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  1. 1. Institut für Biophysik Fachrichtung Physik Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden Practical Course: Giant Unilamellar Vesicles Kirsten Bacia, Jakob Schweizer 30th September 2005 1
  2. 2. Contents Contents 1 Cell Membrane and Lipids 3 1.1 Functions of cellular membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2 Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1 Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.2 Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.3 Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.5 Lipids in Natural Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Formation of Lipid Structures 6 3 Thermodynamics of Lipid Bilayers 8 3.1 Lipid Bilayer Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2 Lipid phase diagrams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.3 Ternary lipid mixtures exhibiting uid-uid phase separation . . . . . . . . . . . . 12 3.4 Model membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.5 Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.6 Planar membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs) by electroformation 13 4.1 Electroformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.2 General protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.3 Lipid mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 5 Analysis 16 2
  3. 3. 1 Cell Membrane and Lipids 1.1 Functions of cellular membranes One of the most basic constituents of a cell is the membrane that surrounds it and allows it to maintain an internal milieu diering from the external media as sketched in gure 1. Eukaryotes additionally contain various internal membranes that enclose specialized compartments and allow for biochemical reactions to be separated spatially. Thus membranes have to fulll many complex functions. They act as selective barriers by oering little permeability to ions and large molecules and containing incorporated channels and transporters to allow for a controlled exchange of solutes. Gradients of ions, pH and electrical charge across the membrane are exploited to store energy and to allow fast responses to a signal. Signal transduction across the membrane without material transport is accomplished by receptor molecules undergoing conformational changes. Membranes are also home to many enzymes catalyzing biochemical reactions, and they contain molecules that allow recognition between cells. In conjunction with the cytoskeleton, membranes enable cells and their internal compartments to adopt certain shapes, which can be dynamic, such as in cell motility or the growth of tubular structures. Cargo is transported between dierent compartments (or delivered to the outside of the cell) in small vesicles. These processes require mechanisms of vesicle budding and ssion, vesicle transport, recognition between membranes and membrane fusion. The basic building block of membranes is the phospholipid bilayer, into which many more molecules, in particular cholesterol, proteins with lipid anchors and proteins with transmembrane domains (integral membrane proteins) are incorporated. Other membrane proteins can be asso- ciated with these (peripheral membrane proteins). Some lipids and proteins carry carbohydrate modications (glycolipids, glycoproteins). The protein-to-lipid ratio varies depending on the type of membrane, averaging around 1:1 by mass (which corresponds to about 1:50 by number). The uid mosaic model assumes that - apart from some specic, short-range protein-lipid and protein- protein interactions, the protein molecules are distributed randomly in the phospholipid matrix, constituting a 2−dimensional solution of protein in phospholipid [2]. However, it also conceivable that specialized domains are formed within membranes, based on cooperative lipid-lipid interac- tions. According to theraft hypothesis [3], specialized sub-micron scale domains with properties similar to the liquid-ordered phase (see below) are formed, which may functions as platforms in cellular processes like sorting and signalling. 1.2 Lipids 1.2.1 Phospholipids Phospholipids form the main building block of membranes. Unlike storage lipids (fats), which consist of a glycerol and three fatty acid chains, phospholipids only contain two fatty acids. They are derived from either glycerol or sphingosine (see gure 2). Phosphoglycerides (glycerophospho- lipids) contain a phosphatidate, i.e. they have two fatty acids esteried to two carbons of the glycerol and a phosphoric acid group esteried to the third carbon. The phosphate group in turn is esteried to an alcohol, like ethanolamine, choline, serine or inositol. Sphingomyelin is based on sphingosine, which in contrast to glycerol already contains a long hydrocarbon chain. The second hydrocarbon chain is again provided by an ester-linked fatty acid. For the headgroup, the sphingosine is esteried to a phosphoric acid group, which is in turn ester-linked to a choline. The fatty acid chains usually contain an even number of carbon atoms between 14 and 24, where 16 and 18 are most common. They are normally unbranched and either saturated or contain one or more, non-conjugated double bonds in the cis-conguration. Some common names for these acyl chains and some abbreviations for phospholipid headgroups are given in Table 1. 3
  4. 4. 1 Cell Membrane and Lipids Figure 1: Sketch of the eukaryotic cell. The left half of the gure represents a plant cell, the right side an animal cell. The cell is surrounded by the cell membrane of which an enlarged section is shown below. Main element of the cell membrane are phospholipids and cholesterol forming a bilayer of two leaets, in which lipids and cholesterol are arranged in a parallel manner to the membrane normal. Proteins are associated with and incorporated into the membrane. The cell membrane does not constitute a rigid structure but exhibits a complex range of dynamics. It is therefore considered a two-dimensional uid as described by the uidic mosaic membrane model. Inside the cell one can nd many elements of dierent function: C cilia, CH chromatin, D cellular junctions (e.g. desmosomes), EN endocytosis, ER endoplasmic reticulum, EX exocytosis, F actin laments, G Golgi apparatus consisting of cisternae and vesicles, H nuclear envelope, K nuclear pore, LY lysosome , M mitochondrium, MV microvilli, N nucleus, NU nucleolus, P plastids, R free polyribosomes, S storage lipids, T microtubuli, V vacuole (plant cell), W cell wall. The gures are taken from Biophysik by Walter Hoppe [1]. 4
  5. 5. 1.2 Lipids phosphatidic acid (phosphatidyl-) fatty acid (acyl-) O glycerol sphingosine O CH2 CHOH H O CH N CH O CH3 O CH3 fatty acid (acyl-) O CH2 O P O CH2 CH2 N + CH3 fatty acid (acyl-) O CH2 O P O CH2 CH2 N + CH3 O CH3 O CH3 phosphoryl choline phosphoryl choline N-acyl-sphingosine or ceramide (a) DOPC. (b) SM. CH 3 CH 3 CH 3 O H CH 3 O H O H CH 3 O O P O + H H N HO O O H (c) DPPC. (d) Cholesterol. + (CH ) N O N (CH3) 2 3 2 H3 C CH 3 H3 C CH 3 O O CH CH CH O N + N O H O H N O O P O (CH2) 17 (CH2) 17 O N S CH 3 CH 3 O H (e) TRITC-DPPE. (f) DiI-C18. Figure 2: Typical lipids and uorescent analogs (e,f). Number of carbons : Chain name Full name Abbreviation Number of double bonds 12 : 0 lauroyl phosphatidylcholine PC 14 : 0 myristoyl phosphatidylethanolamine PE 16 : 0 palmitoyl phosphatidylserine PS 18 : 0 stearoyl phosphatidylinositol PI 18 : 1 oleoyl Table 1: Chain- and headgroup names Lipid name Abbreviation 1,2-dilauroyl-sn-glycero-3-phosphocholine DLPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] POPS 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] DPPS Table 2: Lipid names and abbreviations. 5
  6. 6. 2 Formation of Lipid Structures Membrane type PC PE PS PI SM Glycol. Chol. Others (a) Human erythrocyte 20 18 7 3 18 3 20 11 plasma membranes (b) Mammalian liver 18 12 7 3 12 8 19 21 plasma membranes (c) Golgi membranes 25 9 3 5 7 0 8 43 Table 3: Lipid composition of natural membranes in weight%. 1.2.2 Glycolipids Animal glycolipids consist of a ceramide moiety (as in sphingomyelin, see Fig. 2(b)) and a sugar moiety (in place of the phosphorylcholine group found in sphingomyelin). Gangliosides, like for instance GM1, have branched chains of several sugar residues. 1.2.3 Sterols Cholesterol (Chol) is a molecule found almost exclusively in eukaryotic membranes, presumably because the molecular oxygen required in its synthesis was not available in the anaerobic atmo- sphere of early prokaryote development. It is strongly preserved among eukaryotes as a membrane constituent, indicating that it plays a key role. In addition, cholesterol is a precursor molecule for the production of bile salts (e.g. glycocholate) which act as detergents in solubilizing food lipids and in the biosynthesis of steroid hormones (progestagens, glucocorticoids, mineralcorti- coids, androgens and estrogens). In contrast to other hormones to which the membrane poses an impermeable barrier (they bind to receptors on the external side), steroid hormones are able to directly cross the membrane and bind to receptor proteins in the cytosol. 1.2.4 Membrane proteins Membrane proteins can be directly integrated into the membrane by exposure of hydrophobic amino acid residues, either in a transmembrane manner (single pass α-helix, multi pass α-helices, β - barrel) or by embedding of an α-helix only in the cytosolic leaet, i.e. in plane with the membrane. Depending on the signal sequence, hydrophobic stretches and charges, transmembrane proteins are inserted with the C-terminus on the cytoplasmic side (Type I) or on the ER / external side (Type II). Other membrane proteins are anchored indirectly by lipidic anchors, i.e. either fatty acids (myristic acid, palmitic acid), prenyl groups (farnesyl, geranylgeranyl) or a C-terminally linked glycolipid (glycosyl-phosphatidyl-inositol-(GPI)-anchor). Finally, a protein can be attached to the membrane by non-covalent complex formation with other membrane proteins. 1.2.5 Lipids in Natural Membranes Lipid compositions of natural membranes depend on cell type and organelle. Typical weight- percentages for (a) a human erythrocyte plasma membranes, (b) a mammalian liver plasma mem- branes and (c) Golgi membranes is listed in the table 3. Note that 20 weight-% of cholesterol corresponds to approx. 50 mol-% cholesterol for the given molecular masses. 2 Formation of Lipid Structures Aggregation of lipids is a self-assembly process, driven by lipid interactions such as van der Waals, hydrophobic, electrostatic interactions and hydrogen bonding. In general, the presence of an individual hydrophobic molecule in an aqueous environment is en- tropically highly unfavorable. Water molecules are forced to build H-bonds around the hydropho- bic molecule, forming so called clathrate-structures, which represent high order of H2 O-molecules 6
  7. 7. k1 kN N=1 X 1 m 0 1 N XN m0 N (a) H2 O-molecules (red) in next neighborhood to (b) Monomers and aggregates of hydrophobic the hydrophobic molecule (grey) have less possi- molecules are in equilibrium exchange bilities to establish H-bonds than water molecules (black) surrounded by other hydrophilic particles. This situation is therefore entropically unfavored. Figure 3: Principle of hydrophobic force. and therefore low entropy [4], as depicted in gure 3(a). To decrease the amount of clathrate structures, hydrophobic molecules tend to aggregate. Consider now the self-assembly process of lipids: Membrane lipids have a polar, hydrophilic headgroup and two long exposed hydrocarbon chains that are extremely hydrophobic, meaning a large energy value is required to transfer them from a hydrophobic environment (organic solvent, arrangement with other lipids in a micelle or lipid bilayer) to an aqueous environment. According to ∆G = µ0 0 singledispersed − µmicellar = −RT ln(XCM C ), (1) this large dierence in chemical potentials provokes that the critical micelle concentration which is a measure of the lipid concentration at which 50% of the freely dispersed lipid molecules have associated into micelles, is very small for phospholipids. A typical value for a two-chain, 16 carbon phospholipid is ∆G ≈ 75 kJ/mol ≈ 30RT , and critical micelle concentrations of two-chain phospholipids are typically below 10−12 M. Amphiphilic molecules thus have a strong tendency for aggregation. However, it depends on lipid shapes and lipid packing if micelles or bilayers are formed. Pertinent amphiphile ge- ometry can be approximated by three param- eters: 1. The optimal surface area ao occupied by the headgroup. (Due to electrostatic re- pulsion between neighboring molecules, this may depend strongly on pH and solution ionic strength for charged lipids.) 2. The length of the hydrocarbon chains l, which sets an upper limit to micelle size. Figure 4: For a small shape factor lipids form mi- (Voids are energetically very costly.) celles. Intermolecular Surface Forces [5]. 3. The molecular volume of the hydrocar- bon tail v . In brief, the nal shape of a lipid aggregate is mainly determined by th so-called shape factor of the constituting lipids: v (2) a0 lc 7
  8. 8. 3 Thermodynamics of Lipid Bilayers (a) Lipid molecule. (b) Micelle. (c) Lipid bilayer. (d) Vesicle. Figure 5: Lipid structures. The simplest lipid structures are spherical aggregates with their chains pointing towards the center and their heads forming the sphere surface, so-called micelles. The sketch of a micelle given in gure 4 also shows the inuence of the packing properties. It is evident that micelle-forming lipids must have a large headgroup area a0 and a short hydrocarbon chain volume v . For a micelle 1 the shape factor has to be smaller than 3 . Such lipids are called cone-shaped lipids. For bilayer structures small headgroup areas and a bulky hydrocarbon chain volume is required, i.e. cylinder-shaped lipids. The shape factor should be then: 1 v 1 (3) 2 a0 l c Bilayers are usually composed of lipids with two hydrocarbon chains. In a planar bilayer, the hydrophobic chains in the middle of the structure are well accommodated as they are in a hydrophobic environment and the hydrophilic heads face an aqueous interface. But at the edge of such a planar bilayer the hydrocarbon chains would be exposed to water (5(c)). Therefore the structure will tend to shield these edges from the aqueous environment by connecting open edges with each other. This will result in vesicles, spherical structures comparable to soap bubbles (see gure 5(d)). The strong internal lateral pressure inhibits the formation of pores unless the pores are stabilized, e.g. by peptides or cone-shaped surfactants. The lipid bilayer is thus held together very eciently by noncovalent, but cooperative interactions. Some further lipid aggregate structures, the corresponding shape factor and lipid representatives are given in gure 6. 3 Thermodynamics of Lipid Bilayers 3.1 Lipid Bilayer Phases Lipids can adopt a variety of phases, but the focus here will be only on the lamellar (2-dimensional, i.e. bilayer) phases. Phase transitions can be induced in various ways. Most frequently, thermal transitions are studied. Although a variety of lamellar gel phases with dierent packing structures have been identied by x-ray crystallography (Lβ has the carbon chains perpendicular to the bilayer plane, Lβ is with the carbon chains tilted, LβI is an interdigitated phase and Pβ has a 8
  9. 9. 3.1 Lipid Bilayer Phases Figure 6: Lipids form dierent aggregate structures depending on their shape factor. Figure is taken from Intermolecular Surface Forces [5]. 9
  10. 10. 3 Thermodynamics of Lipid Bilayers (a) Gel phase (Lβ ): There is (b) Liquid-crystalline phase (c) Liquid-disordered phase both lipid chain conformational (Lα ): The phospholipids show (Lo ): THe lipid chains are and translational order. both lipid chain conformational ordered due to interactions with and translational disorder the cholesterol (depicted as el- (lateral diusibility). lipses), but there is translational disorder, allowing for lateral diusion. Figure 7: Lipid bilayer phases. ripple structure), only the common characteristics of gel phases need to be considered here: In the gel phase, the hydrocarbon chains are ordered in all-trans conguration and there is long-range translational order, impeding lateral movement. For this reason, it has also been termed the solid-ordered phase (So ). Upon increase of the temperature above the melting point, Tm , the entropy term becomes dominating, resulting in the liquid-crystalline phase, Lα . The Lα phase is characterized both by low conformational order in the carbon chains (lower internal order) and by low translational order (lower packing order, higher translational diusion). For this reason, it has more recently also been termed the liquid-disordered phase (Ld ). Headgroup area, which is on the order of 0.5 nm2 increases by approximately 15 to 30 % upon melting. The addition of cholesterol (the most common type of sterol) results in a loss of cooperativity of the gel to liquid-crystalline transition. NMR studies indicate that this is due to the introduction of another equilibrium phase, the liquid-ordered phase (Lo ), in which there is still high conformational order like in the gel phase, but the translational order is already lost (high translational diusion) as in the Lα phase ([6], [7], [8]). Theoretical studies [9] predict that the order-disorder transition of the carbon chains and the order-disorder transition of the packing do not necessarily need to be coupled, thus supporting that an Lo phase is formed. However, experimentally, the Lo phase has not been found in single-lipid, but only in binary lipid systems that contain certain sterols. 3.2 Lipid phase diagrams Lipid phase diagrams depict which lipid phases exist in equilibrium for a combination of thermo- dynamic parameters, like temperature, pressure (if applicable) and composition. They can only be constructed for simple systems. Binary lipid mixtures can already show quite complex phase diagrams and ternary mixtures even more so. The Gibbs Phase Rule is applied to determine the number of degrees of freedom F for a system of C components, exhibiting P dierent phases: P + F = C + 2. Since only lipid phases forming in excess water are considered here as biologically most relevant, the water is omitted: It is not counted as a component and there is no degree of freedom for the water concentration. Furthermore, pressure can usually be considered as given (except in monolayer systems), so that the number of remaining degrees of freedom is: F =C −P +1 (4) It is impossible to produce a phase diagram of as complex a lipid mixture as found in native membranes. In principle, according to the Gibbs Phase Rule, a huge number of phases could be allowed. What appears to happen is that especially for eukaryotic membranes containing cholesterol, the transition or the transitions smear out into a broad transition or no measurable 10
  11. 11. 3.2 Lipid phase diagrams temperature fluid Tm(A) e u rv e sc i du nc liq u is te ex e rv cu co us T li d so solid Tm(B) 0 xfluid(T) xsolid(T) 1.0 x(A) composition Figure 8: Phase diagram of a binary lipid mixture of saturated phosphatidylcholines: The diagram shows a typical schematic phase diagram for a binary mixture of saturated phosphatidylcholines that dier only in their chain lengths, for example A = DPPC and B = DMPC [22,24]. The lipids show complete miscibility in the solid (gel) and the uid (liquid-crystalline) phase. However, due to their dierent melting temperatures (here: Tm (A) = 41 ◦ C, Tm (B) = 24 ◦ C), there is a solid-uid coexistence region. Upon cooling a mixture (depicted by the cross and the arrows), a DPPC-enriched mixture starts to solidify at the liquidus curve, increasing the DMPC content of the remaining liquid. Hence in the coexistence region, DPPC-enriched solid is in equilibrium with DMPC-enriched liquid. At a chosen temperature T , the compositions of the phases xuid and xsolid are xed. They can be read from where the horizontal dotted line, a so-called tie-line, intersects the liquidus and the solidus curves. The relative amounts of liquid and solid can be calculated from material conservation, expressed as the lever rule. Finally, the solidus curve is reached and the residual liquid (now approaching pure DMPC) solidies. 11
  12. 12. 3 Thermodynamics of Lipid Bilayers transition at all [10]. (A prerequisite for compiling a phase diagram is that there are distinguishable phases, and transitions between them are approximately rst-order.) Nevertheless, studying simple systems with identiable phases is hoped to give insight into phenomena that could be pertinent to real cell membranes. Even when there is no real cooperative phase transition over the whole membrane, there could be local ones, such as the formation of lipid phase domains (rafts) or lipid melting in the vicinity of membrane proteins. 3.3 Ternary lipid mixtures exhibiting uid-uid phase separation A mixture consisting of three major lipids, DOPC, sphingomyelin (SM) and cholesterol, has been studied as a model for domain (raft) formation in cellular membranes. For a certain range of compositions, lipid bilayers formed from this mixture exhibit a coexistence of two uid (liquid) phases, the liquid-disordered (Ld = Lα ) and the liquid-ordered (Lo ) phase (area indicated in Fig. 9). This phase separation was visualized in Giant Unilamellar Vesicles (GUVs) using uorescent markers that specif- ically label one of these phases. The task of this practical lab course is to produce Giant Unilamellar Vesicles from a 1:1:1 mixture of DOPC, SM and cholesterol and to visualize Figure 9: Phase coexistence and diusional mobil- the phase separation. ity in lipid bilayers consisting of DOPC, SM and The two uid phases have been further char- cholesterol at room temperature. The numbers de- acterized by measuring the diusion of a small, note diusion coecients of the probe diIC18 in units lipid-like probe inside these phase domains us- of 10−8 cm2/s (i.e. in µm2/s) [11]. ing Fluorescence Correlation Spectroscopy (FCS) ([11], Fig. 9). Diusion measurements by FCS are treated in other practical labs. 3.4 Model membranes 3.5 Vesicles To obtain vesicles (gure 5(d)), lipids are usually dissolved in an organic solvent and the solvent is evaporated using a nitrogen stream or vacuum, so that a thin lipid lm is produced on a glass surface (vial). The lipid lm is hydrated with an aqueous solution, where the temperature should be above the melting temperature Tm of the highest melting lipid in the mixture. Formation of vesicles may be supported by perturbations like shaking, temperature cycling or applying an alternating electric eld. The sizes, shapes and lamellarity of the vesicles obtained depend both on the type of lipids and the detailed protocol. Giant unilamellar vesicles (GUVs) have a diameter on the order of 1 to 300 µm. They are produced either by the protocol described above with minimal perturbation in the hydration step [12] or, with a generally much better yield of unilamellar vesicles, by using the electroformation approach [15]. Multilamellar vesicles (MLV) are quickly generated by the general protocol described above. Starting from MLVs, large unilamellar vesicles (LUVs, 100 to 1000 nm) with a narrow size distribution around a desired value are produced by freeze-thaw cycling the vesicles, followed by extrusion, i.e. pressing the vesicle suspension repeatedly through a membrane of dened pore size. Small unilamellar vesicles (SUVs, 20 to 50 nm in diameter) have very large curvature and most of their lipids are in the outer leaet. They are prepared by extrusion through membranes with smaller pore size (30 nm) or by supplying energy to the MLV suspension by sonication. Extrusion and sonication have to be performed above the highest lipid Tm. Liposomes of small size (SUVs, LUVs) can also be produced from detergent-lipid micelle solutions by dilution, dialysis, sequestration of detergent onto beads or gel 12
  13. 13. 3.6 Planar membranes Figure 10: Principle of lipid swelling (taken from ltration chromatography. This method allows incorporation of membrane proteins that are so hydrophobic that they require detergents for solubilization. 3.6 Planar membranes When lipids are deposited on a water surface and laterally compressed they form a monolayer with the hydrophilic headgroup facing the water and the hydrophobic tail facing the air. Mono- layers provide regular and stable structures and, importantly, their composition can be accurately controlled [15]. Also, lateral pressure as a thermodynamic parameter can be measured and ad- justed. However, monolayers do not directly mimic biomembranes, since they lack the second leaet. Sequential transfer of two monolayers (tails facing each other) to a solid support lead to the formation of a supported bilayer [16]. Alternatively, spontaneous spreading and fusion of small unilamellar vesicles on a hydrophilic surface is employed [17]. Supported planar bilayers and monolayers can suer from artifacts due to interactions with the support. 4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs) by electroformation 4.1 Electroformation Applying a low-voltage electric eld can promote the formation of truly unilamellar vesicles. For GUV electroformation, a solution of lipids in organic solvent is dried on conductive electrodes instead of a glass surface (platinum wires or conductively coated coverslips). The hydration step is then performed in the presence of an electric (normally alternating) eld. The electroformation method was developed by Angelova and others and has become widely used for preparing GUVs [15, 18, 19, 20, 21]. The alternating eld induces the formation of unilamellar vesicles upon swelling of lipid layers in an aqueous environment (gure 10). The disadvantage with this method is that GUV yield and size decrease strongly when ions (salt) are present in the aqueous solution. 4.2 General protocol Lipids are dissolved in chloroform:methanol (2:1) at 10 mg/ml total concentration and ≈ 5 µl of this solution is spread on the conductive surface of an ITO-coated coverslip. For lipid mixtures 13
  14. 14. 4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs) by electroformation U = 1.2 V n = 10 Hz conductive tape ITO-coverslip lipid H2O ITO-coverslip (a) Capacitor-type conguration for electroforma- tion. (b) Flow chamber for electroformation. Figure 11: Electroformation concept. involving high-Tm lipids like sphingomyelin, coverslips are preheated to safely above the Tm (heat- ing block at ≈ 65 ◦ C). Two ITO-coverslips are then assembled into a capacitor-type conguration (Fig. 11(a)) in a ow chamber (Fig. 11(b)). Vacuum grease is used for sealing. The chamber is lled with water or sucrose solution (≈ 300 µl chamber volume). ITO-coverslips are connected to a pulse generator via pieces of conductive tape and an alternating voltage (U = 1.2 V, f = 10 Hz) is applied for 1 to 3 hours. For high-Tm lipids, electroswelling is performed above the Tm on a heating block or in an oven. GUVs form in multiple layers above the lipid-coated coverslip and can be studied directly in the chamber (in situ). 14
  15. 15. 4.3 Lipid mixture 4.3 Lipid mixture We will prepare a lipid mixture of DOPC, C18-sphingomyelin (SM) and Cholesterol (Chol) with a molar ratio of 1:1:1. Furthermore, 0.1 mol% GM1 and 0.1mol% diIC18 are added directly to the lipid mixture (for imaging; for FCS lower concentrations of diIC18 are appropriate). DiIC18 is a uorescent lipid analogue (see gure 2(f)). GM1 is a ganglioside, which is exploited as a binding partner by the B subunit of cholera toxin. After GUV formation, we will add uorescently labeled cholera toxin B subunit (ctxB-Alexa488) as a second marker for domain visualization. The lipid molecular weights are: DOPC: M = 786.15 g/mol SM: M = 731.09 g/mol Chol.: M = 386.7 g/mol GM1: M = 1563.9 g/mol diIC18: M = 933.88 g/mol The stock solutions have concentrations of (subject to change) DOPC: c = 20 mg/ml SM: c = 10 mg/ml Chol.: c = 6 mg/ml GM1: c = 1 mg/ml diIC18: C = 100 µM µM = µmol/l The total lipid content is to be n = 1.36 × 10−6 mol (subject to change) (5) How much volume of each stock solution would be needed? What can be done if volumes are too small to be accurately pipetted? For spreading the lipid on the ITO coverslip, the nal lipid concentration should be roughly 10 mg/ml. In what nal volume should the above amount of lipids be dissolved? 15
  16. 16. 5 Analysis 5 Analysis Giant vesicles will be visualized using light microscopy. Please refer to the literature or the indi- cated websites for background reading on light microscopy: For Innity-Corrected Optical Systems: For Phase Contrast: Read Sections: • Brief Overview of Phase Contrast • Phase Contrast Microscopy • Phase Contrast Microscope Alignment For DIC: Sections: • Brief Overview of DIC Microscopy • Fundamental Concepts in DIC Microscopy • DIC Microscope Conguration and Alignment • Comparison of Phase Contrast and DIC Microscopy For Fluorescence Microscopy: For Laser Scanning Microscopy: 16
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  18. 18. References [20] K. Kottig. Fluoreszenz-Korrelations-Spektroskopie an Lipidvesikeln auf oxidiertem Silizium. PhD thesis, Technische Universität München, 2005. [21] N. Kahya, E. I. Pécheur, W. P. de Boeij, D. A. Wiersma, and D. Hoekstra. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. Biophys. J., 81(3):146474, Sep 2001. 18