DOCSLIB.ORG

Lymphocytes Adhesion Molecules on Human T Redistribution of Antigen

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Lymphocytes Adhesion Molecules on Human T Redistribution of Antigen Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes This information is current as of September 26, 2021. Jason S. Mitchell, Oguz Kanca and Bradley W. McIntyre J Immunol 2002; 168:2737-2744; ; doi: 10.4049/jimmunol.168.6.2737 http://www.jimmunol.org/content/168/6/2737 Downloaded from References This article cites 45 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/168/6/2737.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes1 Jason S. Mitchell, Oguz Kanca, and Bradley W. McIntyre2 The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular ␤ interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell 1 integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an ␤ enrichment of 1 integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of Downloaded from molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is en- ␤ riched in TCR and CD59, while 1 integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, ␤ CD59, CD43, and 1 integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the http://www.jimmunol.org/ normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated. The Journal of Immunology, 2002, 168: 2737–2744. he lymphocyte cell surface membrane consists of micro- as Ras, Lck, Grb-2, phosphatidylinositol-3 kinase, and Fyn (2, scopically and biochemically distinguishable lipid mi- 4–7). Lipid rafts/microdomains are also believed to have a func- T crodomains that result from the assembly of sphingolipids tional significance, because cells with cholesterol depleted from and cholesterol into laterally mobile rafts (1, 2). Sphingolipids lipid microdomains have decreased levels of basal adhesion and contain predominately large, saturated acyl chains that allow them decreased CD3-induced TCR␨ phosphorylation (8, 9). Further- to pack tightly together. Furthermore, phase separations between more, lipid raft/microdomain patching can induce Ca2ϩ flux, by guest on September 26, 2021 lipid microdomains and loosely ordered membrane glycerophos- Z-chain associated-70 kDa protein/linker for activation of T cells/ pholipids give lipid microdomains a high degree of lateral mobility extracellular signal-regulated kinase-2 phosphorylation, and within the membrane (3). These lipid microdomains are also NFAT stimulation (10). The partitioning of these signaling mole- known as lipid rafts or glycolipid-enriched membrane domains cules to laterally mobile sphingolipids has been proposed as a and, due to their insolubility in nonionic mild detergents, they are means for signaling molecules to specifically “traffic” to areas of also called detergent-insoluble glycolipid-enriched domains receptor engagement and initiate a variety of different signaling (DIGs)3 or detergent-resistant membranes (4). Lipid microdomains pathways (1, 3). have a low density, and sucrose gradient ultracentrifugation of Lipid microdomains can be detected with the B-subunit of chol- mild detergent cell lysates can isolate these microdomains into era toxin (CTB), the membrane-binding subunit that binds a major low-density fractions along with the molecules that are associated component of lipid rafts, GM1 gangliosides (11, 12). Costimula- with them. Western blot analysis of these low-density fractions has tion of naive T lymphocytes with anti-CD3 and anti-CD28 beads revealed the presence of many GPI-anchored proteins, such as results in redistribution of GM1 lipids from a diffuse distribution to Thy-1, Ly-6, and CD59, and intracellular signaling proteins such a concentration at the bead contact site, as visualized by FITC- CTB. It was also determined that cross-linking GPI-anchored CD59, a raft/microdomain-associated lymphocyte surface protein, Department of Immunology, University of Texas, M. D. Anderson Cancer Center, or, more importantly, cross-linking of GM1 directly with immo- Houston, TX 77030 bilized CTB provided efficient T cell costimulation (13). The im- Received for publication July 10, 2001. Accepted for publication January 14, 2002. plication of these studies was that costimulation is mediated by The costs of publication of this article were defrayed in part by the payment of page lipid microdomains. Furthermore, the integrin LFA-1 was shown charges. This article must therefore be hereby marked advertisement in accordance to colocalize to membrane rafts/microdomains, and high-avidity with 18 U.S.C. Section 1734 solely to indicate this fact. adhesion could be induced by clustering these membrane microdo- 1 This work was supported by National Institutes of Health Grant CA62596 and Predoctoral Cancer Immunobiology Training Program Grant CA09598. mains (8). Therefore, lipid microdomains may also regulate lym- 2 Address correspondence and reprint requests to Dr. Bradley W. McIntyre, Depart- phocyte intercellular interactions needed for efficient adhesion to ment of Immunology University of Texas, M. D. Anderson Cancer Center, 1515 APCs during immune recognition or for adhesion to endothelial Holcombe Boulevard, Box 180, Houston, TX 77030. E-mail address: bmcintyr@ cells to mediate lymphocyte migration and recirculation. The bio- mail.mdanderson.org chemical evidence available so far has substantially implicated 3 Abbreviations used in this paper: DIG, detergent-insoluble glycolipid-enriched do- main; CTB, cholera toxin B-subunit; HPB-ALL, human peripheral blood acute lym- lipid rafts/microdomains in the normal function of T cells (14–16). phocytic leukemia; IODO-GEN, 1,3,4,6-tetrachloro-3␣,6␣,diphenylglycoluril. However, the information from this type of analysis may be Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 2738 REORGANIZATION OF LYMPHOCYTE SURFACE MICRODOMAINS limited because the use of detergents in these experiments may sucrose gradients and the collected fractions were immunoprecipitated with ␤ partially solubilize some lipid microdomains even at low temper- protein G-agarose beads (Pierce) precomplexed with the anti- 1 integrin atures, disrupting low-affinity interactions and transient associa- mAb 18D3. Polypeptides were eluted by boiling in reducing Laemmli sam- ple buffer and separated by 7.5% SDS-PAGE. Dried gels were exposed to tions (3, 15). Because lipid microdomain trafficking is based on Kodak XR film (Kodak, Rochester, NY) at Ϫ80°C. molecular compartmentalization, fluorescence microscopy has be- come an invaluable tool to visualize lipid microdomain associa- Fluorescence staining and image analysis tions and movements. In this paper we have investigated the spatial In the GM1 internalization experiments, HPB-ALL T cells were stained in relationships of various T cell adhesion and activation molecules complete RPMI 1640 medium with FITC-conjugated CTB (Sigma-Al- in the context of Ab cross-linking of GM1 lipid microdomains. drich) at 25 ␮g/ml for1hat37°C. The cells were then washed twice with Using CTB to follow lipid microdomains and specific fluorescent complete medium, fixed for 45 min with 4% paraformaldehyde, perme- abilized gently with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, mAbs to follow surface molecules, we have found that clustering and stained with specific monoclonal (10 ␮g/ml) and AlexaFluor 594 goat lipid microdomains at physiological temperatures causes a dy- anti-mouse Abs. Finally, the cells were mounted to glass slides with Pro- namic rearrangement of normal T cell microdomain organization long Antifade reagents (Molecular Probes).
Load more

Recommended publications
  • Biological Membranes
    14 Biological Membranes To understand the structure The fundamental unit of life is the cell. All living things are composed of Goal and composition of biological cells, be it a single cell in the case of many microorganisms or a highly membranes. organized ensemble of myriad cell types in the case of multicellular organisms. A defining feature of the cell is a membrane, the cytoplasmic Objectives membrane, that surrounds the cell and separates the inside of the cell, the After this chapter, you should be able to cytoplasm, from other cells and the extracellular milieu. Membranes also • distinguish between cis and trans surround specialized compartments inside of cells known as organelles. unsaturated fatty acids. Whereas cells are typically several microns (μm) in diameter (although • explain why phospholipids some cells can be much larger), the membrane is only about 10 nanometers spontaneously form lipid bilayers and (nm) thick. Yet, and as we will see in subsequent chapters, the membrane is sealed compartments. not simply an ultra-thin, pliable sheet that encases the cytoplasm. Rather, • describe membrane fluidity and how it membranes are dynamic structures that mediate many functions in the is affected by membrane composition life of the cell. In this chapter we examine the composition of membranes, and temperature. their assembly, the forces that stabilize them, and the chemical and physical • explain the role of cholesterol in properties that influence their function. buffering membrane fluidity. The preceding chapters have focused on two kinds of biological molecules, • explain how the polar backbone namely proteins and nucleic acids, that are important in the workings of a membrane protein can be accommodated in a bilayer.
    [Show full text]
  • Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy
    Int. J. Mol. Sci. 2013, 14, 3514-3539; doi:10.3390/ijms14023514 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy Simon J. Attwood 1, Youngjik Choi 2 and Zoya Leonenko 1,2,3,* 1 Department of Physics and Astronomy, University of Waterloo, Waterloo, ON N2L 3G1, Canada; E-Mail: [email protected] 2 Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada; E-Mail: [email protected] 3 Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1, Canada * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-519-888-4567. Received: 31 December 2012; in revised form: 29 January 2013 / Accepted: 1 February 2013 / Published: 6 February 2013 Abstract: Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented.
    [Show full text]
  • Bioactive Dietary Long-Chain Fatty Acids: Emerging Mechanisms of Action 170.106.202.226
    Downloaded from https://www.cambridge.org/core British Journal of Nutrition (2008), 100, 1152–1157 doi:10.1017/S0007114508992576 q The Authors 2008 Review Article . IP address: Bioactive dietary long-chain fatty acids: emerging mechanisms of action 170.106.202.226 Robert S. Chapkin1,2,3*, David N. McMurray1,2,4, Laurie A. Davidson1,2, Bhimanagouda S. Patil1,3, Yang-Yi Fan1 and Joanne R. Lupton1,2 , on 25 Sep 2021 at 18:00:38 1Faculty of Nutrition, Texas A&M University, College Station, TX, USA 2Center for Environmental and Rural Health, Texas A&M University, College Station, TX, USA 3Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX, USA 4Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, College Station, TX, USA (Received 1 February 2008 – Revised 28 March 2008 – Accepted 31 March 2008 – First published online 20 May 2008) , subject to the Cambridge Core terms of use, available at The plasma membranes of all eukaryotic cells contain heterogeneous self-organising intrinsically unstable liquid ordered domains or lipid assem- blies in which key signal transduction proteins are localised. These assemblies are classified as ‘lipid rafts’ (10–200 nm), which are composed mostly of cholesterol and sphingolipid microdomains and therefore do not integrate well into the fluid phospholipid bilayers. In addition, caveolae represent a subtype of lipid raft macrodomain that form flask-shaped membrane invaginations containing structural proteins, i.e. caveolins. With respect to the diverse biological effects of long-chain PUFA, increasing evidence suggests that n-3 PUFA and perhaps conjugated fatty acids uniquely alter the basic properties of cell membranes.
    [Show full text]
  • Lipid Raft Formation and Peptide-Lipid Interactions in Myelin Model Membranes
    Wilfrid Laurier University Scholars Commons @ Laurier Theses and Dissertations (Comprehensive) 2012 Lipid raft formation and peptide-lipid interactions in myelin model membranes Ashtina R. Appadu Wilfrid Laurier University, [email protected] Follow this and additional works at: https://scholars.wlu.ca/etd Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Appadu, Ashtina R., "Lipid raft formation and peptide-lipid interactions in myelin model membranes" (2012). Theses and Dissertations (Comprehensive). 1124. https://scholars.wlu.ca/etd/1124 This Thesis is brought to you for free and open access by Scholars Commons @ Laurier. It has been accepted for inclusion in Theses and Dissertations (Comprehensive) by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. Lipid raft formation and peptide-lipid interactions in myelin model membranes By Ashtina Appadu B.Sc. Biochemistry/Biotechnology, Wilfrid Laurier University, 2009 THESIS Submitted to the Department of Chemistry In partial fulfillment of the requirements for Master of Science in Chemistry Wilfrid Laurier University 2012 Ashtina Appadu ©2012 Abstract Multiple sclerosis (MS), a demyelinating disease affecting 75,000 Canadians and almost 400,000 Americans, is one of the most prevalent diseases in young adults. Unfortunately, there exist no known cures to date and the pathways involved in the progression of the disease remain relatively obscure. The demyelination process triggered by the onset of MS, affects the lipid composition of the myelin membrane and causes a loss in viable myelin which can in turn greatly impact the proper functioning of the central nervous system (CNS). The cholesterol content of myelin fluctuates during MS and consequently this could affect the fluidity as well as the lipid microdomain profile in the membranes.
    [Show full text]
  • Carbohydrate-Dependent Signaling from the Phosphatidylglucoside-Based Microdomain Induces Granulocytic Differentiation of HL60 Cells
    Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells Yasuko Nagatsuka*†, Miki Hara-Yokoyama‡, Takeshi Kasama§, Masataka Takekoshi¶, Fumiko Maeda¶, Seiji Ihara¶, Shigeyoshi Fujiwaraʈ, Eriko Ohshima**, Kumiko Ishii††, Toshihide Kobayashi††, Kazufumi Shimizu*, and Yoshio Hirabayashi†** *Department of Microbiology, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan; ‡Department of Hard Tissue Engineering and §Laboratory of Biochemical Analysis, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; ¶Department of Molecular Life Science 2, Tokai University School of Medicine, Isehara 259-1193, Japan; ʈDepartment of Infectious Diseases, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo 154-8567, Japan; and ††Sphingolipid Functions Laboratory, Supra-Biomolecular System Research Group, Frontier Research System and **Neuronal Circuit Mechanisms Research Group, Brain Science Institute, The Institute of Physical and Chemical Research, Wako-shi, Saitama 351-0198, Japan Communicated by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, April 24, 2003 (received for review October 3, 2002) Glycosphingolipids form glycosphingolipid signaling microdo- (PhGlc), that is present in DIM fractions from the promyelocytic mains. Here, we report an unrecognized type of phosphatidylglu- cell line HL60. This lipid was first isolated from human umbilical coside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cord red blood cells (RBCs) as an antigen detected by a human cells with rGL-7, which preferentially reacts with PhGlc, induced monoclonal antibody (GL-2) against blood group antigen, i. differentiation of HL60 cells. This was manifested by the appear- Studies using GL-2 unexpectedly revealed that one of the target ance of nitroblue tetrazolium-positive cells together with CD38 antigens was not GSL, but rather an alkaline-labile lipid.
    [Show full text]
  • Regulation of Monoamine Transporters and Receptors by Lipid Microdomains: Implications for Depression
    www.nature.com/npp REVIEW ARTICLE Regulation of monoamine transporters and receptors by lipid microdomains: implications for depression Joanne J. Liu1,7, Adrienne Hezghia1,8, Saame Raza Shaikh2, Joshua F. Cenido 1,9, Ruth E. Stark 3,4, J. John Mann1,5,6 and M. Elizabeth Sublette1,5 Lipid microdomains (“rafts”) are dynamic, nanoscale regions of the plasma membrane enriched in cholesterol and glycosphingolipids, that possess distinctive physicochemical properties including higher order than the surrounding membrane. Lipid microdomain integrity is thought to affect neurotransmitter signaling by regulating membrane-bound protein signaling. Among the proteins potentially affected are monoaminergic receptors and transporters. As dysfunction of monoaminergic neurotransmission is implicated in major depressive disorder and other neuropsychiatric conditions, interactions with lipid microdomains may be of clinical importance. This systematic review evaluates what is known about the molecular relationships of monoamine transporter and receptor regulation to lipid microdomains. The PubMed/MeSH database was searched for original studies published in English through August 2017 concerning relationships between lipid microdomains and serotonin, dopamine and norepinephrine transporters and receptors. Fifty-seven publications were identified and assessed. Strong evidence implicates lipid microdomains in the regulation of serotonin and norepinephrine transporters; serotonin 1A, 2A, 3A, and 7A receptors; and dopamine D1 and β2 adrenergic receptors. Results were conflicting or more complex regarding lipid microdomain associations with the dopamine transporter, D2, D3, and D5 receptors; and negative with respect to β1 adrenergic receptors. Indirect evidence suggests that antidepressants, lipid-lowering drugs, and polyunsaturated fatty acids may exert effects on depression and suicide by altering the lipid milieu, thereby affecting monoaminergic transporter and receptor signaling.
    [Show full text]
  • Fatty Acids As Gatekeepers of Immune Cell Regulation
    Review TRENDS in Immunology Vol.24 No.12 December 2003 639 Fatty acids as gatekeepers of immune cell regulation Parveen Yaqoob Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Whiteknights PO Box 226, Reading, RG6 6AP, UK Fatty acids have diverse roles in all cells. They are PUFA, EPA and DHA, and are the chief sources of these important as a source of energy, as structural com- fatty acids. ponents of cell membranes, as signalling molecules and Fatty acids have diverse roles in all cells. They are as precursors for the synthesis of eicosanoids. Recent important as a source of energy, as structural components research has suggested that the organization of fatty of cell membranes and as signalling molecules. In addition, acids into distinct cellular pools has a particularly arachidonic acid and EPA can both serve as precursors for important role in cells of the immune system and that the synthesis of eicosanoids. In 1978, Meade and Mertin forms of lipid trafficking exist, which are as yet poorly [1] published the first, and somewhat speculative, review understood. This Review examines the nature and regu- addressing the role of fatty acids in the immune system. In lation of cellular lipid pools in the immune system, their the 25 years that have elapsed since, there have been delivery of fatty acids or fatty acid derivatives to specific significant developments in our understanding of the locations and their potential role in health and disease. organization of lipids within cells, some of which are particularly relevant to immune regulation.
    [Show full text]
  • A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2 Dmitry Lupyan Mount Sinai School of Medicine
    Virginia Commonwealth University VCU Scholars Compass Physiology and Biophysics Publications Dept. of Physiology and Biophysics 2010 A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2 Dmitry Lupyan Mount Sinai School of Medicine Mihaly Mezei Mount Sinai School of Medicine Diomedes E. Logothetis Mount Sinai School of Medicine, Virginia Commonwealth University Roman Osman Mount Sinai School of Medicine Follow this and additional works at: http://scholarscompass.vcu.edu/phis_pubs Part of the Medicine and Health Sciences Commons From The iopB hysical Journal, Lupyan, D., Mezei, M., Logothetis, D.E., et al., A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2, Vol. 98, Page 240. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. Reprinted with permission. Downloaded from http://scholarscompass.vcu.edu/phis_pubs/25 This Article is brought to you for free and open access by the Dept. of Physiology and Biophysics at VCU Scholars Compass. It has been accepted for inclusion in Physiology and Biophysics Publications by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. 240 Biophysical Journal Volume 98 January 2010 240–247 A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2 Dmitry Lupyan,† Mihaly Mezei,† Diomedes E. Logothetis,†‡ and Roman Osman†* †Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York; and ‡Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia ABSTRACT Phosphoinositides like phosphatidylinositol 4,5-bisphosphate (PIP2) are negatively charged lipids that play a pivotal role in membrane trafficking, signal transduction, and protein anchoring. We have designed a force field for the PIP2 headgroup using quantum mechanical methods and characterized its properties inside a lipid bilayer using molecular dynamics simulations.
    [Show full text]
  • Lipid Rafts in Arabidopsis Thaliana Leaves
    Lipid rafts in Arabidopsis thaliana leaves Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universitat¨ Wurzburg¨ vorgelegt von Fatih Demir aus Mannheim Wurzburg¨ 2010 Pr¨ufungskomission Eingereicht am: 30. September 2010 Vorsitzender: Prof. Dr. Thomas Dandekar 1. Gutachter: Prof. Dr. Rainer Hedrich 2. Gutachter: Prof. Dr. Gregory Harms Tag des Promotionskolloquiums: Doktorurkunde ausgeh¨andigtam: iii Acknowledgments ? First, I would like to thank Prof. Dr. Rainer Hedrich for giving me the opportunity to carry out this research project in a vibrant and stimulating working group with great chances and responsibilities. And for his support whenever I needed it. ? Prof. Dr. Gregory Harms for being my second referee and bearing my contempt in writing an English PhD thesis as a non-native speaker. ? My supervisors Dr. Ines Kreuzer for the supervision of the project and being critical on lipid raft topics and Dr.es J¨org& Yvonne Reinders for teaching me mass spectrometry and the advantages of having a working pre-column which was not stiffed with lipids of all kinds. All the mass spectrometric measurements and data were acquired by a Demir-Reinders cooperation. I also greatly appreciate the technicians and PhD students in the Reinders lab in Regensburg who performed the practical sample preparation for mass spectrometry. ? J¨orgBlachutzik conducted microscopy and revealed all the nice co-localization data which appeared in our publication and also in this thesis { thanks for the bright spots on a dark background. ? My molecular biology capacities, Dr. Dietmar Geiger & his group members for the gener- ation of binary fusion constructs of ABI1, CPK21 and SLAH3.
    [Show full text]
  • Lipid Phase Separation in Phospholipid Bilayers and Monolayers Modeling the Plasma Membrane
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1512 (2001) 317^328 www.bba-direct.com Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane Saame Raza Shaikh, Alfred C. Dumaual, Laura J. Jenski, William Stillwell * Department of Biology, Indiana University-Purdue University at Indianapolis, 723 W. Michigan Street, Indianapolis, IN 46202-5132, USA Received 28 September 2000; received in revised form 5 April 2001; accepted 5 April 2001 Abstract It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techni- ques are employed, pressure^area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At XSDPE v 0.15 in SM, a more horizontal region emerges in the pressure^ area isotherms indicating `squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of `squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase.
    [Show full text]
  • Hydrodynamic Effects on Spinodal Decomposition Kinetics in Lipid
    Hydrodynamic effects on spinodal decomposition kinetics in lipid bilayer membranes Jun Fan and Tao Han Department of Mechanical and Aerospace Engineering, Princeton University, Princeton NJ 08544 Mikko Haataja Department of Mechanical and Aerospace Engineering, Princeton Institute for the Science and Technology of Materials (PRISM), and Program in Applied and Computational Mathematics (PACM), Princeton University, Princeton NJ 08544 (Dated: August 11, 2010) Abstract The formation and dynamics of spatially extended compositional domains in multicomponent lipid membranes lie at the heart of many important biological and biophysical phenomena. While the thermodynamic basis for domain formation has been explored extensively in the past, domain growth in the presence of hydrodynamic interactions both within the (effectively) two-dimensional membrane and in the three-dimensional solvent in which the membrane is immersed has received little attention. In this work, we explore the role of hydrodynamic effects on spinodal decom- position kinetics via continuum simulations of a convective Cahn-Hilliard equation for membrane composition coupled to the Stokes equation. Our approach explicitly includes hydrodynamics both within the planar membrane and in the three-dimensional solvent in the viscously dominated flow regime. Numerical simulations reveal that dynamical scaling breaks down for critical lipid mixtures due to distinct coarsening mechanisms for elongated versus more isotropic compositional lipid do- mains. The breakdown in scaling should be readily observable in experiments on model membrane systems. PACS numbers: 87.14.Cc, 87.15.A-, 87.16.D- 1 I. INTRODUCTION Lipid bilayer membranes are ubiquitous in mammalian cells, and facilitate the interaction between a cell and its surroundings. Furthermore, lipid membranes are employed in impor- tant biomedical applications, such as vehicles for targeted drug delivery.
    [Show full text]
  • Dynamic, Yet Structured: the Cell Membrane Three Decades After the Singer–Nicolson Model
    REVIEW Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model G. Vereb*†, J. Szo¨ llo˝si*†‡, J. Matko´ §, P. Nagy*, T. Farkas¶,L.Vı´gh¶,L.Ma´ tyus*, T. A. Waldmannʈ, and S. Damjanovich*‡** *Department of Biophysics and Cell Biology and ‡Cell Biophysical Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, H-4012, Debrecen, Hungary; §Department of Immunology, Lora´ndEo¨ tvo¨s University, H-1117, Budapest, Hungary; ¶Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, H-6701, Szeged, Hungary; and ʈMetabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1374 Contributed by T. Farkas, April 29, 2003 The fluid mosaic membrane model proved to be a very useful metric distribution in the membrane bilayer all indicate a hypothesis in explaining many, but certainly not all, phenomena molecular heterogeneity and the possible formation of mem- taking place in biological membranes. New experimental data brane microdomains (12). show that the compartmentalization of membrane components can be as important for effective signal transduction as is the Advanced Cell Biophysical and Molecular Biological fluidity of the membrane. In this work, we pay tribute to the Methodology Provides Quantitative Data on the Static and Singer–Nicolson model, which is near its 30th anniversary, honor- Dynamic Organization of Membranes ing its basic features, ‘‘mosaicism’’ and ‘‘diffusion,’’ which predict Membrane dynamics, i.e., the ever changing mobility and prox- the interspersion of proteins and lipids and their ability to undergo imity relationships of lipid and protein molecules in the plasma dynamic rearrangement via Brownian motion.
    [Show full text]