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Institut für BiophysikFachrichtung PhysikFakultät Mathematik und Naturwissenschaftender Technischen Universität Dresden

Practical Course:Giant Unilamellar Vesicles

Kirsten Bacia, Jakob Schweizer

30th September 2005

1

Contents

Contents1 Cell Membrane and Lipids 3

1.1 Functions of cellular membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2.1 Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2.2 Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.2.3 Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.2.4 Membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.2.5 Lipids in Natural Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2 Formation of Lipid Structures 6

3 Thermodynamics of Lipid Bilayers 83.1 Lipid Bilayer Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.2 Lipid phase diagrams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.3 Ternary lipid mixtures exhibiting �uid-�uid phase separation . . . . . . . . . . . . 123.4 Model membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.5 Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.6 Planar membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs) by electroformation 134.1 Electroformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.2 General protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.3 Lipid mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

5 Analysis 16

2

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 tomaintain an internal milieu di�ering from the external media as sketched in �gure 1. Eukaryotesadditionally contain various internal membranes that enclose specialized compartments and allowfor biochemical reactions to be separated spatially. Thus membranes have to ful�ll many complexfunctions. They act as selective barriers by o�ering little permeability to ions and large moleculesand 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 andto allow fast responses to a signal. Signal transduction across the membrane without materialtransport is accomplished by receptor molecules undergoing conformational changes. Membranesare also home to many enzymes catalyzing biochemical reactions, and they contain molecules thatallow recognition between cells. In conjunction with the cytoskeleton, membranes enable cellsand their internal compartments to adopt certain shapes, which can be dynamic, such as in cellmotility or the growth of tubular structures. Cargo is transported between di�erent compartments(or delivered to the outside of the cell) in small vesicles. These processes require mechanisms ofvesicle budding and �ssion, vesicle transport, recognition between membranes and membranefusion.

The basic building block of membranes is the phospholipid bilayer, into which many moremolecules, in particular cholesterol, proteins with lipid anchors and proteins with transmembranedomains (integral membrane proteins) are incorporated. Other membrane proteins can be asso-ciated with these (peripheral membrane proteins). Some lipids and proteins carry carbohydratemodi�cations (glycolipids, glycoproteins). The protein-to-lipid ratio varies depending on the typeof membrane, averaging around 1:1 by mass (which corresponds to about 1:50 by number). The�uid mosaic model assumes that - apart from some speci�c, 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 conceivablethat specialized domains are formed within membranes, based on cooperative lipid-lipid interac-tions. According to the�raft� hypothesis [3], specialized sub-micron scale domains with propertiessimilar to the liquid-ordered phase (see below) are formed, which may functions as platforms incellular processes like sorting and signalling.

1.2 Lipids

1.2.1 Phospholipids

Phospholipids form the main building block of membranes. Unlike storage lipids (fats), whichconsist of a glycerol and three fatty acid chains, phospholipids only contain two fatty acids. Theyare derived from either glycerol or sphingosine (see �gure 2). Phosphoglycerides (glycerophospho-lipids) contain a phosphatidate, i.e. they have two fatty acids esteri�ed to two carbons of theglycerol and a phosphoric acid group esteri�ed to the third carbon. The phosphate group in turnis esteri�ed to an alcohol, like ethanolamine, choline, serine or inositol. Sphingomyelin is basedon sphingosine, which in contrast to glycerol already contains a long hydrocarbon chain. Thesecond hydrocarbon chain is again provided by an ester-linked fatty acid. For the headgroup, thesphingosine is esteri�ed 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 oneor more, non-conjugated double bonds in the cis-con�guration. Some common names for theseacyl chains and some abbreviations for phospholipid headgroups are given in Table 1.

3

1 Cell Membrane and Lipids

Figure1:

Sketchofthe

eukaryoticcell.

Theleft

halfofthe�gure

representsaplant

cell,theright

sidean

animalcell.

Thecellis

surroundedby

thecell

mem

braneofwhich

anenlarged

sectionisshown

below.Main

elementofthe

cellmem

braneare

phospholipidsandcholesterolform

ingabilayeroftwo

lea�ets,inwhich

lipidsandcholesterolare

arrangedin

aparallelm

annertothe

mem

branenorm

al.Proteinsareassociated

withand

incorporatedinto

themem

brane.Thecellm

embrane

doesnot

constitutearigid

structurebut

exhibitsacom

plexrange

ofdynamics.

Itis

thereforeconsidered

a�two-dim

ensional�uid�as

describedby

the�uidic

mosaic

mem

branemodel.

Insidethe

cellonecan

�ndmany

elements

ofdi�erentfunction:

Ccilia,C

Hchrom

atin,Dcellular

junctions(e.g.

desmosom

es),ENendocytosis,ER

endoplasmic

reticulum,EX

exocytosis,Factin

�laments,G

Golgiapparatusconsisting

ofcisternaeand

vesicles,Hnuclear

envelope,Knuclear

pore,LYlysosom

e,M

mitochondrium

,MV

microvilli,N

nucleus,NU

nucleolus,Pplastids,R

freepolyribosom

es,Sstorage

lipids,Tmicrotubuli,V

vacuole(plantcell),W

cellwall.The�gures

aretaken

fromBiophysik

byWalter

Hoppe

[1].

4

1.2 Lipids

O

O

CH2

CH

O

O

CH2

O P O

O

O

CH2

CH2

N+

CH3

CH3

CH3

cholinephosphoryl

glycerolfatty acid (acyl-)

phosphatidic acid (phosphatidyl-)

fatty acid (acyl-)

(a) DOPC.

N+

CH3

CH3

CH3

O P O

O

O

CH2

CH2

CH2

CHOH

CHNH

O

sphingosine

cholinephosphorylN-acyl-sphingosine or ceramide

fatty acid (acyl-)

(b) SM.

O

O

O

O

O P

O

O

ON

+

H

(c) DPPC.

H

HH

HO

H

CH

CH

CH

CH

CH

H

3

3

3

3

3

(d) Cholesterol.

N O

N

O

O

O

O

O

O

PH

O O

H

S

N

O

O

N+

H

(CH )3 2

(CH )3 2

(e) TRITC-DPPE.

N+

CH CH

CHH C

CH

N

HC

CH

CHH C33

(CH )2 17

3

(CH )2 17

3

33

(f) DiI-C18.

Figure 2: Typical lipids and �uorescent analogs (e,f).

Number of carbons :Number of double bonds

Chain name

12 : 0 lauroyl14 : 0 myristoyl16 : 0 palmitoyl18 : 0 stearoyl18 : 1 oleoyl

Full name Abbreviation

phosphatidylcholine PCphosphatidylethanolamine PEphosphatidylserine PSphosphatidylinositol PI

Table 1: Chain- and headgroup names

Lipid name Abbreviation1,2-dilauroyl-sn-glycero-3-phosphocholine DLPC1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPC1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] POPS1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] DPPS

Table 2: Lipid names and abbreviations.

5

2 Formation of Lipid Structures

Membrane type PC PE PS PI SM Glycol. Chol. Others(a) Human erythrocyteplasma membranes

20 18 7 3 18 3 20 11

(b) Mammalian liverplasma membranes

18 12 7 3 12 8 19 21

(c) Golgi membranes 25 9 3 5 7 0 8 43

Table 3: Lipid composition of natural membranes in weight%.

1.2.2 GlycolipidsAnimal glycolipids consist of a ceramide moiety (as in sphingomyelin, see Fig. 2(b)) and a sugarmoiety (in place of the phosphorylcholine group found in sphingomyelin). Gangliosides, like forinstance GM1, have branched chains of several sugar residues.

1.2.3 SterolsCholesterol (Chol) is a molecule found almost exclusively in eukaryotic membranes, presumablybecause 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 membraneconstituent, indicating that it plays a key role. In addition, cholesterol is a precursor moleculefor the production of bile salts (e.g. glycocholate) which act as detergents in solubilizing foodlipids and in the biosynthesis of steroid hormones (progestagens, glucocorticoids, mineralcorti-coids, androgens and estrogens). In contrast to other hormones to which the membrane poses animpermeable barrier (they bind to receptors on the external side), steroid hormones are able todirectly cross the membrane and bind to receptor proteins in the cytosol.

1.2.4 Membrane proteinsMembrane proteins can be directly integrated into the membrane by exposure of hydrophobicamino 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 lea�et, i.e. in plane with the membrane.Depending on the signal sequence, hydrophobic stretches and charges, transmembrane proteins areinserted with the C-terminus on the cytoplasmic side (Type I) or on the ER / external side (TypeII). 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 linkedglycolipid (glycosyl-phosphatidyl-inositol-(GPI)-anchor). Finally, a protein can be attached to themembrane by non-covalent complex formation with other membrane proteins.

1.2.5 Lipids in Natural MembranesLipid 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 cholesterolcorresponds to approx. 50 mol-% cholesterol for the given molecular masses.

2 Formation of Lipid StructuresAggregation of lipids is a self-assembly process, driven by lipid interactions such as van derWaals, 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 H2O-molecules

6

(a) H2O-molecules (red) in next neighborhood tothe hydrophobic molecule (grey) have less possi-bilities to establish H-bonds than water molecules(black) surrounded by other hydrophilic particles.This situation is therefore entropically unfavored.

k1

kN

XN

mN

0NX

1m

1

0N=1

(b) Monomers and aggregates of hydrophobicmolecules are in equilibrium exchange

Figure 3: Principle of hydrophobic force.

and therefore low entropy [4], as depicted in �gure 3(a). To decrease the amount of clathratestructures, hydrophobic molecules tend to aggregate.

Consider now the self-assembly process of lipids: Membrane lipids have a polar, hydrophilicheadgroup and two long exposed hydrocarbon chains that are extremely hydrophobic, meaning alarge 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 = µ0singledispersed − µ0

micellar = −RT ln(XCMC), (1)

this large di�erence 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 moleculeshave associated into micelles, is very small for phospholipids. A typical value for a two-chain, 16carbon phospholipid is ∆G ≈ 75 kJ/mol ≈ 30RT , and critical micelle concentrations of two-chainphospholipids are typically below 10−12 M.

Amphiphilic molecules thus have a strong

Figure 4: For a small shape factor lipids form mi-celles. Intermolecular & Surface Forces [5].

tendency for aggregation. However, it dependson lipid shapes and lipid packing if micelles orbilayers 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, thismay depend strongly on pH and solution ionicstrength for charged lipids.)2. The length of the hydrocarbon chains

l, which sets an upper limit to micelle size.(Voids are energetically very costly.)

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 factorof the constituting lipids:

v

a0lc(2)

7

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 thecenter and their heads forming the sphere surface, so-called micelles. The sketch of a micelle givenin �gure 4 also shows the in�uence of the packing properties. It is evident that micelle-forminglipids must have a large headgroup area a0 and a short hydrocarbon chain volume v. For a micellethe shape factor has to be smaller than 1

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:

12

<v

a0lc< 1 (3)

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 ahydrophobic environment and the hydrophilic heads face an aqueous interface. But at the edgeof such a planar bilayer the hydrocarbon chains would be exposed to water (5(c)). Therefore thestructure will tend to shield these edges from the aqueous environment by connecting open edgeswith 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 poresare stabilized, e.g. by peptides or cone-shaped surfactants. The lipid bilayer is thus held togethervery e�ciently 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 Bilayers3.1 Lipid Bilayer PhasesLipids 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, thermaltransitions are studied. Although a variety of lamellar gel phases with di�erent packing structureshave been identi�ed by x-ray crystallography (Lβ has the carbon chains perpendicular to thebilayer plane, Lβ′ is with the carbon chains tilted, LβI is an interdigitated phase and Pβ′ has a

8

3.1 Lipid Bilayer Phases

Figure 6: Lipids form di�erent aggregate structures depending on their shape factor. Figure is takenfrom Intermolecular & Surface Forces [5].

9

3 Thermodynamics of Lipid Bilayers

(a) Gel phase (Lβ): There isboth lipid chain conformationaland translational order.

(b) Liquid-crystalline phase(Lα): The phospholipids showboth lipid chain conformationaland translational disorder(lateral di�usibility).

(c) Liquid-disordered phase(Lo): THe lipid chains areordered due to interactions withthe cholesterol (depicted as el-lipses), but there is translationaldisorder, allowing for lateraldi�usion.

Figure 7: Lipid bilayer phases.

ripple structure), only the common characteristics of gel phases need to be considered here: In thegel phase, the hydrocarbon chains are ordered in all-trans con�guration and there is long-rangetranslational order, impeding lateral movement. For this reason, it has also been termed thesolid-ordered phase (So).

Upon increase of the temperature above the melting point, Tm, the entropy term becomesdominating, resulting in the liquid-crystalline phase, Lα. The Lα phase is characterized both bylow conformational order in the carbon chains (lower internal order) and by low translationalorder (lower packing order, higher translational di�usion). For this reason, it has more recentlyalso been termed the liquid-disordered phase (Ld). Headgroup area, which is on the order of0.5 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 ofthe gel to liquid-crystalline transition. NMR studies indicate that this is due to the introduction ofanother equilibrium phase, the liquid-ordered phase (Lo), in which there is still high conformationalorder like in the gel phase, but the translational order is already lost (high translational di�usion)as in the Lα phase ([6], [7], [8]). Theoretical studies [9] predict that the order-disorder transitionof the carbon chains and the order-disorder transition of the packing do not necessarily need tobe 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 diagramsLipid 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 onlybe constructed for simple systems. Binary lipid mixtures can already show quite complex phasediagrams and ternary mixtures even more so. The Gibbs Phase Rule is applied to determine thenumber of degrees of freedom F ] for a system of C components, exhibiting P di�erent phases:P +F ] = C +2. Since only lipid phases forming in excess water are considered here as biologicallymost relevant, the water is omitted: It is not counted as a component and there is no degree offreedom 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 nativemembranes. In principle, according to the Gibbs Phase Rule, a huge number of phases couldbe allowed. What appears to happen is that especially for eukaryotic membranes containingcholesterol, the transition or the transitions smear out into a broad transition or no measurable

10

3.2 Lipid phase diagrams

0 1.0

tem

pe

ratu

re

T (A)m

T (B)m

T

compositionx(A)

x (T)fluid x (T)solid

fluid

coexis

tence

liquidus

solid

us

curve

curv

e

solid

Figure 8: Phase diagram of a binary lipid mixture of saturated phosphatidylcholines: The diagram showsa typical schematic phase diagram for a binary mixture of saturated phosphatidylcholines that di�er only intheir chain lengths, for example A = DPPC and B = DMPC [22,24]. The lipids show complete miscibility inthe solid (gel) and the �uid (liquid-crystalline) phase. However, due to their di�erent 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-enrichedsolid is in equilibrium with DMPC-enriched liquid. At a chosen temperature T , the compositions of thephases x�uid and xsolid are �xed. They can be read from where the horizontal dotted line, a so-calledtie-line, intersects the liquidus and the solidus curves. The relative amounts of liquid and solid can becalculated from material conservation, expressed as the �lever rule�. Finally, the solidus curve is reachedand the residual liquid (now approaching pure DMPC) solidi�es.

11

3 Thermodynamics of Lipid Bilayers

transition at all [10]. (A prerequisite for compiling a phase diagram is that there are distinguishablephases, and transitions between them are approximately �rst-order.) Nevertheless, studying simplesystems with identi�able phases is hoped to give insight into phenomena that could be pertinentto real cell membranes. Even when there is no real cooperative phase transition over the wholemembrane, 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 separationA mixture consisting of three major lipids,

Figure 9: Phase coexistence and di�usional mobil-ity in lipid bilayers consisting of DOPC, SM andcholesterol at room temperature. The numbers de-note di�usion coe�cients of the probe diIC18 in unitsof 10−8 cm2

/s (i.e. in µm2/s) [11].

DOPC, sphingomyelin (SM) and cholesterol,has been studied as a model for domain (�raft�)formation in cellular membranes. For a certainrange of compositions, lipid bilayers formedfrom this mixture exhibit a coexistence of two�uid (liquid) phases, the liquid-disordered (Ld

= Lα) and the liquid-ordered (Lo) phase (areaindicated in Fig. 9). This phase separationwas visualized in Giant Unilamellar Vesicles(GUVs) using �uorescent markers that specif-ically label one of these phases. The task ofthis practical lab course is to produce GiantUnilamellar Vesicles from a 1:1:1 mixture ofDOPC, SM and cholesterol and to visualizethe phase separation.

The two �uid phases have been further char-acterized by measuring the di�usion of a small,lipid-like probe inside these phase domains us-ing Fluorescence Correlation Spectroscopy (FCS)

([11], Fig. 9). Di�usion measurements by FCS are treated in other practical labs.

3.4 Model membranes3.5 VesiclesTo obtain vesicles (�gure 5(d)), lipids are usually dissolved in an organic solvent and the solventis evaporated using a nitrogen stream or vacuum, so that a thin lipid �lm is produced on a glasssurface (vial). The lipid �lm is hydrated with an aqueous solution, where the temperature shouldbe above the melting temperature Tm of the highest melting lipid in the mixture. Formationof vesicles may be supported by perturbations like shaking, temperature cycling or applying analternating electric �eld. The sizes, shapes and lamellarity of the vesicles obtained depend both onthe type of lipids and the detailed protocol. Giant unilamellar vesicles (GUVs) have a diameter onthe order of 1 to 300 µm. They are produced either by the protocol described above with minimalperturbation in the hydration step [12] or, with a generally much better yield of unilamellarvesicles, by using the electroformation approach [15]. Multilamellar vesicles (MLV) are quicklygenerated 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 producedby freeze-thaw cycling the vesicles, followed by extrusion, i.e. pressing the vesicle suspensionrepeatedly through a membrane of de�ned pore size. Small unilamellar vesicles (SUVs, 20 to 50nm in diameter) have very large curvature and most of their lipids are in the outer lea�et. Theyare prepared by extrusion through membranes with smaller pore size (30 nm) or by supplyingenergy to the MLV suspension by sonication. Extrusion and sonication have to be performedabove the highest lipid Tm. Liposomes of small size (SUVs, LUVs) can also be produced fromdetergent-lipid micelle solutions by dilution, dialysis, sequestration of detergent onto beads or gel

12

3.6 Planar membranes

Figure 10: Principle of lipid swelling (taken from www.avantlipids.com)

�ltration chromatography. This method allows incorporation of membrane proteins that are sohydrophobic that they require detergents for solubilization.

3.6 Planar membranesWhen lipids are deposited on a water surface and laterally compressed they form a monolayerwith 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 accuratelycontrolled [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 secondlea�et. Sequential transfer of two monolayers (tails facing each other) to a solid support leadto the formation of a supported bilayer [16]. Alternatively, spontaneous spreading and fusion ofsmall unilamellar vesicles on a hydrophilic surface is employed [17]. Supported planar bilayers andmonolayers can su�er from artifacts due to interactions with the support.

4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs)by electroformation

4.1 ElectroformationApplying a low-voltage electric �eld can promote the formation of truly unilamellar vesicles. ForGUV electroformation, a solution of lipids in organic solvent is dried on conductive electrodesinstead of a glass surface (platinum wires or conductively coated coverslips). The hydration stepis then performed in the presence of an electric (normally alternating) �eld. The electroformationmethod 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 swellingof lipid layers in an aqueous environment (�gure 10). The disadvantage with this method is thatGUV yield and size decrease strongly when ions (salt) are present in the aqueous solution.

4.2 General protocolLipids are dissolved in chloroform:methanol (2:1) at 10 mg/ml total concentration and ≈ 5 µl ofthis solution is spread on the conductive surface of an ITO-coated coverslip. For lipid mixtures

13

4 Experiment: Preparing Giant Unilamellar Vesicles (GUVs) by electroformation

conductivetape ITO-coverslip

ITO-coverslip

U = 1.2 V

H O2lipid

n = 10 Hz

(a) Capacitor-type con�guration 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 con�guration(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 apulse generator via pieces of conductive tape and an alternating voltage (U = 1.2V, f = 10 Hz)is applied for 1 to 3 hours. For high-Tm lipids, electroswelling is performed above the Tm on aheating block or in an oven. GUVs form in multiple layers above the lipid-coated coverslip andcan be studied directly in the chamber (in situ).

14

4.3 Lipid mixture

4.3 Lipid mixtureWe will prepare a lipid mixture of DOPC, C18-sphingomyelin (SM) and Cholesterol (Chol) witha molar ratio of 1:1:1. Furthermore, 0.1 mol% GM1 and 0.1mol% diIC18 are added directly to thelipid 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 bindingpartner by the B subunit of cholera toxin. After GUV formation, we will add �uorescently labeledcholera 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 roughly10 mg/ml. In what �nal volume should the above amount of lipids be dissolved?

15

5 Analysis

5 AnalysisGiant vesicles will be visualized using light microscopy. Please refer to the literature or the indi-cated websites for background reading on light microscopy:

For In�nity-Corrected Optical Systems:

http://micro.magnet.fsu.edu/primer/anatomy/in�nityintro.html

For Phase Contrast:

http://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phaseindex.htmlRead Sections:

• Brief Overview of Phase Contrast

• Phase Contrast Microscopy

• Phase Contrast Microscope Alignment

For DIC:

http://micro.magnet.fsu.edu/primer/techniques/dic/dichome.htmlSections:

• Brief Overview of DIC Microscopy

• Fundamental Concepts in DIC Microscopy

• DIC Microscope Con�guration and Alignment

• Comparison of Phase Contrast and DIC Microscopy

For Fluorescence Microscopy:

http://www.microscopyu.com/articles/�uorescence/�uorescenceintro.html

For Laser Scanning Microscopy:

http://www.olympusconfocal.com/theory/confocalintro.html

16

References

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[4] Bray D. Alberts, B. Molecular Biology of the Cell. Garland Publishing, 1994.

[5] Jacob Israelachvili. Intermolecular & Surface Forces. Academic Press Limited, 1991.

[6] H. W. Meyer, K. Semmler, and P. J. Quinn. The e�ect of sterols on structures formed inthe gel/subgel phase state of dipalmitoylphosphatidylcholine bilayers. Mol. Membr. Biol.,14(4):187�93, 1997.

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[9] M. Nielsen, L. Miao, J. H. Ipsen, M. J. Zuckermann, and O. G. Mouritsen. O�-lattice modelfor the phase behavior of lipid-cholesterol bilayers. Phys. Rev. E. Stat. Phys. Plasmas. Fluids.Relat. Interdiscip. Topics, 59(5 Pt B):5790�803, May 1999.

[10] P.L. Yeagle. The Structure of Biological Membranes. CRC Press, 1991.

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[13] K. Akashi, H. Miyata, H. Itoh, and K. Kinosita. Formation of giant liposomes promoted bydivalent cations: critical role of electrostatic repulsion. Biophys. J., 74(6):2973�82, Jun 1998.

[14] K. Akashi, H. Miyata, H. Itoh, and K. Kinosita. Preparation of giant liposomes in physiologicalconditions and their characterization under an optical microscope. Biophys. J., 71(6):3242�50,Dec 1996.

[15] M. I. Angelova and D. S. Dimitrov. Liposome electroformation. Faraday Discuss.Chem. Soc.,81(81):303�311, 1986.

[16] L. K. Tamm and H. M. McConnellL. Supported phospholipid-bilayers. Biophys. J., 47(1):105�113, 1985.

[17] E. Sackmann. Supported membranes: scienti�c and practical applications. Science,271(5245):43�8, Jan 1996.

[18] D.S. Dimitrov and M.I. Angelova. Lipid swelling and liposome formation mediated by electric�elds. Bioelectrochem. Bioenerg., 19(2):323�336, 1988.

[19] Soelau S. Meleard Ph. Faucon J.F. Bothorel P. Angelova, M.I. Preparation of membraneproteins into giant unilaemellar vesicles via peptide-induced fusion. Prog. Colloid. Polym.Sci., 89:127�131, 1992.

17

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. Reconstitutionof membrane proteins into giant unilamellar vesicles via peptide-induced fusion. Biophys. J.,81(3):1464�74, Sep 2001.

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