{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Campus":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Degree":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","DegreeGrantor":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","Extent":"http:\/\/purl.org\/dc\/terms\/extent","FileFormat":"http:\/\/purl.org\/dc\/elements\/1.1\/format","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","GraduationDate":"http:\/\/vivoweb.org\/ontology\/core#dateIssued","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","Program":"https:\/\/open.library.ubc.ca\/terms#degreeDiscipline","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","Rights":"http:\/\/purl.org\/dc\/terms\/rights","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Medicine, Faculty of","@language":"en"},{"@value":"Biochemistry and Molecular Biology, Department of","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Campus":[{"@value":"UBCV","@language":"en"}],"Creator":[{"@value":"Eastman, Simon J.","@language":"en"}],"DateAvailable":[{"@value":"2008-12-20T01:06:12Z","@language":"en"}],"DateIssued":[{"@value":"1992","@language":"en"}],"Degree":[{"@value":"Doctor of Philosophy - PhD","@language":"en"}],"DegreeGrantor":[{"@value":"University of British Columbia","@language":"en"}],"Description":[{"@value":"It is well established that biological membranes maintain an asymmetric\r\ntransbilayer distribution of component molecules, including lipids. The mechanisms by\r\nwhich this lipid asymmetry is established and maintained are not well understood. In\r\naddition, little is known concerning the biological significance of lipid asymmetry. This\r\nthesis employs large unilamellar vesicle (LUV) model membrane systems to examine the\r\nability of transmembrane pH gradients (\u0394pH) to generate lipid asymmetry and investigate\r\nthe consequences of lipid asymmetry in membrane fusion phenomena.\r\nThe first area of investigation demonstrates that transmembrane pH gradients can\r\ninfluence the inter-vesicular exchange of stearylamine and oleic acid. Vesicles\r\ncontaining stearylamine are shown to aggregate immediately with vesicles containing\r\nphosphatidylserine and disaggregation occurs as stearylamine equilibrates between the\r\ntwo vesicle populations. Despite visible flocculation during the aggregation phase,\r\nvesicle integrity is maintained. It is also shown that stearylamine is the only lipid to\r\nexchange, fusion does not occur and vesicles are able to maintain a pH gradient. When\r\nstearylamine is sequestered to the inner monolayer in response to a transmembrane pH\r\ngradient (inside acidic) aggregation is not observed and diffusion of stearylamine to\r\nacceptor vesicles is greatly reduced. The ability of \u0394pH-dependent lipid asymmetry to\r\nmodulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced\r\nto transfer from one population of vesicles to another by maintaining a basic interior pH\r\nin the acceptor vesicles. It is also shown that the same acceptor vesicles can deplete\r\nserum albumin of bound fatty acid.\r\nThe second area of investigation concerns asymmetric transbilayer distributions\r\nof dioleoylphosphatidic acid (DOPA) induced by transmembrane pH gradients. A fluorescent assay is developed employing 2-(p-toluidinyl)naphthalene-6-sulfonic acid\r\n(TNS) as a probe of lipid asymmetry. The kinetics of DOPA transport are shown to be\r\nconsistent with the transport of the uncharged (protonated) form. Transport of the neutral\r\nspecies can be rapid, exhibiting half-times for transbilayer transport of approximately 25\r\ns at 45\u00b0C. These studies also indicate that the transport of DOPA is associated with a\r\nlarge activation energy (28 Kcal\/mol).\r\nThe third area builds on the ability to generate LUVs with an asymmetric\r\ndistribution of DOPA and concerns studies on the ability of lipid asymmetry to regulate\r\nCa2+ stimulated fusion of LUV systems. It is shown that for LUVs composed of\r\nDOPC:DOPE:PI:DOPA (25:60:5:10 mol\/mol) rapid and essentially complete fusion is\r\nobserved by fluorescent resonance energy transfer techniques when Ca2+ is added.\r\nAlternatively, for LUVs with the same lipid composition but when DOPA has been\r\nsequestered to the inner monolayer, due to the presence of a pH gradient (interior basic),\r\nlittle or no fusion is observed upon addition of Ca2+ It is demonstrated that the extent of\r\nCa2+induced fusion correlates with the amount of exterior DOPA. It is also shown that\r\nLUVs containing only 2.5 mol% DOPA, but when all the DOPA is in the outer\r\nmonolayer, can be induced to fuse to the same extent and with the same initial rate as\r\nLUVs containing 5 mol% DOPA. These results strongly support a regulatory role for\r\nlipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid\r\nbilayers are largely determined by the properties of one monolayer.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/3277?expand=metadata","@language":"en"}],"Extent":[{"@value":"2078937 bytes","@language":"en"}],"FileFormat":[{"@value":"application\/pdf","@language":"en"}],"FullText":[{"@value":"STUDIES OF THE GENERATION AND FUNCTION OF PHOSPHOLIPIDASYMMETRYbySIMON J. EASTMANBSc. Biochemistry, Carleton University, 1986A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BIOCHEMISTRYWe accept this thesis as conformingto the required standardOctober, 1991\u00a9 Simon Eastman, 1991THEIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of BiochemistryThe University of British ColumbiaVancouver, CanadaDate November 4. 1991(Signature)DE.6 (2\/88)ABSTRACTIt is well established that biological membranes maintain an asymmetrictransbilayer distribution of component molecules, including lipids. The mechanisms bywhich this lipid asymmetry is established and maintained are not well understood. Inaddition, little is known concerning the biological significance of lipid asymmetry. Thisthesis employs large unilamellar vesicle (LUV) model membrane systems to examine theability of transmembrane pH gradients (ApH) to generate lipid asymmetry and investigatethe consequences of lipid asymmetry in membrane fusion phenomena.The first area of investigation demonstrates that transmembrane pH gradients caninfluence the inter-vesicular exchange of stearylamine and oleic acid. Vesiclescontaining stearylamine are shown to aggregate immediately with vesicles containingphosphatidylserine and disaggregation occurs as stearylamine equilibrates between thetwo vesicle populations. Despite visible flocculation during the aggregation phase,vesicle integrity is maintained. It is also shown that stearylamine is the only lipid toexchange, fusion does not occur and vesicles are able to maintain a pH gradient. Whenstearylamine is sequestered to the inner monolayer in response to a transmembrane pHgradient (inside acidic) aggregation is not observed and diffusion of stearylamine toacceptor vesicles is greatly reduced. The ability of ApH-dependent lipid asymmetry tomodulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be inducedto transfer from one population of vesicles to another by maintaining a basic interior pHin the acceptor vesicles. It is also shown that the same acceptor vesicles can depleteserum albumin of bound fatty acid.The second area of investigation concerns asymmetric transbilayer distributionsof dioleoylphosphatidic acid (DOPA) induced by transmembrane pH gradients. A11fluorescent assay is developed employing 2-(p-toluidinyl)naphthalene-6-sulfonic acid(TNS) as a probe of lipid asymmetry. The kinetics of DOPA transport are shown to beconsistent with the transport of the uncharged (protonated) form. Transport of the neutralspecies can be rapid, exhibiting half-times for transbilayer transport of approximately 25s at 45\u00b0C. These studies also indicate that the transport of DOPA is associated with alarge activation energy (28 Kcal\/mol).The third area builds on the ability to generate LUVs with an asymmetricdistribution of DOPA and concerns studies on the ability of lipid asymmetry to regulateCa2 stimulated fusion of LUV systems. It is shown that for LUVs composed ofDOPC:DOPE:PI:DOPA (25:60:5:10 mol\/mol) rapid and essentially complete fusion isobserved by fluorescent resonance energy transfer techniques when Ca2+is added.Alternatively, for LUVs with the same lipid composition but when DOPA has beensequestered to the inner monolayer, due to the presence of a pH gradient (interior basic),little or no fusion is observed upon addition of Ca2. It is demonstrated that the extent ofCa2+ induced fusion correlates with the amount of exterior DOPA. It is also shown thatLUVs containing only 2.5 mol% DOPA, but when all the DOPA is in the outermonolayer, can be induced to fuse to the same extent and with the same initial rate asLUVs containing 5 mol% DOPA. These results strongly support a regulatory role forlipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipidbilayers are largely determined by the properties of one monolayer.1111.6.2 Fusion of Model Membranes .411.6.3 Molecular Mechanism(s) of Membrane Fusion 431.7 Thesis Outline 47Chapter 2 Intervesicular Exchange of Lipids Influence of Transmembrane pHGradients 482.1 Introduction 482.2 Materials and Methods 512.2.1 Lipids and Chemicals 512.2.2 Vesicles 512.2.3 Turbidity Experiments to Monitor Vesicle Aggregation 522.2.4 Stearylamine Exchange Monitored by Ion Exchange Chromatographyusing DEAE-Sephacel 532.2.5 Oleic Acid Exchange 542.3 Results 552.3.1 Stearylamine Exchange 552.3.2 Effect of a Transmembrane ApH on Stearylamine Exchange 582.3.3 Fatty Acid Exchange Between Membranes 592.3.4 Exchange of Fatty Acids Between Vesicles and BSA 622.4 Discussion 67Chapter 3 Transbilayer Transport of Phosphatidic Acid in Response to a TransmembranepH Gradient 713.1 Introduction 713.2 Materials and Methods 733.2.1 Lipids and Chemicals 733.2.2 Preparation of Large Unilamellar Vesicles 733.2.3 Induction of Transbilayer Transport of Acidic Phospholipids 733.2.4 Detection of Phosphatidyiglycerol Asymmetry by Periodate Oxidation 743.2.5 Detection of Asymmetry Using TNS 753.2.6 Measurement of the Internal pH of LUVs 753.2.7 Kinetic Analysis of Phosphatidic Acid Transport 763.3 Results 793.3.1 TNS Fluorescence Assay of Asymmetry 793.3.2 Comparison of the TNS Assay to a Chemical Assay 803.3.3 Kinetic Analysis of PA Transport 833.3.4 Influence of pH and Temperature on PA Transport 853.3.5 Transport of DOPA to the Outer Monolayer 863.3.6 Response of Various Phospholipids to a Transmembrane ApH 893.4 Discussion 93VChapter 4 Influence of Lipid Asymmetry on Fusion Between LargeUnilamellar Vesicles .964.1 Introduction 964.2 Materials and Methods 974.2.1 Lipids and Chemicals 974.2.2 Preparation of Large Unilamellar Vesicles 984.2.3 Detection of Fusion 984.2.4 Induction of DOPA Asymmetry 994.2.5 31P NMR Studies 1014.2.6 Freeze Fracture Electron Microscopy 1014.3 Results 1024.3.1 Vesicle Composition 1024.3.2 Effect of Ca2 on LUV Fusion 1034.3.3 Effect of DOPA Content on LUV Fusion 1034.3.4 Effect of DOPA Asymmetry on LUV Fusion 1064.3.5 Fusion of LUVs with DOPA Exclusively on the Outer Monolayer. . 1084.3.6 Polymorphic Phase Preferences 1104.3.7 Freeze-Fracture Studies of Vesicle Fusion 1104.4Discussion 114Chapter 5 Summary 117References 123VTABLE OF CONTENTSAbstract iiTable of Contents ivList of Figures viiList of Tables xAbbreviations xiAcknowledgments .xiiiDedication xivChapter 1 Introduction 11.1 The Structure and Function of Biological Membranes 11.2 Model Membranes 51.2.1 Monolayers 61.2.2 Planar Bilayers 61.2.3 Liposomes 71.3 Properties of Membrane Lipids 111.3.1 Structure 111.3.2 Gel-Liquid Crystalline Phase Transitions 141.3.3 Acid-Base Properties 151.3.4 Lipid Polymorphism 181.3.4.1 Factors Affecting Lipid Polymorphism 181.3.4.2 The Function of Lipid Polymorphism in BiologicalMembranes 211.4 Lipid Transport and Exchange 211.4.1 Extracellular Fatty Acid Transport 221.4.2 Intracellular Fatty Acid Transport 261.5 Membrane Asymmetry 261.5.1 Methods of Detecting Lipid Asymmetry 261.5.2 Phospholipid Asymmetry in Biological Membranes 271.5.3 Lipid Asymmetry in Model Membranes 331.5.4 Biological Significance of Lipid Asymmetry 351.6 Membrane Fusion 361.6.1 Methods of Detecting Membrane Fusion 371.6.1.1 Lipid MixingAssays 381.6.1.2 Mixing of Aqueous Contents 39iv1.6.2 Fusion of Model Membranes .411.6.3 Molecular Mechanism(s) of Membrane Fusion 431.7 Thesis Outline 47Chapter 2 Intervesicular Exchange of Lipids : Influence of Transmembrane pHGradients 482.1 Introduction 482.2 Materials and Methods 512.2.1 Lipids and Chemicals 512.2.2 Vesicles .512.2.3 Turbidity Experiments to Monitor Vesicle Aggregation 522.2.4 Stearylamine Exchange Monitored by Ion Exchange Chromatographyusing DEAE-Sephacel 532.2.5 Oleic Acid Exchange 542.3 Results 552.3.1 Stearylamine Exchange 552.3.2 Effect of a Transmembrane ApH on Stearylamine Exchange 582.3.3 Fatty Acid Exchange Between Membranes 592.3.4 Exchange of Fatty Acids Between Vesicles and BSA 622.4 Discussion 67Chapter 3 Transbilayer Transport of Phosphatidic Acid in Response to a TransmembranepH Gradient 713.1 Introduction 713.2 Materials and Methods 733.2.1 Lipids and Chemicals 733.2.2 Preparation of Large Unilamellar Vesicles 733.2.3 Induction of Transbilayer Transport of Acidic Phospholipids 733.2.4 Detection of Phosphatidylglycerol Asymmetry by Periodate Oxidation 743.2.5 Detection of Asymmetry Using TNS 753.2.6 Measurement of the Internal pH of LUVs 753.2.7 Kinetic Analysis of Phosphatidic Acid Transport 763.3 Results 793.3.1 TNS Fluorescence Assay of Asymmetry 793.3.2 Comparison of the TNS Assay to a Chemical Assay 803.3.3 Kinetic Analysis of PA Transport 833.3.4 Influence of pH and Temperature on PA Transport 853.3.5 Transport of DOPA to the Outer Monolayer 863.3.6 Response of Various Phospholipids to a Transmembrane ApH 893.4 Discussion 93VChapter 4 Influence of Lipid Asymmetry on Fusion Between LargeUnilamellar Vesicles .964.1 Introduction 964.2 Materials and Methods 974.2.1 Lipids and Chemicals 974.2.2 Preparation of Large Unilamellar Vesicles 984.2.3 Detection of Fusion 984.2.4 Induction of DOPA Asymmetry 994.2.5 31P NMR Studies 1014.2.6 Freeze Fracture Electron Microscopy 1014.3 Results 1024.3.1 Vesicle Composition 1024.3.2 Effect of Ca2 on LUV Fusion 1034.3.3 Effect of DOPA Content on LUV Fusion 1034.3.4 Effect of DOPA Asymmetry on LUV Fusion 1064.3.5 Fusion of LUVs with DOPA Exclusively on the Outer Monolayer. . .1084.3.6 Polymorphic Phase Preferences 1104.3.7 Freeze-Fracture Studies of Vesicle Fusion 1104.4Discussion 114Chapter 5 Summary 117References 123viLIST OF FIGURESFigure 1Freeze - Fracture Electron Micrographs of LUVs Produced by Extrusion 10Figure 2The Structure of a Phospholipid and Commonly Occurring Headgroups 13Figure 3Gel to Liquid-Crystalline Phase Transition 16Figure 4Polymorphic Phase Behavior of Lipids 20Figure 5Fatty Acid Transport Into the Interstitial Space 24Figure 6Proposed Mechanisms of Fatty Acid Transport Across Cell Membranes 25Figure 7Phospholipid Asymmetry in Mammalian Plasma Membranes 28Figure 8Fluorescence Assays to Monitor Membrane Fusion 40Figure 9Mechanism of Membrane Fusion Procceding Via Intermediates of the Bilayer toHexagonal H11 Phase Transition 46Figure 10Mechanism of Net Acidic Lipid Transport in Response to a Transmembrane pHGradient 50Figure 11Turbidity Measurements of Vesicle Aggregation .56Figure 12Characterization of Vesicle Elution from DEAE-Sephacel Columns 57Figure 13Effect of Transmembrane ApH on Vesicle Aggregation 60Figure 14Effect of a Transmembrane ApH on Stearylamine Exchange 61viiFigure 15Oleic Acid Exchange in Aggregating Systems 63Figure 16Oleic Acid Exchange in Non-Aggregating Systems 65Figure 17Exchange of Oleic Acid Between BSA and Vesicles 66Figure 18Standard Curve of TNS Fluorescence as a Function of DOPA Concentration inDOPC\/DOPA LUVs 81Figure 19Influence of a Transmembrane pH Gradient on TNS Fluorescence 82Figure 20Comparison of the TNS Assay for Lipid Asymmetry to a Chemical Assay (PeriodateOxidation) for DOPC\/DOPG LUVs 84Figure 21Transbilayer Transport of DOPA in Response to a Transmembrane pH Gradient(Inside Basic) 87Figure 22Influence of the External pH on the Rate of the Transbilayer Transport of DOPA. 88Figure 23Temperature Dependence of ApH Driven DOPA Asymmetry 90Figure 24Transport of DOPA to the Outer Monolayer in Response to a Transmembrane pHGradient (Interior Acidic) 91Figure 25Effect of a Transmembrane ApH on the Transbilayer Distributions of Various AcidicPhospholipids 92Figure 26.Effect of Ca2 Concentration on the Fusion of LUVs Containingl0mol%DOPA 104Figure 27Effect of DOPA Concentration on Vesicle Fusion 105Figure 28Modulation of Membrane Fusion by Lipid Asymmetry 107viiiFigure 29Effect on Fusion of DOPA Transport to the Outer Monolayer 109Figure 30Polymorphic Phase Preferences of Non-Fusogenic Vesicles (no DOPA) andFusogenic Vesicles (10 mol% DOPA) in the Absence and Presenceof Excess Ca2 112Figure 31Freeze-Fracture Electron Micrographs of LUVs in the Absence andPresence of Ca2 113Figure 32Possible Structure of Vesicles Exhibiting an Asymmetric Distribution of AcidicPhospholipids 120ixLIST OF TABLESTable 1Gel-Liquid Crystalline Phase Transition of Some Representative Lipids 17xABBREVIATIONSMLV Multilamellar vesicleFATMLV Frozen and thawed multilamellar vesicleSUV Small unilamellar vesicleLUV Large unilamellar vesicleLUVET Large unilamellar vesicle by extrusuiontechniquesCMC Critical micellar concentrationLipidsFA Fatty acidsSA StearylamineOA Oleic acidPC PhosphatidylcholinePA Phosphatidic acidPE Phosphatidylethanol amineP1 PhosphatidylinositolPS PhosphatidylserinePG PhosphatidyiglycerolCL CardiolipinDOPE Dioleoylphosphatidyl ethanolamineDOPC DioleoylphosphatidylcholineDPPC DipalmitoylphosphatidylcholinexiDOPA Dioleoylphosphatidic acidDOPS DioleoylphosphatidylserineRh-PE N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamineNBD-PE N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dioleoylphosphatidylethanolamineTransmembrane electrochemical potentialApH Transmembrane pH gradientFl11 Hexagonal phaseNMR Nuclear magnetic resonanceESR Electron spin resonanceQELS Quasi-elastic light scatteringHEPES N-(2-hydroxyethyl)piperazine-N\u2019-2-ethanesulfonic acidMES 2-(N-morpholino)ethanesulfonic acidEPPS N-(2-hydroxyethyl)piperazine-N-3-propanesu1fonic acidPIPES Piperazine-N,N\u2019-bis(2-ethanesulfonic acid)TNS 2-(p-toluidinyl)naphthalene-6-sulfonic acidATP Adenosine 5\u2019-triphosphateBSA Bovine serum albuminEGTA Ethyleneglycol-bis-(3-aminoethyl ether)-N,N,N\u2019,N\u2019tetraacetic acidTX-100 Triton-x-100xl\u2019ACKNOWLEDGEMENTSThere are a great many people to whom I am indebted for helping me throughoutmy graduate studies. Firstly I would like to thank all the members of the Cullis lab.There are too many people in the lab to mention everyone, although I probably would nothave made it through the past five years without a great deal of help from many of theseanonymous people. Pool games and beer at the Grad centre are good remedies for almostany ailment. I would especially like to thank Mick Hope for being my co-supervisor anda constant source of knowledge and Kim Wong who is constantly helping everybody inthe lab, usually all at the same time. To all the friends I have made in the department, Ithank you for all the great times had in the past five years. I would like to thank mybrothers David, Michael and Nigel, my sister Nicola and my mom and dad for giving methe confidence to pursue graduate studies and for all the support they have provided methroughout the years. I would also like to thank the members of my newest family for alltheir generosity and for making me feel so welcome. Everyone who knows me, knowsthat Brenda is at least half the reason that I am in a position to finish my graduate studiesand I can not thank her enough for everything she has done for me. Finally, I would liketo thank Pieter Cullis for all his help both in editing this thesis and for being such anextraordinary supervisor. Pieter not only provides his students with excellent ideas andsupport throughout their research but provides an atmosphere in which people can enjoyscience and become good friends.xli\u2019TO MY MOM AND DAD,MY FAMILYANDMY WIFE BRENDAxivCHAPTER 1INTRODUCTION1.1 THE STR UCTURE AND FUNCTION OF BIOLOGICAL MEMBRANESBiological membranes act as highly selective permeability barriers and define theboundaries of a cell or organelle. Specific membrane proteins form pores, channels ortransporters to regulate the flow of ions, metabolites and other molecules betweencompartments, thereby controlling the intracellular environment and regulatingintracellular communication. In addition, membranes are involved in communicationbetween cells, express immunogenetic determinants and participate in a host of otherfunctions which include processes such as procaryotic DNA replication, proteinbiosynthesis, protein secretion, bioenergetics and hormonal responses (Gennis, 1989;Houslay & Stanley, 1982)Biological membranes are comprised mainly of lipids and proteins. The basiclipid bilayer structure of membranes was first proposed in 1925 by Gorter and Grendel.This model was modified by Danielli and Davson in 1935 who postulated that proteinscoated the surface of the lipid bilayer. To date, biological membranes are bestcharacterized by the fluid mosaic model (Singer & Nicholson, 1972) in which proteinsare either embedded in a liquid crystalline lipid bilayer (integral membrane proteins) orattached to the surface of the membrane by ionic interactions or hydrogen bonding(peripheral proteins). The liquid crystalline nature of the membrane, characteristic of allbiological membranes (see Section 1.3.2), is manifested by the rapid lateral diffusion oflipids and many proteins in the plane of the bilayer and the slow transbilayer movementof these molecules. Typically, a phospholipid will have a diffusion coefficient in the1order of 10-8 cm2\/sec in a pure lipid bilayer. This corresponds to a net average velocityof 2 pm\/sec. For proteins in biological membranes, the lateral diffusion coefficient canrange from approximately 10-10 cm2\/sec to values of D < 10-12 cm\/sec where theprotein is considered to be essentially immobile (Gennis, 1989). The rapid lateraldiffusion of lipids in the bilayer contrasts with a very slow rate of transbilayer movementwhich involves the penetration of the polar lipid headgroups into the hydrocarbon interiorin order to cross the membrane. The rate of the transbilayer movement of lipids isdependent on many factors such as the nature of the polar headgroup and the possibleexistence of specific lipid transport proteins (\u201cflippases\u201d or \u201ctranslocases\u201d) in certainbiological membranes (see Sections 1.5.2 and 1.5.3). Thus, in metabolically activemembranes such as the cytoplasmic membrane of E. coli (Donohue-Rolfe & Schaechter,1980) or the endoplasmic reticulum (Bishop & Bell, 1985), the half-time of transbilayerlipid transport has been reported to be on the order of seconds or minutes, whereas thehalf-time for transbilayer lipid transport in pure phospholipid bilayers is on the order ofdays to months (Roseman et al., 1975; Low & Zilversmit, 1980).The lipid component provides the permeability barrier of biological membranesand an appropriate environment for the function of membrane proteins. However, thereis a great diversity of lipids in biological membranes. The erythrocyte membranecontains more than 100 different molecular species (van Deenen & de Gier, 1974), forexample. It is difficult to explain such diversity if the only function of lipids is to forman inert liquid-crystalline bilayer permeability barrier, which could be satisfied by asingle lipid species. It has become increasingly evident that membrane lipids providemore than just a backbone structure for the membrane and in fact are involved in many ofthe functional aspects of membranes. These include signal transduction (Majerus et al.,21987; Berridge, 1987), the production of metabolic energy (Bass, 1988), participation inbiosynthetic pathways (Jackson et a!., 1984), regulation of cell growth (Spiegel &Fishman, 1987), receptor recognition (Cheresh et a!., 1987; Hoekstra, 1990), andmembrane fusion (Duzgunes, 1985; Burger & Verkleij, 1990) among others.A general feature of biological membranes is the asymmetric transbilayerdistribution of the constituents. Proteins maintain an absolute asymmetry in membranes(Op den Kamp, 1979), such that every copy of a particular protein has the sameorientation with respect to the bilayer. This asymmetry is achieved during biosynthesisand is preserved during the lifetime of the membrane. Protein asymmetry providesfunctional asymmetry for the membrane and results in the vectorial transport of solutesacross the membrane (Houslay & Stanley, 1982), for example. Phospholipids alsomaintain an asymmetric transbilayer distribution in biological membranes, but thisasymmetry is not absolute (see Section 1.5). Phospholipids exhibit a preferentialdistribution across the bilayer, with the choline containing lipids predominating on theouter monolayer while the aminophospholipids predominate on the inner monolayer ofplasma membranes (Houslay & Stanley, 1982).In addition to maintaining an asymmetric distribution of their components,biological membranes maintain a transmembrane electrical potential (Aip) which isgenerated by the vectorial transport of ions across the bilayer without the offsettingmovement of another ion. Furthermore, transmembrane ion gradients are present acrossmost membranes. For example, by way of active transport (pumps) plasma membranesmaintain low Na\/K ratios inside cells while high Na\/K ratios exist in the intercellularfluid. Typically the Na+ and K+ gradients across animal cell membranes involve adifference in concentration of 10 to 15 fold, although this value can be much greater in3specialized tissues such as the avian salt gland (Finean et al., 1984). Other pumps keepthe intracellular [Ca2]low with respect to the extracellular medium with gradients in therange of to 1O observed across mammalian membranes (Finean et a!., 1984). Inaddition, transmembrane pH gradients exist across many biological membranes includinglysosomes, mitochondria, chioroplasts and endocytic vesicles (Rottenberg, 1979). Themagnitude of the pH gradient across organelles can range from less than 1 unit to greaterthan 3 units. For example the pH gradient (tXpH = pH1 - pH0) across rat livermitochondria has been determined to be in the range of 1 pH unit (+ 0.7 (Rottenberg,1973) to + 1.4 units (Nicholls, 1974)), whereas the pH gradient across chioroplasts underhigh light conditions can be as large as - 3.5 units (Rottenberg & Grunwald, 1972).The consistent observation of lipid asymmetry in eukaryotic plasma membranessuggests it is crucial to cell viability. The mechanism(s) by which this asymmetry isgenerated and maintained is a matter of debate, as are the functional consequences. It ispossible that transmembrane ion gradients may play a role in these processes. In thisthesis, the importance of ion gradients, notably pH gradients, in the generation of lipidasymmetry is investigated employing model membrane liposomal systems. The role oflipid asymmetry in biological processes such as lipid exchange and membrane fusion hasalso been studied.This chapter provides an overview of the characteristics of lipids and membraneswhich are relevant to the studies undertaken in this thesis. Since model membranes wereexclusively used in this thesis, a brief section is provided to discuss the methods bywhich various model systems are produced and the characteristics of each system. Thebasic physical and chemical properties of lipids are discussed in Section 1.3. Thephysical and chemical properties of lipids are important for understanding how4transmembrane ion gradients can affect the transbilayer distribution of lipids and how inturn this transbilayer asymmetric distribution of lipids can affect the properties of themembrane itself. For example, neutral forms of acidic lipids are transported across thebilayer at a far greater rate than charged lipids. Remaining sections introducebackground aspects of membrane biochemistry investigated in this thesis. Lipid transportand exchange is discussed as the effect of lipid asymmetry on this process is the subjectof Chapter 2. The asymmetric transbilayer distribution of membrane components,specifically lipids, is a major focus of this thesis and is reviewed in Section 1.5. Finally,an overview of membrane fusion is provided in Section 1.6, which serves as anintroduction to the studies on the influence of lipid asymmetry on membrane fusion,summarized in Chapter 4.1.2 MODEL MEMBRANESModel membranes have been developed to study the properties of pure lipids,lipid mixtures and reconstituted lipid-protein mixtures. Model membrane systems haveprovided much useful information about the structure and function of biologicalmembranes since the characteristics of individual components of membranes can bedetermined using these systems. In addition, the environment of the model systems canbe easily manipulated in order to determine the effects of specific factors. Thecomplexity of biological membranes often precludes such studies aimed at understandingthe physical properties and functional roles of individual components.Three basic types of model membranes exist. These are monolayers, planarbilayers and liposomes. These systems are briefly discussed in the following sections.51.2.1 MonolayersAt an air-water interface, phospholipids form an oriented monolayer with thepolar portions in contact with the aqueous phase and the hydrocarbon tails extended intothe air. Monolayer films can be compressed and the resistance to compression measured.Studies of the compression pressure versus the surface area (occupied by the film)provides information about the molecular packing of lipids and lipid-protein interactions.Possibly the best-known observation derived from monolayers studies is thecondensation effect of cholesterol and phospholipid, where the area occupied by a typicalmembrane phospholipid molecule and a cholesterol molecule in a monolayer is less thanthe sum of their molecular areas in isolation (Demel & de Kruijff, 1976). Monolayershave also been used to study the physical chemistry of lipid headgroups and theenzymology of soluble proteins that act at the lipid-water interface (Gennis, 1989).These systems have restricted uses and are obviously not useful for studyingcharacteristics of lipid bilayers, such as transbilayer lipid asymmetry.1.2.2 Planar BilayersPlanar bilayers can be formed by painting a concentrated solution of lipid in anorganic solvent across a small orifice in a nonpolar partition between two aqueouscompartments. The solvent tends to collect at the perimeter of the orifice, leaving abilayer film across the center (Fettiplace et al., 1974). Planar lipid bilayers have provento be excellent systems to study pores, channels and transporters of charged moleculesbecause they allow for electrical measurements across the bilayer through use ofelectrodes in the buffered compartments. Various proteins can be incorporated into themembranes if they are soluble in the solvent used to dissolve the lipid. Limitations with6these systems arise due to the presence of the hydrocarbon solvent which may affect thenormal properties of the lipid bilayer being examined. Furthermore, the relatively smallamount of bilayer present largely precludes studies on the topology of the membraneitself.1.2.3 LiposomesA liposome is a lipid bilayer structure which encloses an aqueous volume and caneither consist of multiple bilayers in a series of concentric shells, referred to as amultilamellar vesicle (MLV), or a single-walled unilamellar vesicle.MLVs, were first described by Bangham et al. (1965) and can be simply preparedby drying down a solution of lipid in organic solvent to a thin film on the wall of a vessel,followed by the addition of an aqueous buffer and agitation of the system. The vesiclesproduced are heterogeneous in size (0.5 - 10 !m) and have a low ratio of trapped aqueousvolume to lipid (\u2014 0.5 L\/imol) although this ratio can be greatly increased (5 - 10L\/[Lmo1) by subjecting the MLVs to freeze-thaw cycles (Mayer et al., 1985).Furthermore, only a small amount of the total lipid is present in the outer monolayer andthus exposed to the external medium (Hope et al., 1986). MLVs are very useful instudies of the structural properties of lipids such as lipid polymorphism and the factorsthat modulate these preferences, since the regular arrays of bilayers in MLVs are suitedfor X-ray studies and the relatively large size of MLVs makes structural and motionalanalysis by nuclear magnetic resonance (NMR) more straightforward than in smallersystems.Unilamellar vesicles are the most commonly used model membrane systems andare usually classed as small unilamellar vesicles (SUVs), with diameters ranging between725 to 40 nm, and large unilamellar vesicles (LUVs) which typically have diameters of 50to 200 nm. There are several ways to produce unilamellar vesicles. SUVs are usuallyproduced by the sonication of MLVs to form limit size vesicles (Huang, 1969). Thesevesicles can also be produced using a French press (Barenholz et a!., 1979). SUVsystems are comparatively simple to prepare and and are relatively homogeneous in sizefor defined lipid compositions. However, these systems have small interior aqueousvolumes so that the trapping efficiencies obtained are poor and the small radii ofcurvature associated with SUVs can perturb the physical properties of the lipids beingstudied (Cullis et al., 1985).LUVs can be produced from organic solvents (Szoka & Papahadjopoulos, 1980)by injecting a solution of lipids in ethanol or ether into an aqueous medium and removingthe organic solvent by heating the solution above the boiling point of the solvent.Alternatively the solvent can be removed by dialysis or gel filtration. Another procedurefor the production of vesicles from organic solvents, the reverse phase evaporationprocedure (REV), involves dissolving the lipids in an organic solvent, such as ether, andforming an emulsion with the appropriate aqueous buffer. The organic solvent issubsequently removed under vacuum resulting in a thick gel of hydrated lipid which canbe diluted and sized by extrusion through polycarbonate filters (Szoka &Papahadjopoulos, 1978). Vesicles produced using reverse phase evaporation procedurescan exhibit large trapping efficiences. Difficulties often occur in the preparation ofvesicles from organic solvents due to the differing solubilities of lipid species in varioussolvents. Furthermore, residual solvents can affect the physical characteristics of thelipids.8A second approach to the formation of unilamellar vesicles is to dissolve lipids ina detergent and then to remove the detergent by dialysis (Mimms et al., 1981) or gelfiltration. Detergents with a low critical micellar concentration (CMC) can often beremoved employing hydrophobic beads (Hope et a!., 1986; Gennis, 1989). However,these vesicles are particularily useful in the study of membrane proteins since solubilizedproteins can be reconstituted into the vesicles during the dialysis period. These systemshave low trapping efficiencies and suffer from similar problems as the REV method, thatis differential solubilities of various lipid species in detergents and the presence ofresidual detergents.A third method of producing LUVs is to repeatedly extrude MLVs at intermediatepressures through polycarbonate filters of defined pore size. These vesicles aresometimes referred to as LUVETs (large unilamellar vesicles by extrusion techniques).This process has many advantages over the previously mentioned methods. First, theprocess is very rapid, secondly, there are no residual solvents or detergents present,thirdly, a relatively high trapping efficiency is obtained ( 30%) and finally, it is usuallypossible to produce homogeneously sized vesicle populations. The size of theunilamellar vesicles generated by extrusion can range from approximately 50 - 200 nmdepending on the pore size of the polycarbonate filters used. Larger vesicles can beprepared by this procedure but a proportion of the vesicles are multilamellar. The abilityto generate homogeneous populations of vesicles by extrusion techniques is illustrated inFigure 1, where freeze-fracture electron micrographs show vesicles produced by theextrusion of egg-PC through polycarbonate filters of various pore sizes ranging from 30-400 nm. The LUVs used to undertake the studies described in this thesis were allproduced by the extrusion technique.9Figure 1Freeze - Fracture Electron Micrographs of LUVsProduced by ExtrusionVesicles were prepared by extruding frozen and thawed egg-PC MLVs, at aconcentration of 100 mM lipid, 20 times through polycarbonate filters of various poresizes. (A) 400 nm, (B) 200 nm (C) 100 nm, (D) 50 nm and (E) 30 nm. The bar in panelA represents 150 nm and all panels exhibit the same magnification (Mayer et al., 1986).101.3 PROPERTIES OF MEMBRANE LIPIDSLipids have many characteristic properties which include differences in theirchemical diversity, their gel-liquid crystalline phase behavior, their polymorphic phasepreferences under various conditions and their acid-base characteristics among others.These topics are briefly reviewed in the following sections.1.3.1 StructureBy definition, lipids are water-insoluble biological molecules that are highlysoluble in organic solvents such as chloroform. This is a rather general definition andincludes an extremely diverse class of molecules.The most abundant class of lipids in most biological membranes are thephospholipids (see Figure 2). These lipids are composed of a hydrophilic head grouplinked to the sn3 position of glycerol-3-phosphate via a phosphate ester. Two acyl chainsare attached to the sn1 and sn2 positions via ester linkages. Figure 2 illustrates thediversity of polar head groups which defines the classes of phospholipid, the mostcommon being the phosphatidyicholines (PC), phosphatidylethanolamines (PE),phosphatidylserines (PS), phosphatidylglycerols (PG), phosphatidic acids (PA),phosphatidylinositols (P1) and the diphosphatidyiglycerols or cardiolipins (CL). Thesehead groups differ with respect to their charge, polarity, size, chemical reactivity andhydrogen bonding capacity and therefore have a large influence on the properties of lipidbilayers. For example, substitution of ethanolamine for choline produces drasticdifferences in the gel-liquid crystalline phase transition temperature (see Section 1.3.2)and the polymorphic phase preferences of these lipids (see Section 1.3.4).11Sphingolipids are another important class of membrane lipids. Ceramide, whichis the precursor to all sphingolipids, is composed of a fatty acyl chain linked to the aminogroup of sphingosine which is an alcohol with a long-chain hydrocarbon tail. Theaddition of phosphocholine to the C-i hydroxyl of ceramide produces sphingomyelinwhich is a major component of nervous tissue and many plasma membranes but only aminor constituent of intracellular membranes. Other sphingolipids include cerebrosides,which are formed by the attachment of one or more carbohydrate groups to the C-ihydroxyl of ceramide, and gangliosides, which contain several sugar residues and one ormore sialic acid residues attached to the C-i hydroxyl of ceramide.A third important class of membrane lipids are the sterols. Cholesterol, the majorlipid in this class, is found almost exclusively in the plasma membranes of mammaliancells, where it can comprise up to 45 mol% of the total lipid. Cholesterol has a largeapolar region and a hydroxyl group constituting the polar domain. Cholesterol is buriedin the nonpolar hydrocarbon area of bilayers with the hydroxyl function exposed to theaqueous surface. In addition to these lipids, there are a number of other lipid componentsin membranes which comprise only minor quantities of the total lipid. These includefatty acids, lyso-phospholipids, plasmalogens and diglycerides among others. Althoughthese lipids are only present in small quantities, they can play major roles in membranefunction. For example small amounts of diacylgicerols have been shown to destabilisebilayers and induce fusion (Siegel et a!., 1989). Furthermore, phosphorylated derivativesof phosphatidylinositol (P1), a phospholipid present usually only in very small quantitiesin biological membranes, are important in transmembrane signalling.12Figure 2The Structure of a Phospholipid and CommonlyOccurring Headgroups0223\u2018IPhospliatychone0Ca2\/jic \u2014AV\u2022 C =0CH3NK3V\u2014CH2CHCOO \u2014\u2014CH2H(O )CHO\u2014 CH2ac-OH\u2014 CH2CH3 H OHOHH HEltianaarnine(PhospatidylehanoIamine)Ser.ne(P1osphatidyiserine)Gefo(PhosphaIidygIyceioI)GeroT(Dphospha{idylgIycero 01 cardohpin)My0n0sd0(Phosphatidyhnositol)131.3.2 Gel-Liquid Crystalline Phase TransitionsPhospholipids can exist in a frozen \u201cgel\u201d state or a fluid \u201cliquid-crystalline\u201d statedepending on the temperature (Figure 3A). The transition of a bilayer from the \u201cgel\u201dphase (crystalline) to the liquid-crystalline phase occurs at a characteristic temperature(Ta) for a specific lipid species. This transition temperature is dependent upon the natureof the lipid headgroup, the acyl chains and the environment in which the lipid isdispersed. Below the transition temperature (T the acyl chains adopt an extended all-trans configuration. As the temperature approaches T, the acyl chains start to develop\u201ckinks\u201d due to the 1200 rotation of C-C bonds to form gauche isomers (see Figure 3B).As the temperature rises above T the average number of gauche isomers per acyl chainincreases. The \u201ckinks\u201d in the acyl chains decrease the length of the acyl chains andincrease the distance between the individual molecules. Thus, the lipid bilayer decreasesin thickness but undergoes lateral expansion during the transition from the gel to liquid-crystalline states. The acyl chains markedly effect the transition temperature (See Table1). For example, in a given lipid species, the temperature at which the gel to liquid-crystalline phase transition occurs increases with increasing acyl chain length and ishigher for saturated acyl chains than for unsaturated acyl chains.The lipid headgroup is also a major determinant of the transition temperature.Charge repulsion between adjacent negatively charged phospholipids can cause lateralexpansion of the bilayer, favouring the liquid-crystalline state. Thus, the phase transitiontemperature for dipalmitoylphosphatidic acid (DPPA) drops from 66\u00b0C at pH 6 (1negative charge) to 43\u00b0C at pH 12 where the PA carries two negative charges (Marsh,1990). Divalent cations can also serve to increase the transition temperature by reducingthe charge repulsion between negatively charged lipids. For example, the gel to liquid14crystalline phase transition temperature of 1,2-(14:0) PG increases from 26\u00b0C to 81\u00b0Cupon binding of 1 Ca2molecule for every 2 PG molecules (Marsh, 1990). Theconformation of the phospholipid headgroup can also affect the phase transition byperturbing the packing of the bilayer in the crystalline state.The significance of the ability of lipids to adopt the gel phase is unknown,although it has been suggested that lateral inhomogeneities (ie. areas of gel-state domainswithin a liquid-crystalline bilayer) may play roles in various physiological processes. Onthis note it should be mentioned that there is no evidence for gel-state lipid componentsin eucaryotic membranes at physiological temperatures (Cullis & Hope, 1985).1.3.3 Acid-Base PropertiesThe polar regions of lipids contain various ionizable groups (phosphate, carboxylgroups and amino groups) which have weak acid or base characteristics. Fatty acids,phosphatidylinositol (P1), phosphatidylserine (PS), phosphatidylglycerol (PG),cardiolipin (CL) and phosphatidic acid (PA) are acidic molecules that carry a netnegative charge at physiological pH.The state of ionization of phospholipids and fatty acids in a membrane can inprinciple be described by an equilibrium characterized by a given dissociation constant.However, the behavior of an ionizable group in solution can differ greatly from itsbehavior at the surface of a membrane. The charge on the lipid molecules generates anelectrostatic surface potential which causes the redistribution of cations and anions at thelipid\/water interface. Thus a membrane containing acidic (negatively charged)phospholipids will attract cations to the lipid water interface, increasing the concentrationof H+ ions at the interface, for example, resulting in a lower pH at the interface than in15Figure 3Gel to Liquid-Crystalline Phase Transition(A) A phosphatidyicholine bilayer in the gel phase exhibiting acyl chains tilted withrespect to the bilayer normal (L) undergoing a phase transition the the liquid-crystallinestate (LcJ. (B) A fatty acid chain in the all trans state and the distortion of the acyl chainupon the introduction of a \u201ckink\u201d due to the formation of a gauche isomer (from Houslay& Stanley, 1982).Acy) chain Acyl chainsA ordered Polar headgroup disorderedRRRR1Crystalline state Liquid-crysta)line statesolid fluidL13i LB99(A) (B)All trans First-order Kink (2G1)...GTG..16LipidTable 1Gel-Liquid Crystalline Phase Transition of SomeRepresentative LipidsPhosphatidyicholines1,2-(16:0)1,2-(18:0)1-(16:0)-2-(18:0)1-(16:0)-2-(18:lcA9)1,2-(16:1 cA9)1,2-(18:1 cA9)41.554.54920-36-19Phosphatidylethanolamines1,2-(18:0) 741,2-(18:lcA9) -16Phosphatidyiglycerols1,2-(18:0) 54.575.1,2-(18:lcA9) -18Phosphatidylserines1,2-(18:0)1,2-(18:lcA9)Phosphatidic Acid1,2-(16:0) 661,2-(18:lcA9) 8pH7.0pHl.0pH7.0pH2.0pH7.0pH9.5Note - mean temperatures, Data obtained from Small, 1986; Houslay & Stanley,1982 and Marsh, 1990.Transition Temperature(T)I\u00b0CConditions70.79.-11-10.17the bulk solution. This in turn is reflected by apparent pKa values that are higher formembrane associated molecules than those free in bulk solution. For example the PKafor various fatty acids in a lipid bilayer has been reported to be between 7.0 - 7.5 which ismuch higher than the pK of 4.8 reported for free fatty acids (Tocanne & Tessie,1990) .The pK of the phosphate groups of the various phospholipids listed above varyfrom <1 to approximately 4 depending on the ionic conditions and the detection methodsused. Phosphatidic acid and cardiolipin have two ionizable groups. The PKa for the twophosphate groups on cardiolipin has been reported to be -1 (Marsh, 1990) while the pK2for phosphatidic acid is between 8 and 9 (Tocanne and Tessie, 1990).1.3.4 Lipid PolymorphismLiquid crystalline lipids can adopt different phases upon hydration depending onmany factors such as the lipid species and its environment (Tilcock, 1986). These phasesinclude bilayers (La), micelles and the inverted hexagonal phase (H11). Lipids capable ofadopting non-bilayer phases in isolation appear to be common to all biologicalmembranes.1.3.4.1 Factors Affecting Lipid PolymorphismIndividual species of lipids found in membranes can adopt different structures(micellar, bilayer or hexagonal H11) depending on the nature of the lipid headgroup, thesize and degree of unsaturation of the hydrocarbon tail, temperature, pH, the degree ofhydration, the ionic strength of the medium, the presence of divalent cations and thepresence of other lipids or proteins (Cullis et al., 1986). The phase adopted can bepredicted by the \u201cshape\u201d properties of the lipid in question. Briefly, lipids with a18headgroup that has a larger cross-sectional area than that of the hydrophobic end(\u201cinverted cone\u201d shape) will form micelles, lipids with approximately equal cross-sectional areas for the polar and non-polar regions (\u201ccylindrical\u201d shape) will formbilayers and lipids with headgroups with small cross-sectional areas with respect to thehydrophobic end (\u201ccone\u201d shape) will adopt the hexagonal H11 phase (see Fig. 4A). Anyfactor that either increases the cross-sectional area of the headgroup or hydrophobicregion of the lipid will alter the effective shape of the molecule and can therefore changethe phase adopted by the lipid (see Fig. 4B). For example an increase in the degree ofunsaturation of the fatty acyl chain of a phospholipid will increase the effective cross-sectional area of the hydrophobic tail and drive the structure towards the hexagonalphase. Similarily an increase in temperature increases the effective cross-sectional areaof the hydrophobic tails by increasing the the number of gauche isomers in the acylchains. Protonation of acidic phospholipids such as PS decreases the charge repulsionbetween molecules thereby reducing the effective cross-sectional area of the headgroupswhich serves to drive structures towards the hexagonal H11 phase. Certain divalentcations will act to dehydrate various phospholipid headgroups, thereby reducing theeffective area of the headgroup. For example PS forms a tight complex with Ca2 thatresults in the formation of cochleate lipid cylinders (Papahadjopoulos et al., 1975). PAand CL can also form a non-bilayer phase (hexagonal H11) in the presence of Ca2+andMg2 (Papahadjopoulos et al., 1976; Vail & Stollery, 1979).19Figure 4Polymorphic Phase Behavior of Lipids(A) Polymorphic phases formed by lipids upon hydration of the lipid at concentrationsabove the critical micellar concentration and the corresponding shapes of the lipids. (B)Factors influencing the bilayer to hexagonal H11 phase transition.LIPID PHASE MOIECUIARSHAPELTSOPHOSPHOUpiOSDETERGENTS VMICELLAR INVERTED CONEPNOSENATIDYLCHO(INESI\u2019IIINGOMYEUN\u2014\u2014 --PHOSPHATIDYLSERINEP**OPHATIDYLINOSITOLPHOSPHATIOYLGLYCEROLPH0SPIIATIOIC ACIDCAEOIOLIPINDTGALACTOSVLDtGLYcTRID\u20acSILAYER CYLINDRICALPNOSPHATIOELETHANOLAUIN(UP4SATI*ATEO)CARDTOLIPIN_C02+pHospIlArloIc ACIO-C2(pH<6.OP1IOSPHATIDIC ACID(pH<3.0IPHOSPHATIDYLSERINE(pH<4.OjMONOGALJCTOSYLDIGLYCERIOEHEXAGONAL (H11J CONEAB0a:a:Da:CazLfRt1ELLARwNCITa:a-\u2014 Da: oa: a:CCCIDICUT w\u2014vHEXAGONALC\u2014zIllI\u20142:0C-)a:UTH201.3.4.2 The Function ofLipid Polymorphism in Biological MembranesBiological membranes are composed of mixtures of lipids, individual species ofwhich adopt different polymorphic phases. Obviously, the lipids present in a biologicalmembrane must maintain the appropriate characteristics required for cell viability. Thatis, they must form a fluid bilayer structure capable of providing a suitable permeabilitybarrier to the flow of ions and metabolites while allowing for transmembranecommunication and membrane protein function among many other demands. It has beensuggested that non-bilayer forming lipids regulate such phenomena as membrane order(Lafleur et al., 1990) the transport of proteins across bilayers (Batenburg & de Kruijff,1988; de Vrije et al., 1990) and the formation of various membrane structures such astight junctions (Kachar & Resse, 1982). However, membrane fusion is the mainbiological phenomenon for which there is extensive evidence to indicate that non-bilayerforming lipids are involved and this is discussed in greater depth in Section 1.6.31.4 LIPID TRANSPORTAND EXCHANGEThe trafficking of lipids within cells and between cells is important in manybiological events including membrane biogenesis and homeostasis and the sorting ofproteins by lipid carrier vesicles. It is now well established that the membranes ofintracellular organelles maintain unique lipid compositions. Since these organellesusually do not synthesize all of the lipids contained in their membranes, the synthesis,sorting and transport of these lipids is crucial for the maintenance of membrane structure.Within cells, transport is regulated by three general mechanisms. These include vesiculartransport, where lipids are transported between membranes by budding and fusing of21vesicles, monomer transport, the transport of single molecules by diffusion through theaqueous phase and lateral diffusion, where molecules exchange between two connectedorganelles (Sleight, 1987). The trafficking of lipids throughout cells is complex and anarea of much current research (van Meer, 1989; Pagano, 1990; Voelker, 1990).The process of lipid trafficking would be expected to be influenced by thetransbilayer distribution of lipids. For example, lipids located on one monolayer of a cellor organelle membrane would not be available for monomer transport on the oppositeside. In the case of vesicular transport the fusion processes could be affected by thetransbilayer distribution of lipids (see Section 1.6 and Chapter 4). Lateral diffusion mayalso be affected by the transbilayer lipid distribution as is the case for tight junctions (seeSection 1.5.4). The effects of the transbilayer distribution of fatty acids on their transport(introduced below) is the subject of Chapter 2.1.4.1 Extracellular Fatty Acid TransportFatty acids provide the main energy source for most mammalian tissues and areessential components of the structural lipids of cell membranes (Bass, 1988). Since fattyacids are hydrophobic, special mechanisms are required to deliver fatty acids through theplasma to the tissue where they are employed. However, the mechanism(s) by whichfatty acids are released from the specific carrier molecules and cross cell membranes enroute to their site of utilization is still a matter of debate.Short chain fatty acids (2-10 carbon atoms long) are absorbed directly through thevilli of the intestinal mucosa, diffuse across the mucosal cytosol and enter the venousblood while remaining in the free fatty acid form. Long-chain fatty acids (12 or morecarbon atoms), on the other hand, are solubilized by bile salts and form micelles. These22micelles are transported to the brush borders or microvilli of the intestinal epithelial cellswhere the fatty acids dissociate from the micelle and diffuse across the epithelial cellmembrane. Inside the epithelial cell, the fatty acids are esterified to form triacylglycerolswhich are integrated with free cholesterol, cholesterol esters, phospholipids and proteinsto form chylomicrons. The chylomicrons are transported through the epithelial cellmembrane into the lacteals of the lymphatic system, which delivers them into the generalcirculation via the thoracic duct (Rawn, 1989). Serum albumins are also responsible forthe transporting a large proportion of fatty acids through the circulation. At the surfaceof endothelial cells, at the fatty acids are hydrolyzed from triglycerides contained inlipoproteins by lipoprotein lipases and pass from the capillary to the interstitial spacethrough the endothelial cells of the capillary wall. Alternatively, as shown in Fig. 5,albumin-fatty acid complexes may pass the endothelium through clefts or directlythrough the capillary endothelial cell by way of plasmalemmal vesicles (Paulussen &Veerkamp, 1990).Upon reaching the target cell the fatty acids must dissociate from the carriermolecule and cross the plasma membrane. The mechanism by which the fatty acidcrosses the membrane is not clear. There is evidence to support passive diffusion of thefatty acid, where the rate of diffusion is limited by the equilibrium distribution of fattyacids between albumin and the plasma membrane (Noy et al., 1986). Other data supportsthe presence of fatty acid transport systems. These include systems where fatty acidseither passively diffuse across the membrane or are transported by membrane proteinsafter the albumin binds to a membrane associated receptor (Stremmel, 1988). It has alsobeen suggested that fatty acids diffuse from albumin into the membrane where they aretransported by plasma membrane fatty acid binding proteins or translocators (see Fig. 6).23Figure 5Fatty Acid Transport Into the Interstitial SpaceTransport of fatty acids (FA) through the capillary endothelial cells into the extracellularfluid followed by transport into the target cell. The fatty acid can cross the endothelialcell either in the free fatty acid form or bound to serum albumin (see Section 1.4.1).PMFABP (Plasma membrane fatty acid binding protein).:tracellular FluidPMFABP A Albumin Receptor24Figure 6Proposed Mechanisms of Fatty Acid Transport AcrossCell MembranesModels of cellular uptake of fatty acids (FA). (A) Passive diffusion through the plasmamembrane. (B) passive diffusion through membrane after albumin (ALB) binds to aspecific receptor that facilitates removal of the fatty acid. (C) An extrinsic fatty acidbinding protein seives to help dissociate FA from serum albumin followed by FAdiffusion across the membrane. (D) Intrinsic fatty acid translocator. (E) Intrinsic fattyacid translocator with an external albumin binding site and an intracellular FABPreceptor (see Section 1.4.1).B C D EAFA251.4.2 Intracellular Fatty Acid TransportOnce delivered to the cytoplasm, fatty acids may be utilized in biosyntheticpathways, in the production of metabolic energy through n-oxidation or they can be reesterified to triacyiglycerols for energy storage. In the cytoplasm, the fatty acids arebound to fatty acid binding proteins (FABPs) which probably participate in theintracellular transport and storage of fatty acids much like albumin does extracellularly(Spener et al., 1989). FABPs have been proposed to promote cellular uptake of fattyacids, protect cellular structures from the detergent effects of fatty acids, modulateenzyme activities and to target fatty acids to specific metabolic pathways (Bass, 1988;Spener et al., 1989; Spener & Mukherjea, 1990; Paulussen & Veerkamp, 1990).1.5 MEMBRANE ASYMMETRYAs previously mentioned (Section 1.1), biological membranes maintain anasymmetric transbilayer distribution of their components. For lipids, this asymmetry isnot absolute and must be maintained during the lifetime of the cell or organelle. In thissection, some of the mechanisms used to establish and maintain lipid asymmetry both inbiological membranes and model membrane systems are briefly reviewed and some ofthe implications that lipid asymmetry has for the functions of biological membranes arediscussed.1.5.1 Methods of Detecting Lipid AsymmetryMost techniques used to detect lipid asymmetry specifically modify one side ofthe lipid bilayer of a sealed membrane. In order for reliable results to be obtained,several criteria must be met. Firstly, the membrane to be probed must be highly purified.26This is often not the case when studying organelles. Secondly, the isolated vesicles musthave a unique sidedness (ie. either all right side out or all inside out). Thirdly, themembrane must be impermeable to the probe so that only one leaflet of the bilayer isexamined. Fourthly, the probe must not alter the permeability of the membrane andfinally the reagent (enzyme, chemical probe, exchange protein etc.) must be able todetect all exposed membrane components (Op den Kamp, 1979; Houslay and Stanley,1982).The techniques used to determine lipid asymmetry include immunologicaltechniques, modification with chemical reagents, enzymatic degradation of lipids,fluorescent probes, lipid exchange proteins and physical techniques such as NMR andESR among others (Op den Kamp, 1979; Etemadi, 1980; Houslay & Stanley, 1982;Schroeder, 1985; Gennis, 1989).1.5.2 Phospholipid Asymmetry in Biological MembranesBretscher (1972) first reported that phospholipids were asymmetrically distributedacross erythrocyte plasma membranes. For normal human erythrocytes, it has beendetermined that all the phosphatidylserine is located on the inner monolayer along with80% of the total phosphatidylethanolamine. The outer monolayer consists mainly of thecholine containing lipids phosphatidyicholine and sphingomyelin, and glycolipids (seeFigure 7). In the twenty years since this finding, a large number of membranes have beenreported to show asymmetric distributions of phospholipids. Some examples includebacterial membranes (Barsukov et a!., 1977; Donohue-Rolfe & Schaechter, 1980;Goldflne et a!., 1982), intestinal (Barsukov et al., 1986) and renal brush- bordermembranes (Venien & Le Grimellec, 1988), beef heart mitochondrial inner membranes27Figure 7Phospholipid Asymmetry in Mammalian PlasmaMembranes(A) Human erythrocyte membrane, (B) rat liver blood sinusoidal membrane, (C) rat livercontinuous plasma membrane, (D) pig platelet plasma membrane and (E) VSV envelopederived from hamster kidney BHK-21 cells. From Cullis & Hope, 1985Outer MonorA 8 C E100 Ii II 1 100go -SF PCPC80 PC PC I s 80I-I sp70 7060 lji 60PC50\u00f7 lips5040 40P131) 30+Sp20P6\u00f720\u2022 10 10010203040 4050 II 5060 I 6070 j80 8090 go1(t) 100Inner monolayer0\u2014I28(Krebs et a!., 1979), cardiac sarcolemma (Post et a!., 1988), viral membranes (Rothmanet al., 1976; Schafer et al., 1974), platelets (Sanchez-Yague & Llanillo, 1986) and plasmamembranes from many other sources as well as many subcellular organelle membranes.Initially, this phospholipid asymmetry was believed to be a static phenomenon,similar to protein asymmetry, where the asymmetry was established during biosynthesisand maintained due to an extremely slow rate of transbilayer movement. However,evidence of the rapid transport of phospholipids in various biological systems (Zilversmit& Hughes, 1977; de Kruijff et al., 1979; van den Besselaar et al., 1979; Hutson &Higgins, 1982) seemed to argue against a purely static mechanism of maintainingasymmetry. Furthermore, reports in 1984 by Seigneuret and Devaux indicating that spinlabelled analogues of aminophospholipids were specifically transported by an ATPdependent mechanism in human erythrocytes provided evidence that asymmetry wasactively maintained. Bishop and Bell (1985) reported a similar finding forphosphatidylcholine analogues in rat liver microsomes, where the transport ofphosphatidylcholine was found to be rapid and saturable and could be inhibited bystructural analogues of PC or by proteases. These studies indicate that lipids can betransported across membranes by specific protein translocases. Different mechanismsare probably involved since the transport of PC and its analogues is independent ofmetabolic energy and has been classified as facilitated diffusion, whileaminophospholipids are transported by an ATP-dependent active transport system(Zachowski & Devaux, 1990; Devaux, 1991).Further evidence for the presence of aminophospholipid translocases has beenprovided by Schroit et a!., (1987), who reported rapid movement of fluorescent analoguesof PS and PE across erythrocyte membranes. PS translocation has also been inferred29from morphological changes in erythrocytes after the incorporation of PS into the outermonolayer. The transport of PS was shown to be dependent on the concentration ofMg2 ions and ATP and also on the state of protein sulfhydryl groups (Daleka & Huestis,1985). Connor and Schroit (1987) have shown that a photoactivatible analogue of PSpreferentially bound to a 30 kDa protein in the erythrocyte membrane. Latter workindicated pyridyldithioethylamine (PDA), an inhibitor of the PS translocase, bound to a30 - 31 kDa protein and co-migrated with band 7 from the erythrocyte membrane(Connor & Schroit, 1988). Other researchers have reported that the aminophospholipidtranslocase may be a 115-130 kDa vanadate-sensitiveMg2+ATPase (Damiana et al.,1987). Zachowski et al. (1986), showed that PE and PS compete for the same translocasewith PS having a higher affinity for the translocase. More recent studies have providedevidence for translocases in various other membranes such as human platelets (Sune etal., 1987) and in chromaffin granules (Zachowski et al., 1989). Isolation andreconstitution would be the ideal method of positively identifying translocases but thiscould be very difficult as it has been suggested that the translocase is present in a verylow copy number in erythrocytes (< 1000 copies\/cell) (Devaux, 1991). A translocase hasbeen reconstituted into liposomes from total protein extracts of rat liver microsomes butthis has not served to purify or identify the translocase (Backer & Dawidowicz, 1987).Although there is now a large body of evidence to support the existence ofphospholipid translocases, other factors may also be involved in the development andmaintenance of phospholipid asymmetry. For example, it has been suggested that thecytoskeleton may play a role in maintaining phosphatidylserine asymmetry (Bevers et al.,1987; Verhallen et al., 1987; Comfurius et al., 1989). This relationship has been inferredfrom experiments which indicate that the breakdown of the cytoskeleton during platelet30activation is accompanied by the appearance of PS on the outer monolayer of platelets(Verhallen et al., 1987) and experiments which indicate a reversible specific binding ofPS to the cytoskeleton (Comfurius et al., 1989). Williamson et al., 1987 have also statedthat a kinetic analysis of the data collected by Devaux and co-workers on PS transportacross erythrocyte membranes indicates that the translocase is actually a bidirectionalflip-flop enzyme and could not in itself maintain aminophospholipid asymmetry.However, other research has provided evidence to suggest that the cytoskeleton is notrequired to maintain PS asymmetry. For example, Middelkoop et al. (1989) have shownthat treatment of erythrocytes with diamide to crosslink cytoskeletal proteins did notaffect the phospholipid asymmetry but the asymmetry was lost if ATP was depleted.Also, studies with vesicles formed from heat treated erythrocytes show that the vesiclesare capable of maintaining aminophospholipid asymmetry as long as ATP is present atsufficient concentrations. This indicates that cytoskeletal proteins are not required forasymmetry since the vesicles contain only about 25% of the spectrin associated witherythrocytes and are devoid of the other cytoskeletal proteins (Calvez et al, 1988).In contradiction to these findings, recent research by Connor & Schroit (1990)argues that an endofacial protein (probably a cytoskeletal component) is required tomaintain PS asymmetry. This argument is based on several observations. Firstly,diamide did not react with the sulfhydryl group of the 32 kDa protein which they havetentatively identified as the PS transporter, but did inhibit PS transport. If the 32 kDaprotein they have identified as the PS transporter is the only protein involved in PStranslocation then diamide should not affect PS transport if it does not react with thisprotein. Secondly, oxidative cross-linking and inhibition of PS transport by diamide wasreversible by intracellular glutathione but PDA inhibition (PDA binds the sulfhydryl of31the 32 kDa protein) was not. This suggests that the inhibition of PS transport is a resultof these chemicals reacting with two different proteins. Finally, it was claimed thatoxidation of either the PDA sensitive site or the diamide sensitive site after the PS wastransported to the inner monolayer, did not promote the movement of PS from the innerto the outer monolayer, suggesting that the PS was stabilized on the inner monolayer. Itis stated that this finding is inconsistent with other data indicating that an ATP-dependentoutward movement of PS occurs in RBCs and that cytoskeletal proteins are not involvedin the transport of exogenously added PS analogues. Thus, the role of the cytoskeleton inmaintaining phospholipid asymmetry in RBC and other plasma membranes is obviouslystill a matter of debate.Other factors may also be involved in the development and maintenance ofphospholipid asymmetry. For example the addition of gramicidin to erythrocytes greatlyincreases the rate of transbilayer movement of phosphatidyicholine. However,formylated gramicidin does not affect the rate of PC flip-flop (Classen et al., 1987). Thishas been correlated to the ability of gramicidin to induce non-bilayer (H1 phases inmembranes and the inability of the formylated gramicidin to induce this phase (Killian &de Kruijff, 1986). It is also possible that membrane potentials play a role in developinglipid asymmetry. Donohue-Rolfe and Schaechter (1980) have shown that the transport ofphosphatidylethanolamine across the cytoplasmic membrane of E. coli is extremelyrapid. This transport is not affected by depletion of ATP or the inhibition of protein orlipid synthesis, but is markedly reduced if the proton-motive force is depleted. Thecomplexity of biological membranes makes determining the role of various mechanismsto induce and maintain lipid asymmetry a difficult task.32In addition to the complications of different mechanisms acting to induce lipidtransport in biological membranes, the discovery that phospholipid transport in biologicalmembranes is rapid has rendered the results of some techniques of determining lipidasymmetry questionable. If the rate of transport of the lipids is fast with respect to themethod used to determine asymmetry, then the results of the assay could be invalid.Also, techniques which perturb the membrane could result in the rapid redistribution oflipids (Etemadi, 1980; Devaux, 1991). This dynamic aspect of lipid asymmetry alongwith the difficulties of preparing pure membrane fractions from eucaryotic cells couldexplain the often different extents of asymmetry reported by various laboratories for thesame membranes. Although the rapid redistribution of lipids under some conditions maylead to difficulties in quantifying the exact extent of lipid asymmetry, the research to dateis unambiguous in indicating that lipid asymmetry is a general feature of most or allbiological membranes.1.5.3 Lipid Asymmetry in Model MembranesModel systems have been used to study both the mechanisms by whichasymmetry is generated and maintained, and the functions of lipid asymmetry inbiological membranes. Liposomes exhibiting lipid asymmetry have been produced bymany procedures including enzymatic or chemical modifications of external lipids, forexample phospholipase digestion (de Kruijff & Baken, 1978; Low & Zilversmit 1980),treatment of vesicles containing phosphatidylserine with PS-decarboxylase (Schroit,1986) and the conversion of phosphatidylethanolamine to its amidine derivative usingisethionyl acetimidate (Roseman et al., 1975). Lipids have also been incorporated intothe exterior monolayer of vesicles by spontaneous transfer of various lipids such as33trisialogangliosides (Feigner et ai., 1981) and phospholipids with fluorescent moleculesattached to one of the acyl chains (Pagano et al., 1981) or through the use of phospholipidtransfer proteins (Low & Zilversmit, 1980; de Kruijff & Wirtz, 1977). Small unilamellarvesicles (SUVs) prepared by the sonication of mixtures of phospholipids have also beenused to study lipid asymmetry (Barsukov et al., 1980; Bramhall, 1986; Berden et al.,1975; Lentz et al., 1980). In these systems the spontaneous asymmetric distribution oflipids occurs upon sonication, presumably due to the small radius of curvature of thevesicle which favors the packing of specific headgroups at the inner monolayer.Phospholipid transport in these systems has been shown to be very slow, usually with ahalf-time on the order of days (Low & Zilversmit, 1980; Ganong & Bell, 1984).Perturbing the bilayer with enzymes (de Kruijff & Baken, 1978), or due to the presenceof residual detergent (Kramer et al., 1981) increases the rate of lipid transport but not tolevels observed in biological membranes. Divalent cations (Lentz et al., 1982), theincorporation of proteins into membranes (de Kruijff et al., 1978; Gerritsen et al., 1980)and incorporation of lipids into the external bilayer (Barsukov et al., 1980; de Kruijff &Wirtz, 1977) also produce relatively fast rates of transport. In these cases half-times fortransport are usually on the order of hours instead of days.Although the rate of transport of phospholipids in these model membranes isoften less than that observed for biological membranes, especially membranes thatsynthesize lipids, model membranes have indicated the importance of various factors inlipid transport. For example the rate of transbilayer lipid redistribution in vesicles withreconstituted integral membrane proteins is similar to the rate observed in erythrocytesindicating that a base rate of lipid flip-flop in certain membranes may simply be due topacking defects around membrane proteins. Other studies have shown that differences in34the lateral packing due to differences in the acyl chains may play a role in developingasymmetry (Gabriel & Roberts, 1987) and the rate of lipid flip-flop (de Kruijff & Wirtz,1977). The observation that divalent cations can increase the rate of lipid transport ofcertain lipids, for example PG (Lentz et al., 1982) indicates that phospholipids canundergo rapid flip-flop under the appropriate ionic conditions. These results combinedwith evidence exhibiting the accumulation of Ca2 in vesicles containing PA (Nayar etal., 1984; Serham et al., 1981a, 1981b) implicates phospholipids as possible ionophores.Model systems have also been used to show the importance of the phospholipidheadgroup structure in the transbilayer diffusion of lipids (Homan & Pownall, 1988) andwork by Ganong and Bell (1984), employing phosphatidylthioglycerol anddioleoylthioglycerol, has shown that the phospholipid headgroup is the major barrier totransbilayer lipid transport. In addition, future experiments involving the reconstitutionof membrane proteins may eventually lead to the isolation and identification of lipidtranslocases.1.5.4 Biological Significance of Lipid AsymmetryThe fact that all the biological membranes studied to date have shown anasymmetric distribution of their phospholipids and that the transbilayer transport ofcertain lipids appears to be mediated by proteins, strongly indicates that the asymmetry oflipids is important for cell function. Indeed, many functions have been proposed for therole of lipid asymmetry. Dragsten et al. (1981) proposed that the ability of membranecomponents to pass through tight junctions between the apical and basolateral surfaces ofepithelial cells depends upon their being located on the inner monolayer of themembrane. Thus lipids and other molecules which are capable of being transported to35the inner monolayer of the membrane pass freely through tight junctions while proteinsand other non transportable molecules remain segregated on their respective surfaces. Ithas been suggested that lipid asymmetry may be responsible for the maintenance of thediscoid shape of erythrocytes (Seigneuret & Devaux, 1984). Research has shown thaterythrocytes which have lost their asymmetry are recognized and phagocytosed bymacrophages more readily than asymmetric erythrocytes (McEvoy et a!., 1986). Sinceaged erythrocytes show an increased exposure of phosphatidylethanolamine andphosphatidylserine on their outer monolayer (Shulka & Hanahan, 1982), it is possiblethat the loss of asymmetry is responsible for the clearance of aged erythrocytes by splenicmacrophages (Tanaka & Schroit, 1983; Schroit et al., 1985). The role of platelets inblood coagulation has been correlated to the exposure of phosphatidylserine on the outermonolayer of the platelets upon activation (Zwaal, 1978; Bevers et a!., 1987). Otherresearch (see below) has indicated that lipid asymmetry may also play a role inmembrane fusion events (Cullis & Hope, 1988; Tullius et al., 1989; Santini et a!., 1990)especially exocytosis (Nayar et al., 1982; Zachowski et al., 1989). Finally, it has beenspeculated that specific lipids may be necessary for the enzymatic activity of certainproteins (Houslay & Stanley, 1982).1.6 MEMBRANE FUSIONMembrane fusion is a central process in many biological events including endoand exocytosis, fertilization, lipid and protein trafficking and muscle biogenesis. Inbiological systems, membrane fusion is a tightly regulated event and although fusion hasbeen extensively studied, the molecular mechanisms involved in controlling fusion arestill poorly understood. Studies suggest that biological membrane fusion is controlled by36many factors including the presence of various ions, specific proteins and the lipidcomposition of the membrane.There are many examples of the importance of proteins in the regulation ofmembrane fusion in vivo. Exocytosis and fusion of enveloped virus to cell membranesare two examples. The role of proteins is quite different in these two processes. Inexocytosis a group of calcium binding proteins, collectively referred to as annexins,appear to play a major role in regulating fusion by aggregating the appropriatemembranes. However, it has been suggested that the actual fusion event is initiated byother factors that destabilize the lipid bilayer, such as fatty acids and diacylglycerol (Zaks& Creutz 1990). In contrast, it has been proposed that the fusion of enveloped viruses totarget membranes is controlled entirely by the viral glycoproteins. That is, theglycoprotein is responsible for recognition and binding to the target membrane and theinitiation of fusion by destabilizing the lipid bilayer (Hoekstra & Kok, 1989; Hoekstra,1990).Regardless of the mechanism by which the fusing membranes are destabilized,the final fusion event must involve the lipid bilayers. Model membranes are therefore anideal system for studying membrane fusion. A brief discussion on the fusion of modelmembranes and some of the methods used to assay fusion events, follows.1.6.1 Methods of Detecting membrane FusionTechniques that have been used to detect membrane fusion include differentialscanning calorimetry (DSC), nuclear magnetic resonance (NMR) and electron spinresonance (ESR) spectroscopy, electron microscopy, gel filtration chromatography,turbidity measurements and fluorescence techniques (Wilschut & Hoekstra, 1986).37Fluorescence techniques have proved to be among the most useful procedures forstudying fusion and a few techniques will be briefly discussed.1.6.1.1 Lipid Mixing AssaysSeveral lipid mixing assays have been developed to monitor fusion. The mostcommonly used method is based on resonance energy transfer (RET) between twofluorescently labelled lipids, often derivatives of phosphatidylethanolamine, N-(lissaminerhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) and N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylethanolamine (N-NBD-PE) (Struck et al., 1981).Rhodamine is an efficient quencher of NBD fluorescence because its excitationmaximum is close to the emission maximum of NBD. In this assay the fluorescentprobes are incorporated into liposomes in equal concentrations (typically < 1% of thetotal lipid). The labelled vesicles are mixed with an excess of unlabelled targetmembrane and NBD fluorescence is constantly monitored. Fusion is detected as anincrease in NBD fluorescence as the fluorescently tagged lipids diffuse away from eachother upon mixing with the unlabelled lipids. Alternatively, the two lipids can beincorporated into separate populations of vesicles and fusion can be monitored as adecrease in NBD fluorescence due to energy transfer between the fluorescent probesupon lipid mixing. The RET assay has proven to be very useful as the increase in NBDfluorescence is proportional to the amount of fusion under specific conditions. However,since the fluorophores must be incorporated into vesicles, at least one of the membranesmust be artificial.An assay for fusion that does allow monitoring of intact biological membranesrelies on the fluorescent dequenching of octadecyirhodamine B chloride (R18) (Hoekstra38et al, 1984). This fluorophore spontaneously incorporates into lipid bilayers after itsaddition in an ethanolic solution. At high enough concentrations in the intact membranethe R18 effectively quenches its own fluorescence. Upon dilution into unlabelledmembranes due to fusion, the efficiency of quenching is reduced and an increase influorescence is observed (see Fig. 8).1.6.1.2 Mixing ofAqueous ContentsMany assays have been used to detect the mixing of aqueous contents uponfusion. The two most commonly used fluorescent assays are the terbium (Th3j,dipicolinic acid (DPA) assay (Wilschut et al., 1980) and the 1-amino-naphthalene-3,6,8-trisulfonic acid \/ N,N\u2019-p-xylenebis-(pyridinium bromide) (ANTS\/DPX) assay (Ellens etal., 1985).In both cases the respective probes are encapsulated in two separate populationsof vesicles. For theTb3+IDPA assay, the Tb3+ is encapsulated with a low concentrationof citrate to prevent it from binding to negatively charged lipids. If vesicle fusion occursa Tb(DPA)3 complex forms which is highly fluorescent. EDTA is present in theexternal medium to prevent the formation of the fluorescent complex due to leakage fromthe vesicles. This assay is not useful in acidic conditions (pH < 4.0) because theprotonated form of DPA is membrane permeable.The ANTS\/DPX assay, in which the decrease in fluorescence of ANTS uponchelation by DPX is measured, can be used in acidic conditions. Leakage of contents tothe external medium does not affect ANTS fluorescence because the quenching offluorescence by DPX is highly concentration dependent. These assays are restricted tosystems where the internal contents can be exchanged.39Figure 8Fluorescence Assays to Monitor Membrane Fusion(A) Mixing of internal contents (Th3\/DPA). (B) lipid mixing assays using resonanceenergy transfer (RET) and (C) relief of fluorescence self-quenching of R18.AC0401.6.2 Fusion of Model MembranesModel systems ranging in complexity from single cells and permeabilized cells tocell-free systems composed of isolated intracellular membranes to pure lipid bilayershave provided the most information about the molecular mechanism of membrane fusion(Burger & Verkleij, 1990). The most useful systems have been the simplest modelmembranes, that is systems composed of pure lipids. These systems allow for the precisecontrol of the environment of the membrane and therefore individual parameters can betested for their effect on fusion.For example, calcium has been shown to induce fusion of vesicles composed ofacidic phospholipids (Papahadjopoulos et al., 1974, 1976, 1977; Wilschut et al., 1980,1981). Since Ca2 is required for many biological fusion events, the possibility arisesthat the interaction of Ca2with acidic phospholipids may mediate membrane fusion.However, the concentration of Ca2+ required to cause fusion between vesicles composedpurely of acidic phospholipids is usually several orders of magnitude larger than thatobserved in the intracellular milieu (Duzgunes, 1985). This does not necessarily rule outthe role of acidic lipids in intracellular membrane fusion events since areas may existwith transiently high local Ca2 concentrations. In addition, as previously mentioned(Section 1.6) certain proteins (annexins among others) act to aggregate fusingmembranes. These proteins have been shown to greatly reduce the concentration of Ca2+required to induce the fusion of acidic phospholipid vesicles (Papahadjopoulos et al.,1990; Zaks & Creutz, 1990). More likely Ca2 could mediate fusion through otherprocesses acting synergistically to destabilize the bilayer. Furthermore, Ca2+has beensuggested to induce a conformational change in a fusion protein to make it fusogenic41(Papahadjopoulos et a!., 1990). Alternatively, the presence of Ca2 could activatephospholipases to produce free fatty acids or diacyiglycerols, both of which have beenshown to destabilize bilayer structures and cause membrane fusion (Zaks & Creutz,1990). Other enzymatic reactions such as phosphorylation of proteins by protein kinasescould also play a role (Burger & Verkleij, 1990). It is also possible that other ions suchas Zn2 or Mg2 act synergistically with the Ca2 to reduce the concentration of Ca2required to induce fusion (Deleers et al., 1986). It is likely that many of these factors areresponsible for the initiation and regulation of membrane fusion.Other components, besides the presence of divalent cations, involved in theinduction and regulation of membrane fusion may be specific proteins which can eitheraid in fusion by aggregating membranes or may be directly involved in destabilizing thelipid bilayer through hydrophobic interactions (Martinez-Bazenet et al., 1988; Meers eta!., 1988(a); Maezawa et al., 1989; Arvinte et al., 1986). Peptides acting in a similarmanner to proteins (Walter et al., 1986; Suenga et a!., 1989), free fatty acids ordiacylglycerols which can induce the formation of non-bilayer phase structures (Cullis &Hope, 1978; Meers et a!., 1988(b); Siegel et a!., 1989) lipid composition (Duzgunes,1985), lipid polymorphic properties (see Section 1.6.3) (Cullis & Hope, 1988; Ellens etal., 1989) and other factors may also be important in the induction and regulation offusion. Since model systems can address questions that are not easily examined incomplex biological systems they have lead to several proposed mechanisms formembrane fusion.421.6.3 Molecular Mechanism(s) of Membrane FusionIn recent years several mechanisms for membrane fusion which share commonfeatures have been proposed. These include the close apposition of membrane bilayers,the formation of an intermediate structure in which the outer monolayers of the vesiclescoalesce (membrane destabilization), the formation of a pore between the two vesiclesand the mixing of the internal contents as the pore expands. The differences in themechanisms of fusion lie in the nature of the intermediate structures and the parameterswhich cause close membrane apposition and bilayer destabilization. For examplePapahadjopoulos et al., (1990) have presented three pathways for the molecularrearrangements of lipids during membrane fusion. In all three pathways, the initial stepinvolves two intact membranes making contact with each other within a short distancewith partial or complete dehydration at the point of contact. In the next stage, a \u201ccontact-induced defect\u201d is postulated, resulting from the local dehydration and otherintermembrane interactions. The resulting structures are unstable since parts of the acylchains become exposed to the aqueous phase. The second step is considered to be thefirst committed step to fusion and has been termed the \u201chydrophobic contactintermediat&\u2019. In this step the lipids of the outer monolayers can mix. The next stageinvolves the formation of a \u201ccurved bilayer annulus\u201d, which is said to be a short-livedhigh-curvature intermediate forming a water filled channel between the two membranes.Finally, the aqueous channel enlarges, thus removing the areas of high-curvature andproducing a larger vesicle. Difficulties with this model include the fact that thedehydration step, which can be induced by Ca2 in PS containing systems, is notgenerally available for other lipid systems.43The most likely intermediate structures in membrane fusion are the intermediarystructures postulated in the formation of non-bilayer lipid phases (hexagonal H11 andcubic phase) (see Figure 9). The role of inverted micellar intermediates (IMIs) inmembrane fusion was first proposed in the late 1970\u2019s (Cullis et al., 1978; Verkleij et al.,1979; Cullis & Hope, 1979) and this theory has been enhanced by more recent kineticanalyses (Siegel 1986). Siegel\u2019s kinetic analysis shows that inverted micelles have a veryshort lifetime (103 sec range). This value agrees well with the rate of biological fusionevents (also of the i0 sec range) and is compatible with a local-point contact fusionevent, which morphological evidence indicates is the method by which biologicalmembranes fuse (Chandler, 1980, 1984; Heuser et al., 1979; Knoll et al., 1988). The nextstep in membrane fusion involves the formation of interlamellar attachment sites (ILAs)where an aqueous channel is formed between the two membranes (see Fig. 9).Alternatively aggregation of IMIs between apposed bilayers can lead to the formation ofthe H11 phase which involves leakage of vesicle contents (Bentz et al., 1985).The involvement of these intermediary structures in membrane fusion issupported by a large body of evidence. For example, Hope & Cullis (1981) showed thatthe ability of molecules to induce the fusion of erythrocyte phospholipids correlated withtheir ability to induce non-bilayer phases and that chemiclly related molecules that werenon-fusogenic did not induce the formation of non-bilayer phases. In addition, almostany biological membrane studied contains a significant amount of lipid that in isolationwill adopt the hexagonal phase under physiological conditions. (Burger & Verkleij,1990) and other lipids that will adopt this phase under various other conditions, such asthe addition of divalent cations or cis-unsaturated fatty acids.44Other studies of membrane fusion in various systems are also consistent with amodel of fusion involving IMIs. These include studies which show the presence ofparticles in influenza induced fusion events (Knoll et al., 1988) and exocytosis (Schimdtet a!., 1983) by fast-freeze freeze-fracture, the enhancement of envelope virus fusion withmodel membranes by the addition of H11 forming lipid (Van Meer et a!., 1985; White etal., 1983) and the indication of fusion pore formation during exocytosis by capacitancemeasurements (Zimmerberg, 1987, Brenckenridge & Walmers, 1987). Finally as statedearlier, factors which induce the formation of the H11 phase, such as fusion proteins,diacylglycerol and cis-unsaturated fatty acids also tend to induce fusion (Hope & Cullis,1981; Siegel et al., 1989).45Figure 9Mechanism of Membrane Fusion Procceding ViaIntermediates of the Bilayer to Hexagonal H11 PhaseTransitionModel of membrane fusion proceeding via an inverted micellar intermediate (IMI).Formation of the hexagonal Hi phase occurs via aggregation of IMIs (see Section 1.6.3).From Ellens et a!., 1989.gij [3 1H aiIH phaseprecurio rs(leakage)1Isotropic orinverted Cubic H11PhasesIMI461.7 THESIS OUTLINEThe mechanisms by which lipid asymmetry is generated and maintained inbiological membranes are still poorly understood. The functions of lipid asymmetry inbiological membranes are also ill defined. Previous research has indicated that iongradients, specifically pH gradients, can induce lipid asymmetry in model membraneLUV systems. The purpose of this thesis is to study the mechanism by which pHgradients induce this asymmetry for phosphatidic acid and other acidic lipids. Further,the role of lipid asymmetry in such processes as lipid exchange and membrane fusion areexamined.The influence of transmembrane pH gradients on the exchange of simple lipids(stearylamine and fatty acids) between LUVs, and between LUVS and bovine serumalbumin is discussed in Chapter 2. In Chapter 3, the mechanism by which transbilayerpH gradients induce the asymmetric distribution of dioleoylphosphatidic acid (DOPA) isexamined. Additionally, a new fluorescent assay to detect the asymmetric distribution ofacidic phospholipid is described. Finally, in Chapter 4, the ability of lipid asymmetry tomodulate membrane fusion between LUVs is examined.47CHAPTER 2INTERVESICULAR EXCHANGE OF LIPIDS: INFLUENCE OFTRANSMEMBRANE PH GRADIENTS2.1 INTRODUCTIONLipid transport and exchange are important physiological processes. Forexample, the diffusion of fatty acids between micelles, fatty acid binding proteins andcell membranes represents fundamental steps in the catabolism and biosynthesis of lipids.In addition, fatty acids are the major source of metabolic energy of most mammaliantissues. Due to the low solubility of fatty acids in aqueous media and the considerabledaily flux of fatty acids from adipose triglyceride stores into the plasma (\u2014 700 mmol\/dayin the average human) complex mechanisms have evolved in order to transport fattyacids to their site of utilization (Bass, 1988). These include the transport of fatty acidsthrough the plasma in the form of relatively inert triglycerides in lipoproteins or bound tofatty acid binding proteins (FABPs), of which serum albumin is the main example inplasma. Although, there is more than one mechanism for the delivery of fatty acids totarget membranes (see Chapter 1, section 1.4.1), much evidence exists that fatty acidexchange between membranes, or between membranes and binding proteins, occurs viamonomer diffusion from one surface to the other. The rate limiting step in this process isthe release of monomers from the donor surface to the aqueous medium (Ferrel et al.,1985; Roseman & Thompson, 1980; Nicholls & Pagano, 1982; Doody et al., 1980; Noyet al., 1986). Thus the transbilayer distribution of an exchangeable lipid in a lipid bilayershould have a profound effect on the rate at which the lipid is exchanged between two48surfaces as lipids sequestered on the inner monolayer are not available to dissociate intothe external aqueous environment.Previous studies have shown that transmembrane pH gradients can induce theasymmetric transbilayer distribution of simple lipids with acid or base characteristics,such as stearylamine and fatty acids (Hope & Cullis, 1987). A model of the mechanismby which lipids are transported across lipid bilayers in response to transmembrane pHgradients is depicted in Figure 10. The ability of a transmembrane ApH to regulate thetransbilayer distribution of lipids should affect the exchange characteristics of thesesimple lipids.This chapter examines the effects of transmembrane pH gradients on theexchange of simple lipids between model membranes and between membranes andbovine serum albumin (BSA). The results are discussed with respect to the mechanismand modulation of lipid flow both in vitro and in vivo.49Figure 10Mechanism of Net Acidic Lipid Transport in Responseto a Transmembrane pH GradientIf the exterior pH is low a certain proportion of the acidic lipids are protonated (neutral)and freely permeable across the bilayer. However, upon reaching the more basic interiorenvironment, the lipids are deprotonated and negatively charged. The charged form ofthe lipid is relatively impermeable to the bilayer such that the lipids effectively becometrapped on the inner monolayer.Outside Inside\u2022OOC tcoo+ I 1WHOOC COOHII[R-C0010[H], IR-COOiilHii[R-COOH]0 [R-COOH]At equilibrium[R-COOH]0 [R-COOHJ1Thus(R-COO][R\u2014C00]0 [HJI502.2 MATERIALS AND METHODS2.2.1 Lipids and ChemicalsDioleoylphosphatidylcholine (DOPC) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (Rh-PE), N-(7-nitro-2, 1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolamine (NBD-PE) and brain phosphatidylserine (PS) wereobtained from Avanti Polar Lipids (Birmingham, AL).Stearylamine (SA) and oleic acid (OA) were purchased from Sigma (St. Louis)while the [3H} dipalmitoylphosphatidycholine (DPPC) was purchased from NEN. [14C]Oleic acid was obtained from ICN and the [\u20184C] stearylamine was obtained from Dr. J.Wilschut. Lipid compositions of vesicles are expressed as molar ratios.2.2.2 VesiclesLarge unilamellar vesicles were prepared by extrusion through polycarbonatefilters (Hope et a!., 1985) using the Lipex Extruder obtained from Lipex BiomembranesInc., Vancouver, Canada. Lipid mixtures were dried down from chloroform under astream of nitrogen gas and residual chloroform was removed under vacuum for one hour.The appropriate buffer composed of either 150 mM sodium citrate, 10 mM HEPES, 5mM K2S04(pH 7.4) or 150 mM sodium citrate, 5 mM K2S04(pH 4.0) was added to thelipid film and vortexed to prepare a liposomal suspension of approx. 10 p.mol\/ml totalphospholipid. The liposomes were freeze-thawed five times using liquid nitrogen-warmwater cycles in order to increase the trapped volume of the vesicles and to promoteequilibrium solute distributions. These freeze and thawed multilamellar vesicles51(FATMLVs) (Mayer et a!., 1985) were then extruded ten times through two stacked 0.1urn polycarbonate filters (Nuclepore). After preparation, all vesicles were passed downSephadex G-50 columns equilibrated with 10 mM Na2SO4,10 mM HEPES, 5 mMK2S04(pH 7.4). Exchange of the initial hydrating buffer for the low ionic strengthbuffer was necessary to enable binding of charged vesicles to the ion exchange columns.2.2.3 Turbidity Experiments to Monitor Vesicle AggregationVesicles (DOPC\/PS (8:2) mol\/mol or DOPC\/SA (9.5:0.5) mol\/mol) wereprepared as described above by hydrating the lipid films in buffer containing 150 mMcitrate (pH 7.4). After exchanging the external buffer employing G-50 columns theturbidity of both sets of vesicles (at a concentration of 1 umol\/ml total phospholipid) wasmonitored at 550 nm using a Shimadzu UV-160 spectrophotorneter.Vesicles containing PS were then added into a cuvette to a final concentration of0.5 p.mol\/ml. After monitoring the turbidity of the solution for approx. lOs,stearylamine-containing vesicles (0.5 umol\/ml final concentration) were added andmixed. The change in absorbance was then measured over the next 2 mm.To examine the effects of transmembrane pH gradients (ApH) on the aggregationof LUVs, vesicles composed of DOPCIPS(8:2) or DOPC\/SA (9.5:0.5) were prepared asdescribed above except the vesicles containing SA were hydrated in a buffer containing150 mM citrate (pH 4.0). The turbidity of both sets of vesicles were measuredseparately. PS-containing vesicles were added to the cuvette to a final concentration of0.5 p.mol\/ml. After approx. 20 s stearylamine-containing vesicles were added and mixed.Approx. 40 s later valinomycin and nigericin (0.1 and 0.01 umol, respectively) wereadded to the cuvette with mixing.522.2.4 Stearylamine Exchange Monitored by Ion Exchange Chromatography usingDEAE-SephacelVesicles composed of DOPC\/PSINBD-PE (8:2:0.1) containing trace quantities of[3H]DPPC or DOPC\/[14C]SAIRh-PE (9.5:0.5:0. 1) were prepared in 150 mM citrate, 10mM HEPES, 5 mMK2S04buffer (pH 7.4) and passed down Sephadex G-50 columnsequilibrated with 10 mM Na2SO4,10 mM HEPES, 5 mM K2S04 buffer (pH 7.4). Then,1 tmol of each set of vesicles were passed down DEAE-Sephacel (Pharmacia) columnsequilibrated in external buffer (10 mM Na2SO4,10 mM HEPES, 5 mM K2S04(pH 7.4)).After two fractions (approx. 3.5 ml per fraction) were collected, the eluting buffer waschanged (500 mM NaC1, 10 mM HEPES (pH 7.4)) and four more fractions werecollected. After establishing elution profiles for the two vesicle populations, 1 &mo1 ofeach population of vesicles were added together, mixed, and allowed to incubate for 5mm at room temperature (approx. 20\u00b0C). The mixture of vesicles was then applied to aDEAE-Sephacel column and eluted under the same conditions as described above.Radioactive decays were monitored using a Packard Tricarb 2000 CA liquid scintillationcounter. The presence of fluorescent lipid was assayed employing an SLM Aminco SPFSOOC fluorometer.To study the effects of a transmembrane pH gradient on stearylamine exchange,brain phosphatidylserine-containing vesicles([3H]DOPCIPS or DOPC!PS (8:2)) andstearylamine-containing vesicles (DOPC\/[\u20194C]SA or3H]DOPC\/[\u20194C]SA(9.5:0.5)) wereprepared as described above with the exception of the buffers. The PS-containingvesicles were hydrated at pH 7.4 while the SA-containing vesicles were hydrated at pH4.0. Vesicles (1 tmo1 of each population) were mixed([3H]DOPC\/PS + DOPCI[\u20194C]SAor DOPC\/PS +[3H]DOPC\/[14C]SA) in the pH 7.4 buffer and incubated either in the53presence or absence of the ionophores valinomycin and nigericin (0.1 and 0.01 tmol,respectively). After a 5 mm incubation at room temperature, the vesicle populationswere separated on DEAE columns and the fractions were assayed for 3H and 14C.2.2.5 Oleic Acid ExchangeOleic acid containing vesicles (DOPC\/PS\/[\u20194C]OA(8:2:0.5)), DOPC vesicles andDOPC vesicles containing stearylamine (DOPC\/SA (9.5:0.5)) were hydrated at pH 10.0(100 mM Na2SO4,50 mMH3B0,5 mMK2S04)or pH 7.0 (150 mM sodium citrate, 10mM HEPES, 5 mMK2S04)as required. After extrusion, vesicle populations werepassed down G-50 columns equilibrated in pH 7.0 external buffer (20 mM Na2SO4,10mM HEPES, 5 mMK2S04). Vesicle populations (1 !.tmol of each) were mixed eitherwith or without the presence of ionophores, and after a 5 mm incubation at roomtemperature (\u2014 21\u00b0C) the populations were separated on DEAE-Sephacel columns asdescribed above. Fractions from the column were then counted employing a dual labelprogram on a Packard 2000 CA liquid scintillation counter.For experiments monitoring the extraction of fatty acids from BSA, [\u20184C]fattyacid were mixed with BSA at a ratio of 2:1 (F.A.\/BSA). Fatty acid loaded BSA wasadded to 1 mL of 20 mM DOPC vesicles to a final concentration of 5 mg\/mL BSA. 200L of the mixture was applied to a 20 x 1.5 cm Sepharose CL-4B column and eluted withthe external pH 7.0 buffer. The fractions were measured for 3H and 14C counts.All results presented in this thesis are average values obtained from a minimum ofthree experiments, except where noted. In this Chapter, Figures 11 and 13 present resultsfrom a single experiment which are typical of multiple trials.542.3 RESULTS2.3.1 Stearytamine ExchangeThe aggregation-disaggregation behavior of vesicles consisting of DOPC and 20mol% PS and donor vesicles composed of DOPC and 5 mol% stearylamine is shown inFig. 11. The turbidity profiles of vesicles prepared with a transmembrane ApH = 0 (pH7.4 inside and outside) demonstrate that separately PS and stearylamine-containingvesicles are only slightly turbid, giving an absorbance of approx. 0.06 at 550 nm.However, when mixed the turbidity of the solution immediately increases then slowlydecreases until it reaches the original turbidity of the separate vesicles. This process canalso be followed visually. It is logical to suggest that this aggregation-disaggregationphenomenon occurs due to an initial electrostatic attraction giving rise to aggregation ofthe oppositely charged vesicles during which time exchange of the stearylamine occurs,followed by disaggregation of the vesicles after the surface charges of acceptor and donorvesicles have equilibrated. This interpretation is supported by the data shown in Fig 12.Figs. 12A and 12B show the elution profiles from DEAE-Sephacel columns of[3H]DOPC\/PS (8:2) and DOPC\/[14C]SA(9.5:0.5) vesicles, respectively. In Fig. 12C thetwo vesicle populations were mixed and incubated for 5 mm before being applied to theDEAE column. All the tritium counts are associated with the PS-containing vesicles,indicating that there has been no exchange of[3H]DPPC. The [14C] stearylamine countson the other hand are approximately equally distributed between the two vesiclepopulations. These results clearly suggest that only stearylamine is undergoing exchangein this vesicle system. It was also of interest to incorporate the fluorescent energytransfer probes rhodamine-PE and NBD-PE into the stearylamine and PS-containing55Figure 11Turbidity Measurements of Vesicle AggregationTurbidity measurements at 550 nm of (A) (- - -) DOPC,PS (8:2) v\u00e9sicles (1 iimol\/ml),(B) (\u2014 \u2014) DOPC\/stearylainine (9.5:0.5) vesicles (1 imol\/ml) and (C) ( ) amixture of 0.5 tmol\/ml of the two vesicle populations. The results for (A) and (B)overlap. The.arrow indicates the time at which the SA-containing vesicles were added.110.8. 0.6Ui()z0.40(I)0.20.014060 80TIM[ (seconds)56Figure 12Characterization of Vesicle Elution from DEAESephacel ColumnsElution profiles from DEAE-Sephacel columns of (A)[3H]DOPC\/PS (8:2) vesicles, (B)DOPC\/[\u20194C]stearylamine (9.5:0.5) vesicles, (C) a mixture of vesicles in (A) and (B), and(D) a mixture of DOPC\/PS\/NBD-PE (8:2:0.1) and DOPC\/stearylamine\/Rh-PE(9.5:0.5:0.1) vesicles. (.) 3H disintegrations, (D) 14C disintegrations. In panel (D), (.)indicates rhodamine fluorescence (ex. 560, em. 590) and (o) indicates NBD fluorescence(ex. 480, em. 530). The arrow indicates the point at which elution with high ionicstrength buffer was started.jFRACTION57vesicles, respectively, in order to ascertain whether there was evidence of lipidmixing and fusion during aggregation. The results, shown in Fig. 12D, show that theseprobes do not exchange between the acceptor and donor vesicles during the aggregationstage. Moreover, there was no detection of fluorescence energy transfer during theaggregation-disaggregation reaction.2.3.2 Effect of a Transmembrane ApH on Stearylamine ExchangeHaving established that stearylamine rapidly equilibrates between the two vesiclepopulations in the absence of a transmembrane pH gradient, vesicles of DOPC\/SA(9.5:0.5) were prepared with an internal pH of 4.0. Given the external pH of 7.4, a pHgradient of -3.4 units (calculated as inside pH minus outside pH) is then formed acrossthe bilayer. Previous work has shown that such a gradient induces stearylamine toaccumulate at the inner monolayer of the vesicle resulting in a reduction of the outermonolayer concentration of stearylamine by up to three orders of magnitude (Hope &Cullis, 1987). Fig. 13 demonstrates the change in the outer surface charge associatedwith stearylamine-containing vesicles in the presence of a pH gradient (acidic inside).When DOPC\/PS (8:2) and DOPC\/stearylamine (9.5:0.5) vesicles are mixed there is noaggregation, consistent with an absence of a positive surface charge on theDOPC\/stearylamine vesicles.However, dissipating the gradient using a combination of nigericin, a protonionophore, and valinomycin, a K+ ionophore, in the presence of K+ ions allowsstearylamine to equilibrate across the bilayer. A positive surface charge is then restoredand aggregation with the negatively charged PS-containing vesicles is observed as anincrease in turbidity. Subsequently, inter-vesicle exchange of stearylamine takes place,58and the vesicles disaggregate. This mechanism is confirmed in Fig. 14 whichdemonstrates that in the presence of a pH gradient (ApH = -3.4), when stearylamine islocated at the inner monolayer of the vesicle, exchange of stearylamine between vesiclesdoes not occur. Specifically,[3H]DOPC\/PS LUVs were incubated withDOPC\/[14C]stearylamine LUVs (pH 4.0 inside) and after a 5 mm incubation, separationon an ion exchange column shows that 97% of the stearylamine is still associated withthe DOPC vesicles. However, as illustrated in Fig. 14B, in the presence of ionophoresstearylamine equilibrates with the PS-containing vesicles. It should be noted thatexchange of phospholipid label between the vesicle systems was never observed.2.3.3 Fatty Acid Exchange Between MembranesThe results presented above deal with the transfer of stearylamine betweenvesicles. Vesicles exhibiting a positive ApH (basic inside) sequester fatty acids to theinner monolayer (Hope & Cullis, 1987), and consequently fatty acid exchange betweenmembranes should also be influenced by transmembrane proton gradients. In order tomonitor the exchange of fatty acid between two vesicle populations and test thishypothesis we studied the following systems. In the first DOPC\/SA (9.5:0.5) vesicleswere incubated with DOPC\/PS\/[14C] A(8:2:0.5) LUVs. Vesicles containing oleic acid(OA) were prepared with a pH gradient that was basic inside (see Section 2.2.5) yieldinga ApH = 3.0. As we have described above (see Figs. 11 and 13), mixing positivelycharged stearylamine-containing vesicles and negatively charged PS-containing vesiclesresults in immediate aggregation followed by a slower disaggregation due to theequilibration of stearylamine between the vesicle populations. The same phenomenonwas observed for DOPCISA and DOPCIPS\/[14C]OAvesicle systems. However, as59Figure 13Effect of Transmembrane ApH on Vesicle AggregationTurbidity measurements at 550 nm of a mixture of DOPC\/PS (8:2) andDOPC\/stearylamine (9.5:0.5) vesicles with a transmembrane pH gradient (ApH = -3.4)DOPCIPS vesicles were added at t = O;\u2019L addition of DOPC\/stearylamine vesicles; 4,addition of ionophores (nigericin and valinomycin) to collapse the pH gradient.E0tou)C,)TIME (secs..)60Figure 14Effect of a Transmembrane ApH on StearylamineExchangeElution profiles of[3H]DOPCIPS (8:2) vesicles and DOPC\/[14C]stearylamine vesicles.(A) DOPC\/[14Cjstearylamine vesicles have a transmembrane pH gradient (ApH = -3.4)acidic interior. (B) The same vesicle mixture plus the ionophores, nigericin andvalinomycin, used to collapse the transmembrane pH gradient. (.) ,3H disintergrations,(D) 14C disintergrations.UU0 1 2- 3 4 5 6FRACTION61shown in Fig. 15A, the exchange of oleic acid is greatly reduced by the presence of apositive ApH. It is worthwhile pointing out that despite vesicle aggregation andstearylamine exchange the bulk of the oleic acid remains associated with the innermonolayer of the DOPC\/PS\/OA vesicles. This suggests that vesicle aggregation and lipidexchange does not significantly enhance the proton permeability of the PS-containingvesicles, otherwise the ApH would collapse enabling oleic acid to exchange. When theApH is deliberately dissipated employing the ionophores valinomycin and nigericin in thepresence of K,[14C] oleic acid is observed to elute from the DEAE column in bothvesicle fractions indicating equilibration of the fatty acid between DOPC\/SA andDOPC\/PS\/OA vesicles (Fig 15B).The second approach involved monitoring the transfer of fatty acid from onepopulation of vesicles to another in non-aggregating systems as shown in Fig. 16. LUVscomposed of[3H]DOPC were incubated with vesicles of DOPC\/PS![\u20194C]OA 8:2:0.5).The results of Fig. 16A show that in the absence of a ApH oleic acid rapidly equilibratesbetween the two vesicle populations. However, when the DOPC LUVs exhibit a ApH of3.0 units (inside basic) oleic acid preferentially moves into the DOPC vesicles (Fig. 16B).At equimolar concentrations of donor and acceptor vesicles, 85% of the oleic acidtransfers to the DOPC vesicles. At higher ratios of acceptor to donor vesicles more than90% of the oleic acid transfers to the DOPC vesicles.2.3.4 Exchange of Fatty Acids Between Vesicles and BSAThe above experiments clearly demonstrate an ability of transmembrane pHgradients to modulate lipid flow between membranes. However, a large proportion offree fatty acid in vivo is delivered to peripheral tissues bound to serum albumin. In the62Figure 15Oleic Acid Exchange in Aggregating SystemsElution profiles from DEAE-Sephacel columns of mixtures of DOPC\/SA (9.5:0.5) andDOPCIPS\/[\u20194C]OA (8:2:0.5) vesicles. (A) The oleic acid-containing vesicles had a basictransmembrane pH gradient (ApH = 3.0). (B) The vesicles in the presence of ionophoresto collapse the pH gradient. (0) 14C disintergrations. The arrow indicates the point atwhich elution with high ionic strength buffer was started.0.010010 1 2 3 4 5 6 7Fraction63light of our observations on the modulation of fatty acid flow between membranes bytransmembrane pH gradients, it was of interest to see whether albumin could be depletedof fatty acid when incubated with a population of vesicles that exhibit a positive ApH(inside basic). Fig. 17 presents the data from such an experiment. When incubated withvesicles composed of DOPC (ApH = 0) fatty acid equilibrates between protein andmembrane as shown in Fig. 17A. However, in the presence of a positive ApH, net flux offatty acid occurs in the direction of the vesicles, significantly depleting albumin of lipid(see Fig. 17B).64Figure 16Oleic Acid Exchange in Non-Aggregating SystemsElution profiles from DEAE-Sephacel columns of[3H]DOPC, DOPC\/PS\/[\u20194C]OAvesicle mixtures. (A) Neither vesicle population has a transmembrane pH gradient. (B)The[3H]DOPC vesicles have a basic pH gradient (ApH = 3.0). (.) H disitergrations;(D) 1C disitergrations.100a1a0 1 2 3 4 5 6 7FRACTION65Figure 17Exchange of Oleic Acid Between BSA and VesiclesExtraction of[14C]OA from fatty acid loaded bovine serum albumin (BSA) by DOPCvesicles. Separation of vesicles and BSA on Sepharose CL-4B columns. (A)[3H]DOPCvesicles without a transmembrane pH gradient (ApH = 0.0). (B)[3HjDOPC vesicles witha basic transmembrane pH gradient (ApH = 3.0). (.) 3H disitergrations, (o) l4Cdisitergrations. BSA elutes between fractions 18-28.10080604020n10.AApHO.00 5 10 15 20 25 30 35 40FRACTION66DISCUSSIONThe results presented here illustrate the remarkable exchange abilities ofstearylamine and oleic acid and the sensitivity of these exchange characteristics totransmembrane pH gradients across vesicle membranes. Here the mechanismsmodulating these exchange processes and their implications for lipid exchange in vivoare discussed.The aggregation and subsequent disassociation processes observed for positivelycharged (DOPC\/SA) and negatively charged (DOPC\/PS) vesicles provides a graphicillustration of rapid intervesicular exchange of stearylamine. There are three points ofinterest.First, with regard to the exchange mechanism, whereas the initial attractionbetween positively and negatively charged vesicles is sufficiently intense to producevisible flocculation under our experimental conditions, neither membrane fusion norexchange of diacylphospholipids was observed. The lack of phospholipid exchangeindicates that intervesicular mixing between external monolayers, commonly observed infusing systems (Wilschut & Hoekstra, 1986) does not occur and that the bilayers of theseaggregated vesicles remain intact. This is also supported by the observation that theproton permeability barrier for acceptor vesicles is maintained during the aggregationdisaggregation process involving exchange of stearylamine (Fig. 15). Thus, exchange ofstearylamine may be most logically suggested to proceed via intermediary partition of SAinto the aqueous phase separating the vesicles. In the case of oleic acid, it is widelyaccepted that non-mediated lipid exchange occurs via this mechanism, involvingdesorption of monomers from the bilayer into the aqueous solution, and subsequent67diffusion to an acceptor site (Ferrel et al., 1985; Roseman & Thompson, 1980; Nicholls& Pagano, 1982; Doody et al., 1980). This is also supported by the result presented herefor non-aggregating systems (Fig. 16) where oleic acid is observed to rapidly equilibratebetween the two vesicle populations.The second point concerns the non-exchangeable characteristics of the fluorescentlipids rhodamine-PE and NBD-PE. As indicated in Fig. 12, even under conditions ofvesicle aggregation and stearylamine exchange, there is no evidence of lipid mixing asreported by these probe molecules. This provides an additional confirmation ofindications that these fluorescent lipids do not readily exchange between aggregatedsystems (Wilschut & Hoekstra, 1986; Struck et al., 1981), supporting their use as non-exchangeable markers in studies of membrane fusion.The third point concerns the modulation of stearylamine and oleic acid diffusionby transmembrane pH gradients. These phenomena are relatively straight-forward tounderstand in the light of previous observations (Hope & Cullis, 1987) that lipophilicweak bases, such as stearylamine, will partition to the inner monolayer in vesiclesexhibiting a transmembrane pH gradient (inside acidic) (see Fig. 10). Alternatively,lipophilic weak acids such as oleic acid will partition to the inner monolayer when theinterior is basic (Hope & Cullis, 1987). Thus the absence of aggregation and lipidexchange when appropriate pH gradients are employed is a graphic consequence of theApH-dependent lipid asymmetry.Modulation of lipid exchange by transmembrane pH gradients may also occur invivo. For example, the results presented here show that fatty acid can be induced totransfer from one population of vesicles to another by simply maintaining a basic interiorpH in the acceptor vesicles. Such vesicles can also induce fatty acid depletion of bovine68serum albumin. Although serum and cytoplasmic pH values are strictly maintainedwithin narrow limits (Deamer, 1982), in organelles such as the lysosome, which maintainan acidic interior (Hamilton & Cistola, 1986; Hope & Cullis, 1987), fatty acid would beexpected to mainly reside in the outer (cytoplasmic) monolayer. In addition theadsorption of short chain fatty acids in the digestive tract has been associated with thepresence of pH gradients formed by various mechanisms including proton pumps whichcreate acidic microclimates at the epithelial cell surface. Phospholipid asymmetry,common to many plasma membranes (Op den Kamp, 1979) may also play a role.Phosphatidylserine, for example, is negatively charged and frequently located in thecytoplasmic monolayer of cell membranes, resulting in a negative surface potential(Cullis & Hope, 1985). This potential will repel anions from and attract cations to thelipid\/water interface and will result in a significantly lower pH at the cytoplasmicmembrane surface when compared to the exterior bulk pH. Such a gradient could beimportant in enhancing the flow of fatty acids out of adipocytes, for example.The possibility of fatty acid flow from lysosomes into the cytoplasm because ofthe acidic pH gradient across the lysosomal membrane has been discussed previously(Hamilton & Cistola, 1986; Hope & Cullis, 1987). Mitochondria, on the other hand,develop a basic internal pH during oxidative phosphorylation (Nichols, 1982). Given theresults presented here this might be expected to culminate in an accumulation of freefatty acids by mitochondria. However, cells have evolved a complex system by whichfatty acids are first activated and subsequently converted to carnitine derivatives whichare then transported into the mitochondrial matrix. The data described here suggest thatintracellular free fatty acid may be deliberately kept at a very low concentration toprevent these lipids accumulating within organelles, such as the mitochondria, in an69unregulated manner. The potential harm of this type of accumulation is illustrated by theability of low concentrations of lysosomotropic detergents to kill cells (Miller et al.,1983). These molecules are aliphatic amines which accumulate in lysosomes in responseto the pH gradient, disrupt membrane integrity and cause the release of lysosomalenzymes into the cytoplasm.70CHAPTER 3TRANSBILAYER TRANSPORT OF PHOSPHATIDIC ACID IN RESPONSE TOA TRANSMEMBRANE PH GRADIENT3.1 INTRODUCTIONIt is now generally accepted that biological membranes exhibit asymmetrictransbilayer distributions of their components. For certain lipids (glycolipids &sphingolipids) and proteins, this asymmetry is established during synthesis andmaintained throughout the lifetime of the molecule. However, the rate of transbilayertransport of glycerolipids in most biological membranes precludes a purely staticmechanism for the maintenance of asymmetry (see Op den Kamp, 1979; Etemadi, 1980;Devaux, 1991; Chapter 1 Section 1.5). Although a large body of evidence now exists toindicate the existence of specific proteins to translocate phospholipids across variousmembranes (Seigneuret & Devaux, 1984; Bishop & Bell, 1985; Zachowski et al., 1986;Backer & Dawidowicz, 1987; Connor & Schroit, 1988), such proteins have yet to beisolated or positively identified. In addition, translocators for many species of lipids suchas phosphatidic acid have not as yet been reported. It is probable that other mechanismsexist in conjunction with specific translocators in order to generate and maintain theasymmetry of various phospholipids. For example, Zachowski et al. (1985) havesuggested that the asymmetry of the aminophospholipids may provide the driving forcefor PC asymmetry in erythrocytes. Furthermore, it has been suggested that non-bilayerphase lipids, the presence of integral membrane proteins and ion gradients may play rolesin the induction and maintenance of lipid asymmetry.71In order to investigate the mechanisms by which lipid asymmetry is generated andmaintained, model membrane systems are useful. A major emphasis of this studyconcerns the influence of ion gradients, especially pH gradients, on the transbilayerdistribution of lipids in large unilamellar vesicles (LUVs). Previous studies have shownthat the transbilayer distribution of simple lipids with weak acid or base characteristics,such as fatty acids and stearylamine (Hope & Cullis, 1987; Eastman et a!., 1989; Chapter2), and certain acidic phospholipids (phosphatidyiglycerol & phosphatidic acid) (Hope etal., 1989; Redelmeier et al., 1990), can be modulated by the presence of transmembranepH gradients. The kinetics of ApH driven transport of phosphatidyiglycerol has beenexamined in detail (Redelmeier et al., 1990). The results indicated that PG is transportedin the neutral (protonated) form which could exhibit half-times for transbilayermovement on the order of seconds. However, the assays used to study the transbilayerdistribution of PG were either specific for PG (periodate oxidation) or involved the costlysynthesis of 13C labelled phospholipids. A quantitative analysis of the transport ofphosphatidic acid was precluded by the lack of an appropriate assay for PA asymmetry.This chapter describes the use of a new fluorescent assay employing the probe 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) to detect PA asymmetry. TNS is afluorescent probe previously used to report on the surface potential of membranes(McLaughlin & Haray, 1976; Eisenberg et al., 1979; Searle & Barber, 1979). This assayis shown to be useful as a general assay to detect the transbilayer distribution of acidiclipids. Employing TNS, the kinetics of ApH driven DOPA transport were examined,showing that DOPA is transported via the neutral form with an activation energy similarto that observed for PG.723.2 MATERIALS AND METHODS3.2.1 Lipids and Chemicals.All phospholipids were obtained from Avanti Polar Lipids (Peiham, AL) andwere used without further purification. These included dioleoylphosphatidylcholine(DOPC), dioleoylphosphatidic acid (DOPA), dioleoylphophatidylserine (DOPS), bovineliver phosphatidylinositol (P1), and bovine heart cardiolipin (CL). All lipids were in theNa salt form. TNS [2-(p-toluidinyl)naphthalene-6-sulfonic acid] and all buffers wereobtained from Sigma Chemical Co. (St. Louis, MO.).3.2.2 Preparation of Large Unilamellar Vesicles.Large unilamellar vesicles (LUVs) of the desired lipid compositions wereprepared in the appropriate buffers by extrusion techniques as described in section 2.2.2.3.2.3 Induction of Transbilayer Transport of Acidic Phospholipids.Vesicles were prepared in 300 mM EPPS, pH 9.0, and passed down Sephadex G25 columns (Pharmacia) equilibrated in 150 mM Na2SO4\/1mM EPPS, pH 9.0. At thispoint, phosphate assays (Fiske & Subbarow, 1925) were performed on the vesiclepreparations, and the vesicles were diluted to a concentration of 10.5 mM totalphospholipid. Vesicles were then placed in test tubes and preheated to an appropriatetemperature. At time t = 0, an equal volume of a preheated 100 mM citrate buffer, pH4.0, was added to each tube to obtain the desired ApH. The tubes were then incubated forappropriate times, and PA transport was quenched by placing 200 itL of the sample intotest tubes containing 500 iL of ice-cold 100 mM ammonium acetate\/100 mM sodium73citrate, pH 6.0, and the sample was then stored on ice until assayed for asymmetry. Thetransbilayer movement of PA or PG under these conditions is negligible as the pHgradient is reduced by the presence of acetate and transport is extremely slow at 0\u00b0C (seeResults). For \u201czero time\u201d time points, 100 tL samples were removed from the test tubesbefore the citrate (pH 4.0) was added and placed into the ice-cold acetate and citratemixture (500 iL).To measure PA movement to the outer monolayer, the same procedure wasfollowed except that the vesicles were hydrated in 300 mM citrate, pH 4.0. Untrappedbuffer was exchanged for 150 mM Na2SO4I1mM citrate, pH 4.0, employing columnchromatography, and the external pH was subsequently adjusted by the addition of 100mM EPPS, pH 9.0.3.2.4 Detection of Phosphatidyiglycerol Asymmetry by Periodate OxidationThe transmembrane asymmetry of PG was assayed by two methods. The firstmethod involved periodate oxidation as previously described (Lentz et al., 1980; Hope etal., 1989). Briefly, the PG on the external monolayer of the vesicles was oxidized by theaddition of 100 ii.L of freshly prepared 100 mM sodium periodate to each sample. Toassay the total amount of PG in the vesicles, 50 1iL of 200 mM sodium cholate was addedto the sample before the addition of the periodate. The oxidation of the PG wasquenched after 11 mm by the addition of 100 iL of 1 M sodium arsenite in 1 NH2S04.The formaldehyde resulting from the oxidation of the glycerol was detected by theHantzsh reaction (Nash, 1953).743.2.5 Detection of Asymmetry Using TNS.Following the establishment of PG or PA asymmetry, two samples (200 1iL) wereremoved and placed into test tubes. To each of these samples 3 mL of 3 i.M TNS inSmM ammonium acetate \/5 mM HEPES (pH 7.0) was added and the samples were mixed.The fluorescence of the samples was then measured employing either an SLM AmincoSPF 500C or a Perkin Elmer IS-SO fluorometer using an excitation wavelength of 321nm and an emission of 445 nm.To determine asymmetry, two sets of vesicles were utilized. The first containedonly PC while the second contained a well-defined mixture of PC and the acidic lipidbeing assayed. The acidic lipid made up a maximum of 5% of the total lipid since it wasfound that the TNS fluorescence varied linearly with acid lipid content over the range 0-6% PA or PG (see Figure 19). Due to this linearity, the percentage of acidic lipid in theouter monolayer could be calculated according to x = {[f-f(PC)]\/[f0-f(PC } X0,where fis the TNS fluorescence for the sample for which asymmetry is assayed, f(PC) is thefluorescence of the sample containing no acidic phospholipid, f0 is the fluorescenceassociated with the sample prior to induction of asymmetry, and X0 is the mole percentof acidic phospholipid in the LUVs.3.2.6 Measurement of the Internal pH of LUVs.The internal pH was monitored employing entrapped pyranine according to themethod of Rossignol et al. (1982). This first required construction of a standard curve,utilizing the LUVs with an external buffer of 150 mM Na2SO4\/ 1 mM EPPS (pH 9.0)containing 1 mM pyranine and diluted to a concentration of 10 mM total lipid. To thisdispersion was added an equal volume of a buffer with a pH in the range of 5.0- 9.0.75This buffer was capable of buffering over the range pH 5.0- 9.0, containing 150 mMNa2SO4,20 mM MES, 20 mM PIPES, 20 mM HEPES, and 20 mM EPPS. In order toensure that the internal pH was the same as the external pH, 1 tM nigericin and 1 tg\/mLgramicidin was also present. The fluorescence was then monitored at 45\u00b0C) byemploying excitation wavelengths of 405 and 463 nm (emission wavelength 511 nm),and the ratio of Ip to 1405 vs pH was employed to produce the pH titration curve. Thiswas utilized to obtain the internal pH of vesicles with a transmembrane pH gradient(external pH 4.0) by monitoring 1463\/1405 as a function of incubation time at 45\u00b0C.3.2.7 Kinetic Analysis of Phosphatidic Acid Transport.The analysis of the kinetics of DOPA transport follows a model for the transportof acidic phospholipids across lipid bilayers in response to a transmembrane pH gradientpreviously described in Redelmeier et al., 1990. This model assumes that only theneutral (protonated) form of the acidic phospholipid is able to move across themembrane. Thus the net inward flux (net) of phosphatidic acid is expected to be afunction of the concentration gradient of the neutral species across the membrane, themembrane area and the permeability coefficient.net = dN(A)0t0t\/ dt = P Am ([M]0[M41) (1)where N(A)0t0t is the total number of PA molecules in the outer monolayer, [AH] is the(surface) concentration of the neutral form of the acid, P is the permeability coefficient,Am is the area of the membrane and the subscripts o and i represent the outer and innermonolayer respectively.76From the law of the conservation of massN(A)0t0tI Am = (N(A)0+ N(AH)0)\/ Am = [Aj0 + [MI]0 (2)and from the acid dissociation constantK3 = [Aj0 [H]0 I [AH]0 (3)Rearranging equation 3 and substituting into equation 2 for [A-] givesN(A)0t0tI Am = (1 + K3 \/ [H]0) [AH]0 (4)Substituting equation 4 into equation 1 gives(dN(A)0t0tI dt) I Am (1 + K3 I [H]0)d[AH]0I dt (5)If it is assumed that K>> [H]0, [H]1 << [H]0 and that the concentration of PA in theinner and outer monolayers are equal at time t = 0, then(dN(A)0t0tI dt) I Am = (K3 I [H]0)d[AH]0I dt P [AH]0 (6)Therefore:77d[AH]0Idt=-P pL[AHLI;=-kvJq0where(8)An analytical solution to 7 gives:[AH(t)b=[AH(0)Le+C (9)Since [AH(t)J4,Is proportional to [A(t)J(,[Att)J1,= [k(0)L et+ C (10)If ft Is assumed that the exponential decay Is to some equilibrium value [A\u2019(eq)J:[A(eq)J1,= C (11)Substituting equation 11 Into equation 10 glves([k(t)L - [k(eq)]0)I [k(0)], =e (12)hi ([k(t)J1,- [k(eq)]) I [k(0)J, = -kt (13)78Thus a plot of in ([A(t)]0- [A(eq)]0)I [A(O)J0vs t should yield a straight linewith slope k and units of V1.3.3 RESULTS3.3.1 TNS fluorescence Assay of Asymmetry.Asymmetry of acidic phospholipids in LUVs composed of PC\/acidic lipidmixtures can be detected by ion-exchange chromatographic techniques (Hope & Cullis,1987; Hope et al., 1989). Unfortunately, this technique does not provide quantitativemeasures of asymmetry. In the case of PG, 13C NMR studies on13C-labelled varieties(Hope et a!., 1989) or chemical assays specific for PG (Redelmeier et al., 1990) providemore quantitative information. In order to facilitate asymmetry studies on PA and otheracidic phospholipids a more general and flexible assay for the presence of such lipids inthe outer monolayer of LUV systems was required. An obvious approach is to monitorthe surface potential of the outer monolayer, which will reflect the presence of negativelycharged phospholipids. Studies to investigate the utility of TNS, a probe of membranesurface potential introduced by McLaughlin and co-workers (McLaughlin & Haray,1976; Eisenberg et a!., 1979) as a probe of asymmetry were therefore conducted. In thisregard, it should be noted that TNS is a fluorescent lipophilic weak acid (pKa = 4) whichexhibits enhanced fluorescence when associated with a lipid biiayer. Thus, under theassay conditions employed here, the presence of acidic lipids in the outer monolayer ofthe LUVs will result in decreased partitioning of the negatively charge probe into thebilayer and correspondingly reduced fluorescence intensity. This effect is illustrated inFigure 18 for 100 nm DOPC\/DOPA LUVs containing 0 - 10 mol% DOPA. It may be79noted that the decrease in fluorescence intensity with PA content is linear over the rangeo - 6 mol% DOPA, and thus most asymmetry experiments were restricted to this rangefor ease of analysis.Additional control experiments to establish the utility of the TNS assay wererequired, however. This is due to the weak acid characteristics of TNS, which wouldsuggest that it could be accumulated into LUVs exhibiting a basic interior due topermeation of the neutral form. Such accumulation would be expected to result inenhanced fluorescence intensity arising from increased partitioning of the probe into theinterior lipid monolayer due to the small aqueous to lipid volume ratio in the LUVinterior. Behavior corresponding to this effect is shown in Figure 19 for 100 nm DOPCLUVs with a interior pH of 9.0 (300 mM EPPS) and an exterior pH of 4.0. However, it isalso shown in Figure 19 that dissipating the transmembrane pH gradient by raising theexterior pH and adding 100 mM ammonium acetate to the exterior medium eliminatedsuch effects.3.3.2 Comparison of the TNS Assay to a Chemical AssayIn order to further validate the TNS asymmetry assay for acidic phospholipids, a directcomparison with the periodate assay for PG asymmetry was performed in DOPC\/DOPG(95:5, mol\/mol) LUV systems. As shown in Figure 20, a very similar rate and extent ofPG asymmetry were reported by both procedures.80Figure 18Standard Curve of TNS Fluorescence as a Function ofDOPA concentration in DOPC\/DOPA LUVsDOPCIDOPA LUVs (100 nm) were prepared from lipid dispersions containing 0 - 10mol% DOPA employing a protocol similar to that employed for inducing asymmetry (seeSection 3.2.3), with the exceptions that the heating step was omitted and the ammoniumacetate buffer (pH 6.0) was added prior to the citrate buffer (pH 4.0) to avoid generatinga pH gradient. Fluorescence is expressed as a ratio of the fluorescence measured for theDOPA containing vesicles with respect to that observed for pure DOPC LUVs. Errorbars indicate standard deviations from three sets of triplicate results.1.00.8C,C00.6010li0.40.20 2 4 6 8. 10% Phosphatidic Acid81Figure 19Influence of a Transmembrane pH Gradient on TNSFluorescenceInfluence of a transmembrane pH gradient (interior basic) on the fluorescent response ofTNS in DOPC LUVs. Vesicles (100 nm diameter) were prepared in EPPS buffer (pH9.0), and the exterior buffer was exchanged for the Na2SO4buffer (see materials andmethods). A transmembrane pH gradient was then generated by addition of 100 [it ofthe citrate buffer (pH 4.0) to 100 L of the vesicle solution (10.5 mM phospholipid). ThepH gradient was then either quenched or maintained by addition of either 500 jL of theammonium acetate buffer (pH 6.0) or 500 iL more of the citrate buffer (pH 4.0). TheTNS response was then monitored following addition of 10.5 mL of a 3 pM TNSsolution. (A) indicates the response for the unquenched system at 45\u00b0C; (A) the quenchedsystem at 45\u00b0C; (o) the unquenched system at 22\u00b0C; (.) the quenched system at 22\u00b0C.The fluorescence is expressed as a ratio of that observed at t = 0 to that obtained at time t.Error bars indicate standard deviations from three sets of triplicate results.VC.,CVC,C,,VI0DLi0 20 40 60 80Time (miri)823.3.3 Kinetic Analysis of PA Transport.Initial experiments on PA transport in response to ApH were designed to monitorthe time course of ApH-induced PA asymmetry as assayed by TNS and to test theapplicability of the kinetic analysis employed elsewhere [see Section 3.2.7 andRedelmeier et a!. (1990)]. As shown in Figure 21A, the presence of ApH, interior basic(pH0 = 4.0, pH1 = 9.0), in 100 nm DOPC!DOPA (95:5 mol\/mol) LUVs results in thedepletion of DOPA in the outer monolayer to approximately 5% of the original contentafter a 30 mm incubation at 45\u00b0C. This corresponds to a DOPA content in the innermonolayer of 9.3 mol% and an exterior DOPA content of 0.26 mol%, which is nearingthe detection limits of the TNS assay. It is interesting to note that the maximum ApHinduced PA asymmetry detected ([PA]1 I [PA]0= 39) is considerably greater than thatdetected for DOPG under similar conditions, where a maximum inside:outside ratio of 3was obtained (Redelmeier et al., 1990).The kinetic analysis employed here assumes that [PAH]0>.>.[PAH , where PAHrepresents the neutral form of PA. It is therefore important that the pH1 remainssufficiently high to satisfy this condition. Employing entrapped pyranine, as described inthe last part of this section, the pH1 at 30 mm was measured to be 7.3, indicating that[PAH]0\/ [PAH]1 \u2014 50, which satisfies this demand. It may be noted that the decrease inpH1 from pH 9 to pH 7.3 during PA transport cannot be accounted for by the import ofassociated protons. It may be suggested that packing problems resulting from the importof PA may result in shape changes and partial lysis, resulting in release of buffer.However, freeze-fracture studies revealed no difference in shape induced by the lipidasymmetry (results not shown), and the lack of lysis was indicated by the lack of release83Figure 20Comparison of the TNS Assay for Lipid Asymmetry toa Chemical Assay (Periodate Oxidation) forDOPC\/DOPG LUVsDOPG asymmetry assayed by TNS (.) and periodate assay (o) procedures. Vesicles (100nm diameter) containing 5 mol% DOPG (in DOPC) were prepared as indicated undermaterials and methods to exhibit a transmembrane pH gradient (pH1 = 9.0; pH0 = 4.0)and subsequently incubated at 45\u00b0C for the times indicated. For details of the TNS andperiodate assay procedures, see Sections 3.2.4 and 3.2.5. Error bars indicate standarddeviations from three sets of duplicate results.L50N4010 1\u2019oo31o5\u2019o6oTime (mm)84of entrapped radiolabelled citrate during PA import. It is possible that the reduction inpH arose in part due to leakage of internal Na+ ions, which would allow the inwardmovement of protons.As indicated under Materials and Methods, assuming that only the neutral(protonated) form of PA is transported, a plot of in {[[PA(t)]0- [PA(eq)J0\/ [PA(0)]0}vstime should yield a straight line with slope k, where the half-time (t1p for transbilayermovement of the PA is given by t1,,2 = 0.693 k4. As shown in Figure 21B, a very goodlinear fit employing the data of Figure 21A could be achieved employing k and [PA(eq)]0 as variables. This analysis results in a rate constant of 1.67 x 10-i mm4,corresponding to a half-time for transbilayer movement (t112) of DOPA of 4.1 mm at45\u00b0C.3.3.4 Influence of pH and Temperature on PA Transport.The kinetic analysis of PA transport employed here assumes that the neutral(protonated) form of PA is the permeating species. Indeed, it is generally accepted thatweak acids and bases permeate through membranes in the neutral form [see, for example,Gutnecht and Walter (1981b)J. However, it has been reported that weak bases can crosslipid bilayers in the charged form (McLaughlin, 1975) and that fatty acids can act asproton ionophores (Gutnecht & Walter, 1981a) which implies they can move across themembrane in both the charged and uncharged form. As previously shown for PG(Redelmeier et a!., 1990), determination of the rate constant k as a function of exterior pHprovides a method for unambiguously determining whether the neutral form is theprimary permeating species, as k should vary linearly with exterior proton concentrationvia the relation k = [W]0 kn\/Ka if this is the case. As shown in Figure 22A, the rate of85PA transport was found to be strongly dependent on the exterior pH, increasing by nearlyan order of magnitude for every unit pH0 is lowered. A plot of log k vs log [H]0 (Figure22C) reveals a straight line with a slope 0.9 +1- 0.05, strongly indicating that PA ispermeating the membrane in the neutral (protonated) form.The temperature also strongly influenced the rate of DOPA transport. An analysisof transport rates over the range 25 - 45\u00b0C revealed that the transport rate increasednearly 5-fold for every 10CC increase in temperature as shown in Fig. 23A. AnArrhenius plot of the rate constants (Fig 23C) indicated an activation energy for DOPAtransport of 28 kcal\/mol or 117 id\/mo!.3.3.5 Transport of DOPA to the Outer MonolayerThe results to this point demonstrate transport of DOPA from the outer monolayerto the inner monolayer of LUVs with a basic interior. Conversely, it would be expectedthat in LUVs with an acidic interior, PA should move from the inner to the outermonolayer. This behavior is illustrated in Figure 24 where it is shown that the percentageof DOPA in the outer monolayer increases from 50% of the total DOPA to more than85% over a 1 h time course for DOPCIDOPA (95:5, mol\/mol) LUVs with an initialinterior pH of 4.0 and an exterior pH of 9.0.86Figure 21Transbilayer Transport of DOPA in Response to aTransmembrane pH Gradient (Inside Basic)(A) Influence of a pH gradient (interior basic) on the transbilayer distrbution of DOPA inDOPC\/DOPA (95:5 mol\/mol) LUVs. Vesicles were prepared as indicated undermaterials and methods (pH1 = 9.0; pH0 = 4.0) and incubated for the times indicated priorto quenching transport. The amount of DOPA remaining in the outer monolayer wasassayed employing TNS as described in Section 3.2.5. (B) Best fit to these dataemploying the kinetic analysis descrbed in materials and methods. From the slope of thisplot, the rate constant k can be determined to be 1.67 X 10-1 mm4.L>.C0C0V0Ca10Time (miri)15Time (miri)87Figure 22Influence of the External p11 on the Rate of theTransbilayer Transport of DOPAVesicles, DOPC\/DOPA (95:5 mol\/mol) were prepared in 300 mM NaEPPs, pH 9.0 bufferand the exterior buffer was exchanged for 150 mM Na2SO4I1 mM EPPS pH 9.0 (seeSection 3.2.3). The vesicles were then introduced into citrate solutions with varying pH:(o) pH0 = 5.5; (.) pH0 = 5.0; (A) pH0 = 4.5; (A) pH0 =4.0; (a) pH0 = 3.5. Afterincubation at 40\u00b0C for the indicated times, transport was quenched and the amount ofDOPA remaining in the outer monolayer assayed employing TNS. (B) Best fit to thisdata employing the kinetic analysis in materials and methods. (C) Plot of log k vs the logof the external proton concentration.>-aC0aCaKTim. (mtn)Tim. (mm)log [H+10883.3.6 Response of Various Phospholipids to a Transmembrane ApHAs indicated above, the TNS assay for asymmetry should be generally applicableto determine the transbilayer distributions of a variety of acidic lipids in addition to PAand PG. We have therefore employed this assay to determine possible ApH-inducedasymmetry in DOPC systems containing 3 mol% DOPS, 3 mol% bovine heart cardiolipinand 5 mol% bovine liver P1. As shown in Figure 25A, no ApH-induced asymmetry couldbe detected at 45\u00b0C for these phospholipids under conditions similar to those for whichDOPA and egg PG exhibit considerable transbilayer movement. This is in agreementwith preliminary observations described elsewhere (Hope et al., 1989). As shown inFigure 25B, this inability to induce asymmetry for P1, PS and cardiolipin does not arisefrom depletion of the transmembrane pH gradient, as ApH values in excess of 2 units aremaintained over the experimental time course.89Figure 23TeLnperature Dependence of ApH Driven DOPAAsymmetryVesicles, DOPCIDOPA (95:5 mol\/mol), were prepared in 300 mM NaEPPS aspreviously described. The vesicles and the citrate buffer (pH 4.0) were pre-incubated tothe appropriate temperature before establishing the transmembrane DpH. Afterincubation for the times indicated, transport was terminated and the amount of DOPAremaining in the outer monolayer was measured employing TNS (see Section 3.2.5). (o)25\u00b0C; (.) 30\u00b0C; (A) 35\u00b0C; (A) 40\u00b0C; (ci) 45\u00b0C. (B) Best fit of data to the kinetic analysisdescribed in Section 3.2.7. (C) An Arrhenius plot of the rate constants derived from B.0.< 0o-3.0C340 3.30flmi (miti)C\u20146.0\u201465\u20147.0\u20147_SC\u20146.0\u20146.3\u2014LO\u201415\u20143.00 3.10 3.70 3.30103\/r (IC1)90Figure 24Transport of DOPA to the Outer Monolayer inResponse to a Transmembrane pH Gradient (InteriorAcidic)Vesicles, DOPCIDOPA (97.5:2.5 mol\/mol), were prepared in 300 mM citrate (pH 4.0)and the external buffer was exchanged for 150 mM Na2SOdl mM citrate (pH 4.0). Atzero time, outward DOPA transport was initiated by the addition of 100 mM EPPS (pH9.0). Transport was quenched after incubation for the indicated times at 40\u00b0C byaddition of 100 mM ammonium acetate\/100 mM citrate buffer (pH 6.0) precooled to 0\u00b0C.DOPA transport was assayed employing the TNS protocol.951.>-55..\/45. 1 Io 10 20 30 40 50 60Time (mm)91Figure 25Effect of a Transmembrane ApH on the TransbilayerDistributions of Various Acidic Phospholipids(A) Transbilayer distributions of acidic phospholipid following incubation at 45\u00b0C in thepresence of a transmembrane pH gradient (pH1 = 9.0, pH0 = 4.0). The LUVs wereprepared, pH gradients were established, and the amount of acidic lipid in the outermonolayer was assayed as indicated in Section 3.2.5. The lipid compositionscorresponding to various symbols are as follows: (a) DOPCIDOPA (95:5); (A)DOPC\/EPG (95:5); (A) DOPCIDOPS (97:3); (.) DOPC\/bovine heart cardiolipin (97:3);(o) DOPC\/bovine brain P1(95:5). (B) Residual pH gradients detected employingpyranine as a probe of internal pH. Vesicles were prepared as described earlier exceptthat 1 mM pyranine was added to the internal buffer. The internal pH of the vesicles wasmonitored (see Section 3.2.6) by measuring the fluorescence of samples using excitationwavelengths of 405 and 463 nm and an emission wavelength of 511 nm.C>..a0C0IC0C0.00.0aci-aciL5Time (mn)Bx0aC1.CC6SI I I10 20 30 40 50 60 70 80 90Time (mm)923.4 DISCUSSIONThe results of this Chapter establish TNS as a useful probe for determiningtransbilayer distributions of acidic lipids in LUV systems and provide new informationon the mechanism and kinetics of the transbilayer movement of PA. With regard to theTNS assay, the obvious advantages are convenience and generality. Tedious syntheses toachieve13C-labelled or spin-labelled varieties of acidic phospholipids are avoided, thebehavior of the acidic phospholipid itself (rather than a labelled variety) is detected, andthe assay is relatively rapid. A potential disadvantage of the TNS assay is that theasymmetry to be assayed must be relatively stable. This is clearly not a problem for theDOPA asymmetries assayed here. The half-time for transbilayer movement of DOPA at20\u00b0C and pH 6.0 can be estimated from the results of this study as 12.6 days (see below).Such stable asymmetries are not available for other lipids which are weak acids, such asfatty acids, which exhibit much faster transbilayer diffusion rates (Hope & Cullis, 1987).With regard to the mechanism of DOPA transport in response to transmembranepH gradients, the results of this investigation strongly support a first-order processinvolving permeation of the neutral (protonated) form. The linear dependence of the rateconstant on the exterior proton concentration [H+]0provides particularly compellingevidence in this respect. This behavior is consistent with that previously documented foregg phosphatidylglycerol (EPG) and DOPG (Redelmeier et al., 1990) and the generallyaccepted view that weak acids permeate through membranes in the neutral form(Gutnecht & Walter, 1981b). Within this formalism, the rate constant k can be written ask = [H+lokn\/Ka where k is the rate constant for transbilayer movement of the neutralform and Ka is the weak acid dissociation constant. Given that k = 1.67 x 10-1 min1- at9345\u00b0C for DOPA in LUVs with an exterior pH of 4.0, we obtain k = (1.67 x 103)Ka min1, corresponding to k - 1.67 mint(t112 = 25 s), assuming a Ka for DOPA of i-(Tocanne & Teissie, 1990). This is somewhat smaller than, but comparable to, the rateconstant for the neutral form of PG under similar conditions (K = 6 mint;Redelmeier etal., 1990). A more precise comparison is difficult to achieve given the variability in Kavalues reported for PG and PA (Tocanne & Teissie, 1990).The high activation energy (28 kcal\/mol) observed for DOPA transport is similarto that observed for EPG (31 kcal\/mol) and likely reflects requirements related todehydration of the phospholipid headgroup, as discussed elsewhere for PG (Redelmeieret al., 1990). In this regard, the similarity between PA and PG activation energies clearlyestablishes the (protonated) phosphate group as the dominant impediment to transbilayerdiffusion. It should also be noted that the combination of a high activation energy andthe linear dependence on the proton concentration imparts an exquisite sensitivity of therate constant for transbilayer movement of DOPA (and PG) to the experimentaltemperature and pH. Given k = 1.67 x 10-1 mind at 45\u00b0C, pH 4.0, a generalized rateconstant for DOPA can be written ask(T,pH) = 1.67 x i03 -pHexp [44.3(1 - 318jT)] min1where T is temperature (in Kelvin). Thus, at pH 4 and 600C, the rate constant can becalculated to be 1.23 mind (t112 = 34 s), whereas at pH 7.0 and 20\u00b0C k = 3.8 x 10-6 min1(t112 = 127 days). This obviously allows the preparation of LUVs exhibiting stableasymmetric transmembrane lipid distributions following a brief incubation at low pHand\/or high temperature to induce the asymmetry. Aside from the fact that this providesconvenient conditions for assaying asymmetry as indicated above, such systems are ofpotential utility in their own right. Two areas of interest concern the influence of lipid94asymmetry on membrane fusion processes and the influence of lipid asymmetry on thetransbilayer movement of other lipids.The inability to induce asymmetry for other acidic phospholipids (bovine brainP1, bovine heart cardiolipin, and DOPS) is of interest. In the case of DOPS, this can beattributed to the zwitterionic nature even when both acidic functions are protonated. Thelack of response of P1 and cardiolipin is surprising, and likely reflects the influence of thebulky polar inositol group and\/or low phosphate pKa values.In summary, this investigation establishes TNS as a useful probe of asymmetrictransbilayer distributions of acidic phospholipids in LUV systems. Application of thisassay to monitor the ApH-dependent transport of DOPA in LUV systems indicates thatDOPA traverses the membrane in the neutral form which exhibits transbilayerredistribution times which can be on the order of seconds. Finally, the sensitivity of therate constant for transport of pH and temperature allows the generation of systems withrelatively stable asymmetric distributions of PA.95CHAPTER 4INFLUENCE OF LIPID ASYMMETRY ON FUSION BETWEEN LARGEUNILAMELLAR VESICLES4.1 INTRODUCTIONThe asymmetric transbilayer distributions of lipids commonly observed inbiological membranes (see Section 1.5 and Chapter 3) may be expected to play a role inregulating membrane fusion in vivo. Model membranes composed of unsaturatedphosphatidylethanolamine (PE) and phosphatidylserine (PS), approximating the innermonolayer composition of the erythrocyte membrane, for example, fuse readily in thepresence of physiological stimuli such as Ca2 (Hope et al., 1983). Alternatively,vesicles composed of phosphatidylcholine (PC) and sphingomyelin, the outer monolayercomposition, are resistant to fusion. It may therefore be expected that membranes whoseexternal monolayers contain fusogenic lipids such as PE and PS will fuse more readilythan membranes with identical lipid compositions but where the fusogenic lipids arelocalized to the inner monolayer under conditions where the fusion stimulus is in contactwith the outer monolayer. These speculations are supported by several observations. Forexample, Sessions & Horowitz (1981, 1983), have shown that the lipid composition ofthe external leaflet of the plasma membrane of myoblasts, which undergo fusion to formmyotubes, contain more phosphatidylethanolamine and phosphatidylserine than the outermonolayer of the erythrocyte. Further, the concentrations of these lipids in the outermonolayer increases prior to fusion (Santini et al., 1990). It may also be noted thaterythrocytes which have lost lipid asymmetry fuse more readily than erythrocytesexhibiting asymmetric lipid distributions with PE and PS in the inner monolayer (Tullius96et al., 1989). Alternatively, in most cells, the inner monolayer of the plasma membranecontains more PS and PE than the outer monlayer. Since fusion events are nescessary forexocytosis to occur, it would be expected that the inner monolayer of these cells shouldbe capable of undergoing fusion under appropriate conditions. However, cell to cellfusion occurs only in very specific circumstances (some of which are mentioned above)which would be consistent with a lipid composition of the the outer monolayer of themajority of cell types which is relatively resistant to fusion.The regulatory role of lipid asymmetry in fusion has proven difficult toinvestigate, due primarily to the lack of an appropriate model system. However, recentwork has shown that lipid asymmetry can be generated in large unilamellar vesicles(LUVs) by imposing transmembrane pH gradients (Hope et al., 1989; Redelmeier et al.,1990; Eastman et al., 1991, Chapter 3). Here we utilize this phenomenon to investigatethe role of lipid asymmetry in the regulation of Ca2 induced membrane fusion. It isshown that lipid asymmetry can profoundly regulate fusion phenomena between LUVsystems and that the composition of the outer monolayer plays a dominant role indetermining the rate and extent of fusion in the system studied here.4.2 MATERIALS AND METHODS4.2.1 Lipids and ChemicalsDioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine(DOPE), dioleoylphosphatidic acid (DOPA), bovine liver phosphatidylinositol (P1), N-(7-nitro-2, 1,3-benzoxadiazol-4-yl) phosphatidylethanolamine (N-NBD-PE), and N(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) were obtained97from Avanti Polar Lipids (Peiham, AL). TNS [2-(p-toluidinyl) naphthalene-6-sulfonicacid] and all buffers were obtained from Sigma Chemical Co. (St. Louis, MO).4.2.2 Preparation of Large Unilamellar VesiclesVesicles of the appropriate lipid composition were prepared in the appropriatebuffers by extrusion procedures described in Section 2.2.2. During the freeze-thawcycles, the temperature of the water used to thaw the vesicles was maintained below20\u00b0C in order to prevent the vesicles undergoing a phase transition to the hexagonal H11phase due to the presence of DOPE.4.2.3 Detection of FusionVesicle fusion was monitored using resonance energy transfer (RET) as describedby Struck et al (1981) (see Fig. 8B). Briefly, unlabelled vesicles of the appropriate lipidcomposition were mixed with similar vesicles containing 0.7 mol% of each of NBD-PEand Rh-PE, in a 3:1 ratio. The vesicles were prepared in 300 mM HEPES pH 7.5 andwere subsequently run down a Sephadex G-25 column pre-equilibrated with 300 mMsucrose, 1 mM HEPES, pH 7.5. All buffers were adjusted to the appropriate pH witharginine free base. The vesicles were then diluted to a concentration of 10 mM totalphospholipid. A small aliquot of the vesicles (25 L) was added to a cuvette containing1.9 mL of 100 mM sucrose, 50 mM MES, pH 5.5. Fusion was initiated by the injectionof 80 pL of a 200 mM CaC12solution to obtain a final Ca2 concentration of 8 mM. Theincrease in NBD-PE fluorescence due to fluorescence dequenching as the fluorophoresexchanged with lipids on the unlabelled vesicles was monitored using a Perkin Elmer LS50 spectrofluorimeter equipped with a thermostated, stirred cuvette holder. An excitation98wavelength of 465 nm and an emission wavelength of 535 nm were employed and acutoff filter for light less than 530 nm was used to reduce the effects of light scattering.Zero fluorescence was set at the residual fluorescence of the NBD-PE in the labelledvesicles while 100% fluorescence corresponded to complete mixing of the lipids. Usinga 3:1 ratio of unlabelled to labelled vesicles, complete mixing yields a fluorescenceintensity of 75% of the infinitely diluted probe. The fluorescence intensity for theinfinitely diluted probe was obtained by the addition of Triton X-100 to a finalconcentration of 0.1 mM and correcting for its effects on the quantum yield of NBDfluorescence by measuring the decrease in fluorescence of vesicles containing 0.7 mol%NBD-PE after the addition of TX-100 to a 0.1 mM concentration.In order to determine the effect of Ca2+ concentration on the fusion of the LUVs asufficient amount of a stock solution of 200 mM CaC12was added to a cuvette containing100 mM sucrose, 50 mM MES, pH 5.5 to give the desired Ca2 concentration in a finalvolume of 2 mL. A 50 L aliquot of the LUVs containing 10 mol% PA (5 mM totalphospholipid), was injected into the cuvette and the increase in NBD fluorescence due tofusion was monitored as described above.4.2.4 Induction of DOPA AsymmetryLUVs containing 10 mol% DOPA were prepared in 300 mM HEPES, pH 7.5 andpassed down a Sephadex G-25 column equilibrated with 300 mM sucrose, 1 mM HEPES,pH 7.5. In order to initiate the transport of phosphatidic acid to the inner monolayer,these vesicles (10 mM total phospholipid) were mixed with 100 mM sucrose, 20 mMcitrate, pH 4.0 (with arginine). The ability of Ca2 to induce fusion in these systems was99monitored at various time intervals ranging from t = 0 to t = 6 h using the RET fusionassay.The transport of DOPA to the inner monolayer was monitored using TNS asdescribed in Section 3.2.5 with slight modifications. Briefly, unlabelled vesiclescontaining 10 mol% PA were prepared in 300 mM HEPES, pH 7.5 and passed down aSephadex G-25 column equilibrated in 300 mM sucrose, 1 mM HEPES, pH 7.5. In orderto induce DOPA transport, the external pH of the vesicles was reduced by diluting anequal volume of vesicles with a buffer containing 100 mM sucrose, 20 mM citrate, pH4.0. To stop PA transport, 100 p.L of the vesicles were added to 500 tL of ice cold 100mM citrate, 100 mM ammonium acetate, pH 6.0 and the vesicles were stored on ice.Asymmetry was assessed by adding 2 mL of 3 1iM TNS in 5 mM HEPES, 5 mM acetate,pH 7.0 and reading the fluorescence using excitation and emission wavelengths of 321and 445 nm respectively. This value was normalized with respect to vesicles containingno PA (100% fluorescence). The external concentration of PA over time was calculatedby comparing the fluorescence of the samples to a standard curve prepared using vesiclescontaining 0 to 10 mol% PA.For the transport of DOPA to the outer monolayer, LUVs were prepared in 300mM citrate, pH 4.0 (with arginine) and passed down Sephadex G-25 columns equlibratedin 300 mM sucrose, 1 mM citrate,. pH 4.0. Transport of DOPA to the outer monolayerwas initiated by the addition of an equal volume of 100 mM sucrose, 20 mM HEPES, pH7.5. The ability of Ca2 to induce vesicle fusion was monitored using the RET assay.1004.2.5 31P NMR StudiesFrozen and thawed multilamellar vesicles (FATMLVs) were prepared in a 100mM sucrose, 50 mM MES pH 5.5 buffer employing five freeze-thaw cycles. The protondecoupled 81.0 MHz 31P NMR spectra were then recorded employing a Bruker MSL 200spectrometer under the following conditions. The free induction decay (FID) wascollected using a pulse width of 3.7 [Is (90\u00b0) with an interpulse delay of 1.0 is. After1000 scans, the FID was Fourier transformed employing a line broadening function of 25Hz. Sufficient 1 M CaC12was added to the liposomes to bring the Ca2 concentration to100 mM, a value in large excess of the DOPA concentration (100 mM total lipid, 10 mMDOPA). The MLVs were freeze-thawed two additional times to equilibrate the Ca2 andspectra were then re-recorded.4.2.6 Freeze Fracture Electron MicroscopyVesicles used for freeze fracture studies were prepared as for fusion assays.Vesicles (100 mM total lipid) containing either no DOPA or 10 mol% DOPA wereprepared in 300 mM HEPES and passed down Sephadex G-25 columns equlibrated in300 mM sucrose, 1 mM HEPES , pH 7.5. The vesicles were then mixed with an equalvolume of 100 mM sucrose, 20 mM citrate pH 4.0. and incubated for times t = 0 or t =4h at pH 4.0 and then passed down Sephadex G-50 columns equilibrated in 100 mMsucrose, 50 mM MES, pH 5.5.To induce fusion, a small aliquot of 200 mM CaC12was added to the vesicles tobring the Ca2+ concentration to 8 mM. Vesicles were incubated with Ca2+for times t =0, 5, 10, 15, and 30 sec before fusion was stopped. Fusion was stopped by adding 100 [LLof the vesicle solution to a tube containing 25 pL of 200 mM EGTA. Glycerol was then101added to the samples (-10 mM phospholipid) to a final concentration of 25%. Sampleswere then frozen in Freon cooled in liquid nitrogen and fractured. Platinum\/carbonreplicas of the samples were prepared as previously described (Fisher & Branton, 1974).The graphs presented in this Chapter were produced from single experiments butare representative of multiple trials.4.3 RESULTS4.3.1 Vesicle CompositionPrevious studies have shown that vesicles composed of phosphatidic acid ormixtures of PE and PA fuse readily in the presence of Ca2 (Hope et al., 1983). Theincorporation of PC into the membrane increases their stability and prevents Ca2+induced fusion at high PC contents (Duzgunes, 1985). In the studies performed here,various mixtures of DOPC,DOPE,PI and DOPA were tested for their ability to fuse in thepresence of Ca2+. P1 was incorporated to prevent aggregation of vesicles containing littleor no DOPA. It was also chosen because earlier studies have shown that it is nottransported to the inner monolayer of vesicles under conditions where PA is transported(Eastman et al., 1991) and that it is a non-fusogenic lipid in isolation (Sundler andPapahadjopoulos, 1981). PC and PE are zwitterionic lipids that are also not transportedin response to transmembrane pH gradients. It was found that LUVs formed frommixtures of these lipids in the ratios DOPC:DOPE:PI \/ 25:60:5 did not fuse appreciablyin the presence of Ca2,but did when DOPA was present. Further, this system did notfuse in acidic environments which was important in order to allow the generation of lipidasymmetry by the transmembrane ApH.1024.3.2 Effect of (2+ on LUV FusionThe effect of Ca2 on the fusion of symmetric LUVs containing 10 mol% PA(DOPC:DOPE:PI:DOPA \/25:60:5:10) is shown in Figure 26A. The extent and initialrate of fusion are influenced greatly by the concentration of Ca2+up to about 8 mMCa2+, where nearly 100% fusion occurred within 30 sec. Higher concentrations of Ca2+had little further effect on the extent of fusion and led to rapid fusion rates which wereinconvenient to measure. The initial rate of vesicle fusion for vesicles containing 10mol% DOPA as a function of Ca2 concentration is shown in Figure 26B.4.3.3 Effect of DOPA Content on LUV FusionVesicles were prepared with DOPA concentrations ranging from 0 to 10 mol% (in1% increments). These vesicles were tested for fusion in the presence of 8 mM Ca2+using the resonance energy transfer (RET) assay. Fluorescence traces of the LUVscontaining various concentrations of DOPA are shown in Figure 27. The vesiclescontaining no DOPA fuse to only a small extent, whereas the addition of as little as 1%DOPA greatly enhances fusion, The extent of fusion continues to increase withincreasing contents of PA and approaches 100% fusion at concentrations of DOPAgreater than 7 mol% of the total phospholipid.103Figure 26.Effect of Ca2+ Concentration on the Fusion of LUVsContaining 10 mol% DOPAFluorescence traces of the effect of Ca2+ concentration on the fusion of vesiclescomposed of DOPC:DOPE:PI:DOPA (25:60:5:10) (A). Vesicles were preparedas described in Section 4.2.2 and the ability of various concentrations of Ca2 toinduce fusion between the vesicles was monitored using the resonance energytransfer (RET) assay. The initial rate of vesicle fusion at each Ca2+concentrationis shown in (B).0U10\u2022BC\u2022 \/\/78fCa2] (mM)Time (sec)104Figure 27Effect of DOPA Concentration on Vesicle FusionFluorescence traces indicating the effect of DOPA concentration on fusion ofvesicles composed of DOPC:DOPE:PI. Vesicles, both labelled and unlabelled,were prepared containing various amounts of phosphatidic acid (0-10 mol%) asdescribed in Section 4.2.2. The rate and extent of fusion of these vesiclepopulations in the presence of 8 mM Ca2 was monitored employing the RETassay.10090807060D 50403020100300 10 20 40 50 60Time (sec)1054.3.4 Effect of DOPA Asymmetry on LUV FusionPrevious research has shown that the transmembrane pH gradients can induce theasymmetric transbilayer distribution of DOPA in LUVs (Hope et al., 1989; Eastman et al,1991; Chapter 3). In particular, in LUVs with a basic interior, DOPA will move to theinner monolayer to the extent that greater than 95% of the total DOPA is present in thismonolayer. This asymmetry can be readily assayed employing TNS (see Section 4.2.4)and the ability of a transmembrane ApH (pH1 = 7.5 \/ pH0 = 4.0) to induce the inwardmovement of DOPA is shown in Figure 28A. It is important to note that the other lipidsin this system (DOPE,DOPC and P1) are not transported to the inner monolayer of LUVsunder these conditions (Eastman et al., 1991; Chapter 3). The transport of DOPA to theinner monolayer is very rapid with essentially all the external DOPA being transported tothe inner monolayer after a 4 h incubation at room temperature (\u201421\u00b0C).The ability of Ca2 to induce fusion of LUVs containing 10 mol% DOPA wasmonitored at various time intervals during the induction of DOPA asymmetry (Fig. 28B).Immediately after establishing the transmembrane ApH (t = 0) the vesicles fused at thesame rate and to the same extent as vesicles containing 10 mol% DOPA symmetricallydistributed between the two monolayers (ie. no ApH). However, the rate and extent offusion decreased markedly as the external DOPA concentration decreased, indicating thatthe fusion of the LUVs was regulated by the amount of DOPA in the outer monolayer.These results are consistent with the time course of DOPA transport (Fig. 28A) and theconcentration dependance of fusion on the DOPA content of the LUVs (Fig. 28).106Figure 28Modulation of Membrane Fusion by Lipid AsymmetryDetection of the transbilayer transport of DOPA in response to a transmembrane ApHemploying TNS (see methods) (A) and the effect of this transbilayer asymmetry on thefusion of DOPC:DOPE:PI:DOPA (25:60:5:10) vesicles. Vesicles were prepared with aninternal pH of 7.5 as described in Section 4.2.4. A transmembrane ApH was establishedacross the vesicles and the ability of the vesicles to fuse was monitored using the RETassay at various times after the induction of the ipH ranging from t = 0 to t = 4 h (B).L>\u2018U0C010C0a0987654321\u2019A0 30 60 90 120 150Time (miri)180 210 240C0CU30 40Time (sec)1074.3.5 Fusion of LUVs with DOPA Exclusively on the Outer MonolayerThe ability of DOPA transbilayer asymmetry to inhibit fusion of DOPAcontaining LUVs is clearly demonstrated in Fig. 28. These results show that thecharacteristics of one monolayer, the monolayer exposed to the fusion inducing agent,can regulate the fusion characteristics of the bilayer as a whole. In order to study thisability further, LUVs were prepared such that all of the DOPA from the inner monolayerwas transported to the external monolayer. The fusion characteristics of these vesicleswere then compared to vesicles containing symmetrically distributed DOPA at the sameexternal DOPA concentration. This was performed by preparing LUVs containing 2.5mol% DOPA, inducing the asymmetric distribution of the DOPA in response to atransmembrane pH gradient (inside acidic) to achieve 5 mol% DOPA in the externalmonolayer and comparing the rate and extent of fusion of these LUVs to vesiclescontaining 5 mol% DOPA symmetrically distributed between the two monolayers. Theresults of this experiment are shown in Figure 29. After all the DOPA was transportedfrom the inner monolayer to the outer monolayer (final DOPA concentration on outermonolayer \u20144.9 mol%) the vesicles fused at the same rate and to the same extent asvesicles containing 5 mol% DOPA. In contrast, control vesicles containing 2.5 mol%DOPA symmetrically distributed between the leaflets, fused at a slower rate and to alesser extent than the same vesicles with the DOPA asymmetrically located on the outermonolayer.108Figure 29Effect on Fusion of DOPA Transport to the OuterMonolayerFluorescence traces showing the extent of vesicle fusion for vesicles containing2.5 mol% DOPA at times t = 0 (\u2014 _)and t = 4 h ( )after inducing transport ofthe DOPA to the outer monolayer (see Section 4.2.4) and of vesicles containing 5mol% DOPA ( ).C0U807050504030201000 10 20 30 40 50Time (sec)601094.3.6 Polymorphic Phase PreferencesIntermediates in bilayer to hexagonal H11 phase transitions, such as invertedmicelles and interlamellar attachment sites are the most likely intermediates in themembrane fusion process (Cullis & Hope, 1978; Siegel et al., 1989; Ellens et a!., 1989).31p NMR experiments were performed to determine the structural preferences of lipidscomprising the outer monolayer of fusing and non-fusing LUVs in the absence andpresence of excess Ca2. As shown in Figure 30, MLVs composed of DOPC:DOPE:PI(25:60:5) exhibit bilayer spectra both in the presence and absence of Ca2. However,MLVs containing 10% DOPA exhibit bilayer structures in the absence of Ca2 butundergo a transition to the H11 phase in the presence of excess Ca2+ (see Figure 30). Thisis indicated by the transition from a 31P NMR lineshape with a low field shoulder andhigh field peak to a spectra which exhibits reversed asymmetry and was a factor of twonarrower (Cullis & de Kruijff, 1979).4.3.7 Freeze-Fracture Studies of Vesicle FusionFreeze-fracture studies were also carried out, in order to visualize the fusogenicbehavior of vesicles exhibiting symmetric and asymmetric transbilayer lipid distributionsAs shown in Figure 31, LUVs which do not contain DOPA do not fuse appreciablyeither in the absence or presence of Ca2+. This is in agreement with the RET assayresults (see Figure 28). On the other hand, LUVs containing 10 mol% DOPAsymmetrically distributed on both monolayers fuse very quickly in the presence of 8 mMCa2+. After 5 s many large structures are visible and almost all the vesicles appear tohave undergone some degree of fusion. After 30 s all the vesicles have fused into huge110complexes. These results correspond very well with the fluorescence data which shows avery fast initial rate of fusion for the 10% DOPA vesicles and nearly 100% mixing oflipids. In contrast, LUVs containing 10 mol% DOPA, but where the DOPA has beensequestered to the inner monolayer in response to a pH gradient (interior basic) showvery little fusion in the presence of 8 mM Ca2 even after 30 s.111Figure 30Polymorphic Phase Preferences of Non-FusogenicVesicles (no DOPA) and Fusogenic Vesicles (10 mol%DOPA) in the Absence and Presence of Excess Ca231P NMR spectra of freeze-thawed MLVs (FATMLVs) composed ofDOPC:DOPE:PI (25:60:5) in the absence (A) and presence (B) of excess Ca2 orDOPC:DOPE:PI:DOPA (25:60:5:10) in the absence (C) and presence (D) ofexcess Ca2+. Spectra were recorded for the MLVs in the absence of Ca2+ on aBruker MSL 200 NMR spectrometer. Following the addition of excess Ca2 theMLVs were exposed to 2 additional freeze-thaw cycles to equilibrate the Ca2and the spectra were re-recorded.A BC D-25 2l5oPIPPM\u201425PPM112Figure 31Freeze-Fracture Electron Micrographs of LUVs in theAbsence and Presence of CaFreeze fracture electron microgaphs of various vesicle systems. DOPC:DOPE:PI(25:60:5) in the absence of Ca2 (A) and 30 seconds after the addition of 8 mMCa2 (B). DOPC:DOPE:PI:DOPA (25:60:5:10) in the absence of Ca2 (C) and atvarious times after the addition of 8 mM Ca2 [5 sec (D) and 30 sec (E)].DOPC:DOPE:PI:DOPA (25:60:5:10) vesicles which have been exposed to atransmembrane pH gradient for 4 hours in order to induce PA asymmetry. In theabsence of (2+ (F) and at various times after the addition of Ca2 [5 sec (0) and30 sec (H)]. Fusion reactions were terminated during the time course by theaddition of excess EGTA (see Section 4.2.6).113DISCUSSIONThe results of this investigation clearly demonstrates the potential regulatory roleof lipid asymmetry in membrane fusion processes. This is shown by the strongcorrelation between the amount of DOPA in the outer monolayer of the LUVs employedhere and fusion detected by the resonance energy transfer technique and freeze-fractureprocedures. Three aspects of this work which are of interest concern the influence of theinner monolayer on the fusion process, the mechanism of fusion and the relation toregulation of fusion processes in vivo. Each of these points is discussed in turn.The results presented here show that as the amount of DOPA in the outermonolayer of DOPC:DOPE:PI:DOPA (25:60:5:10) LUVs decreases due to net transportof DOPA to the inner monolayer, the fusion rate decreases correspondingly (Fig. 28).This is perhaps not surprising given the strong influence of DOPA content on fusion (Fig.27), however it clearly establishes the importance of the outer monolayer lipidcomposition as a determinant of fusion. A related question concerns the role of the innermonolayer, and whether the composition of the inner monolayer influences thepropensity of the LUV as a whole to fuse. The results of Figure 29 where LUVscontaining only 2.5% DOPA, but where all the DOPA is located in the outer monolayer,fuse at effectively the same rate as symmetric LUVs containing 5 % DOPA suggest thatit does not. The lack of influence of the composition of the inner monolayer, not initiallyinvolved in membrane fusion, on the rate and extent of fusion argues for the validity oflipid asymmetry as a regulatory agent in fusion in vivo and has interesting implicationsfor generating more controlled fusion processes. In particular, fusion between modelmembrane LUV systems in vitro is, in contrast to membrane fusion in vivo, generally a114leaky process. This is at least in part because the stimuli employed to initiate fusion invitro commonly promote hexagonal (H11) phase in the lipid mixture (Burger & Verkleij,1990; Hope & Cullis, 1981). End-stage formation of hexagonal structure is notcompatible with maintenance of a permeability barrier. However, systems exhibitingasymmetric transbilayer distributions of lipid clearly have the potential to be selfregulating and possibly to exhibit leak tight fusion. For example, in asymmetric LUVsystems exhibiting a fusogenic outer monolayer but where the overall lipid mixture willnot support fusion, fusion will presumably proceed until the fusogenic potential of theouter monolayer is dissipated by mixing with inner monolayer lipids. It will be ofparticular interest to determine the leakiness of fusion events in such systems.Regarding the mechanism of fusion, the results presented here are consistent witha fusion process which relies on the ability of component lipids to adopt transitory nonbilayer structures. This is indicated by the fact that MLVs composed of DOPC:DOPE:PI(25:60:5) did not adopt H11 phase in the presence of excess Ca2 and LUVs with the samelipid composition did not fuse appreciably in the presence of Ca2,whereas the additionof 10 % DOPA resulted in the ability of excess Ca2 to induce the H11 phase andstimulated fusion between corresponding LUVs. As has been well discussed elsewhere(Cullis & Hope, 1978; Siegel et al., 1989; Ellens et al., 1989) the actual intermediary infusion is unlikely to be the hexagonal phase per Se. More logical structures include theinverted micelle and interlamellar attachment sites, which are likely intermediaries in thebilayer-to-H11transition process (see Siegel et al., 1989).The regulatory role that lipid asymmetry could play in vivo is clearly of majorinterest. Two types of regulation are clearly possible- a passive form where thepreviously generated static asymmetry determines whether fusion can proceed and a115more active regulation where the local generation of asymmetry promotes or inhibitsfusion. An example of passive regulation could be the stable transbilayer asymmetryobserved for plasma membranes. The presence of phosphatidylcholine and orsphingomyelin in the outer monolayer inhibits fusion with extracellular entities exceptunder exceptional conditions. Alternatively, the inner monolayer composedpredominantly of phosphatidylethanolamine and phosphatidylserine would be expectedto fuse more readily with internal organelles or secretory vesicles in response to localstimuli such as increased Ca2+concentrations.The possibility that lipid asymmetry may actively, locally regulate fusion is morespeculative and clearly requires that lipids can be quickly mobilized from one side of thebilayer to the other as appropriate, or can be rapidly generated on demand. The relativelyslow rates of transbilayer lipid movement in plasma membranes suggests thattransbilayer mobilization would not be a feasible regulatory process in vivo. However, inmembranes such as the endoplasmic reticulum membrane, where transbilayer flip-floprates are rapid (Zilversmit & Hughes, 1977; Bishop & Bell, 1985) it is possible thatfusion could be regulated by such a mechanism. Alternatively, of course, the localenzymatic generation of fusogenic lipids such as diglycerides or phosphatidic acid couldalso result in a similar end.In summary, the results of this investigation clearly demonstrate the potentialregulatory role of lipid asymmetry in membrane fusion phenomena. Further, theproperties of the one monolayer appears to determine the fusogenic tendencies of thebilayer as a whole.116CHAPTER 5SUMMARYThe asymmetric nature of biological membranes is now well established withrespect to both the constituents of the membrane and the environments on either side ofthe membrane. Concerning lipids, the mechanism(s) by which asymmetry is generatedand maintained in biological membranes are still a matter of contention. One possibilityis that transmembrane ion gradients influence the transbilayer distribution of lipids inmembranes. In this thesis, the ability of transmembrane pH gradients (ApH) to modulatelipid asymmetry in model membrane liposomal systems has been addressed.Furthermore, the functional significance of lipid asymmetry has been examined withrespect to lipid exchange and membrane fusion.Previous studies have shown that transmembrane pH gradients can influence thetransbilayer distribution of simple lipids with weak acid or weak base characteristics,such as stearylamine and fatty acids (Hope & Cullis, 1987). In Chapter 2, the ability oftransmembrane pH gradients to modulate the exchange of these simple lipids betweenmembranes is examined. It is shown that when these lipids are symmetrically distributedacross the bilayer, rapid exchange can occur between donor and acceptor membranes.This is demonstrated for both aggregating and non-aggregating systems. However, ifthese lipids are sequestered to the inner monolayer in response to a transmembrane ApH,then little or no exchange occurs between the donor and acceptor membranes. Althoughfatty acids are usually bound to fatty acid binding proteins (FABPs) in the circulation andwithin cells, transmembrane pH gradients may modulate the exchange of free fatty acidsacross various biological membranes. For example, in the intestine, transmembrane pHgradients generated by the outward secretion of H ions by the epithelia, results in the117protonation of the fatty acids and the absorption of the fatty acids by the epithelial cells(Bugaut, 1986). Additionally, organelles with acidic interiors, such as lysosomes, wouldbe expected to accumulate fatty acids in the outer monolayer where the acids could beremoved by fatty acid binding proteins (FABP5) and processed by the cell. The ability oftransmembrane pH gradients to modulate the flow of fatty acids between membranes andbovine serum albumin (BSA) could be significant with respect to the uptake of fatty acidsby cells. For example, it has been speculated that one mechanism by which fatty acidscross cell membranes is by passive diffusion after dissociating from serum albumin. Theresults presented in this thesis indicate that transmembrane pH gradients (inside basic)would favor the transfer of fatty acids from the albumin to the acceptor membrane.Furthermore, this observation suggests that the concentration of free fatty acids ispurposely maintained at very low levels within cells (through binding to FABPs) in orderto prevent the accumulation of these acids in the membranes of organelles with basicinteriors, such as mitochondria.In addition to the ability of transmembrane pH gradients to modulate thetransbilayer distribution of simple lipids, more recent studies have shown that pHgradients can induce the transbilayer distribution of certain acidic phospholipids, such asPG and PA (Hope et al., 1989; Redelmeier et al., 1990). However, the lack of convenientassays to detect the asymmetry of various acidic phospholipids in model membranesprecluded studies into the kinetics and mechanism of lipids other than PG. In Chapter 2,a fluorescence assay which employs TNS to detect the transbilayer distribution of acidicphospholipids is described. Using this assay the kinetics of DOPA transport in responseto a transmembrane ApH is examined. DOPA is shown to be transported in the neutralform with an activation energy of 28 Kcal\/mol.118The ability to generate lipid asymmetry in model membranes allowed studies ofthe effect of lipid asymmetry on membrane fusion, as discused in Chapter 4. It is shownthat vesicles (DOPC:DOPE:PI:DOPA (25:60:5:10)), with a symmetric distribution oflipids fuse rapidly in the presence of Ca2. However, if the DOPA is transported fromthe outer monolayer to the inner membrane then the vesicles become resistant to Ca2+induced fusion. This is consistent with results indicating that similar vesicles preparedwithout DOPA fuse only to a small extent upon addition of Ca2. Further studiesindicate that vesicles containing 2.5 mol% DOPA asymmetrically distributed on the outermonolayer, fuse to the same extent and at the same initial rate as vesicles containing 5mol% DOPA symmetrically distributed between the membranes. These results indicatethat the asymmetric distribution of lipids can have a profound regulatory effect on thefusion characteristics of lipid bilayers and that one leaflet of the bilayer can control thefusion characteristics of the entire bilayer. The experiments presented in this chapter alsosupport the involvement of non-bilayer structures in fusion processes.There are a number of interesting problems which arise from these studies. Themost obvious one concerns the imbalance between the inner and outer monolayers of theLUVs after the inward movement of lipid such as DOPA. In systems containing 10mol% DOPA, for example, translocation of DOPA to the inner monolayer results in aninner monolayer which contains 20% more lipid than the outer monolayer. This is atopological impossibility. However, as indicated elsewhere (Hope et al., 1990) there isno evidence for a compensatory movement of other lipids (ie. PC) to the outermonolayer. It is possible that alternative structures bud off from the inner monolayer asillustrated in Fig. 32. Presently there is no data to support this hypothesis. Freezefracture electron micrographs show no evidence of changes to the vesicle structure.119Figure 32Possible Structure of Vesicles Exhibiting anAsymmetric Distribution of Acidic PhospholipidsIn order to accommodate the extra lipid on the inner monolayer, invaginations couldperhaps be formed similar to the cristae observed in the mitichondrial inner membranebut only involving one monolayer.fflffi120However, it is possible that the structure of the vesicles is affected by the cryo-protectant(glycerol) or the structure may undergo changes during the freezing process. Futureexperiments using cryo-transmission electron microscopy will hopefully be useful indetecting any morphological changes caused during the induction of asymmetry. Cryotransmission electron microscopy should not affect the morphology of the vesicles sinceno cryo-protectants are used and the freezing process is so rapid that there is no time forrearrangements of lipid structures (Burger & Verkleij, 1990).A second interesting problem concerns the ability of lipid asymmetry to regulatenon-leaky membrane fusion. The results of Chapter 4 suggest that LUVs composed of anappropriate lipid composition, could be prepared such that the outer monolayer wasfusogenic under conditions where the fusogenic lipid (eg. DOPA) was locatedexclusively on the outer monolayer, but which were non-fusogenic when the lipid wassymmetrically distributed. Such LUVs would be expected to undergo only a limitednumber of fusion events as it would be expected that the lipids would redistribute (flip-flop) during the fusion events producing vesicles with a non-fusogenic outer monolayerlipid composition. It would be of interest to see if such a regulated fusion eventproceeded without leakage of vesicle contents. Such a system would more closelyresemble the highly regulated, non-leaky fusion events observed in biological systems.LUVs exhibiting asymmetric distributions of lipids and pH sensitive fusioncharacteristics could also be useful in the delivery of drugs to specific cells. Forexample, a lipid which induced fusion in the neutral form, but did not affect the lipidbilayer when it was in a charged form, could be sequestered to the inner monolayer ofvesicles in response to a transmembrane pH gradient. On the inner monolayer the lipidwould be charged and non-fusogenic. However, as the pH gradient across the vesicles121dissipated, the fusogenic lipid would move to the outer monolayer. If the exterior pHwas such that a proportion of the lipid was neutralized, then the LUV would becomefusogenic. 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