet anchored-peptides proximal to clusters of cholera toxin B subunit (CTxB) in chemically fixed CH27 B cells. One peptide, called PM, is anchored through saturated palmitoyl and myristoyl acylations, giving it a strong preference for more ordered lipids, while the second peptide, called GG, is anchored through an unsaturated and branched geranylgeranyl modification that gives it a strong preference for more disordered lipids. We find that the local density of PM is increased in the vicinity of CTxB clusters while the local density of GG is reduced in the vicinity of CTxB clusters when compared to the average density of peptides on the cell surface. These results indicate that clustering of CTxB on the outer leaflet affects the lateral organization of proteins anchored to the inner leaflet in a way that depends on the anchoring structure. Ongoing work is aimed at probing the membrane’s ability to sort proteins based on the phase preference of their membrane anchor in response to biochemical perturbations and changes in temperature, with the goal of distinguishing possible physical bases of this lipidmediated membrane heterogeneity. 435-Pos Board B215 Partition Coefficient of a Transmembrane Peptide, between Lo and Ld Phases: Does the Peptide Distinguish Macro from Nano Domains? Thais A. Enoki1,2, Sarah Kim3, Frederick A. Heberle4, Gerald W. Feigenson1. 1 Cornell University, Ithaca, NY, USA, 2University of Sao Paulo, Sao Paulo, Brazil, 3Johns Hopkins University, Baltimore, MD, USA, 4Oak Ridge National Laboratory, Oak Ridge, TN, USA. We measured the partition coefficient of the transmembrane peptide GWALP23 between liquid-ordered (Lo) and liquid-disordered (Ld) domains