Investigation on the relationship between lipid composition and ...

Investigation

on

the

relationship

between

lipid

composition and structure in model membranes composed

of extracted natural phospholipids

Andreas Santamaria1,2, Krishna C. Batchu1, Giovanna Fragneto1,3, Val¨¦rie Laux1, Michael

Haertlein1, Tamim A. Darwish4, Robert A. Russell4, Nathan R. Zaccai5,+, Eduardo Guzm¨¢n2,6 and

Armando Maestro7,8,*.

1

2.

Institut Laue-Langevin, 71 Avenue des Martyrs, 38042 Grenoble, Cedex 9, France.

Departamento de Qu¨ªmica F¨ªsica, Universidad Complutense de Madrid, Ciudad Universitaria

s/n, 28040 Madrid, Spain.

3.

?cole doctorale de Physique, Universit¨¦ Grenoble Alpes, 38400 Saint-Martin-d¡¯H¨¦res, France.

4.

National Deuteration Facility, ANSTO-Sidney, Lucas Heights, NSW 2234, Australia

5.

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB22 7QQ,

United Kingdom.

6.

Instituto Pluridisciplinar, Universidad Complutense de Madrid, Paseo Juan XXIII 1, 28040

Madrid, Spain.

7.

Centro de F?s? ica de Materiales (CSIC, UPV/EHU) - Materials Physics Center MPC, Paseo

Manuel de Lardizabal 5, E-20018 San Sebasti¨¢n, Spain.

8.

IKERBASQUE¡ªBasque Foundation for Science, Plaza Euskadi 5, Bilbao, 48009 Spain.

+

current address: Domainex, Pampisford, Cambridge CB22 3EG, United Kingdom

* Corresponding author [armando.maestro@ehu.eus]

1

Abstract

Unravelling the structural diversity of cellular membranes is a paramount challenge in life

sciences. In particular, lipid composition affects the membrane collective behaviour, and its

interactions with other biological molecules. Here, the relationship between membrane

composition and resultant structural features was investigated by surface pressure-area isotherms,

Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the

mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in

the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike

synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar. The

structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral

packing, was found to depend strongly on their specific composition, with cholesterol having a

major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquiddisorder phases.

1. Introduction

Lipid molecules constitute the building blocks of cellular membranes. The main constituents

of mammalian membranes are glycerophospholipids, including zwitterionic phosphatidylcholines

(PC) and phosphatidylethanolamines (PE), and lower proportions of anionic phosphatidylserines

(PS) and phosphatidylinositols (PI). The lipid hydrophobic moiety consists of a diacylglycerol that

contains saturated or cis-unsaturated fatty acyl chains of variable extent and unsaturation degree1.

Due to their unique chemistry, including hydrophobic acyl chains and polar headgroups, these

phospholipids spontaneously self-assemble as bilayers with the hydrophobic acyl chains facing

each other and the polar headgroups interacting with the external aqueous phase. In particular, PC

is well known to provide a stable, fluid environment due to its liquid crystalline arrangement, while

PE, due to its relatively small polar headgroup, creates defects in the membrane bilayer that

facilitates fusion as well as the interaction with peripheral membrane proteins2. Finally, PS and PI,

even at low concentrations, provide the negative charge of the membranes, which is a key factor

in the interaction with positively charged patches of proteins2¨C4. Synthetic versions of PC, PE, PS

and PI phospholipids, usually with 16- or 18-carbon acyl chains, which are the most abundant in

cell membranes5,6, have been used to mimic eukaryotic membranes7¨C9. These membranes are,

however, much more complex systems, being characterized by differently unsaturated acyl chains

2

as well as by the presence of sterols and sphingolipids, such as sphingomyelin (SM). Another

important feature of the plasma membrane (PM) is the high levels of cholesterol10,11 (~ 50% in

mol), and the formation of lipid rafts, due to the immiscibility of cholesterol (and saturated lipids)

with unsaturated lipids12. These rafts would help to concentrate proteins as well as lipids locally,

enabling cellular processes such as budding of enveloped viruses and sorting of lipids and proteins

to the Golgi complex12. The endoplasmic-reticulum-Golgi intermediate compartment, ERGIC,

membrane (EM) is also a membrane of biological interest, which contains a lower amount of

cholesterol, as it provides an anchoring point for viral proteins13. The ERGIC is an organelle that

lies between the endoplasmic reticulum and the Golgi complex, mediating trafficking between

them and facilitating the sorting of cargo14.

In the present work, Langmuir lipid monolayers were exploited to accurately replicate the

ERGIC membrane15,16 and the plasma membrane10. Indeed, the structural characterization of these

membranes should yield insights into their function and malfunction. A simpler lipid mixture,

lacking cholesterol and sphingomyelin, was also prepared in order to characterize a monolayer

uniquely composed of three natural phospholipids, PC-PE-PS (Figure 1A shows the molar lipid

compositions). Previously, synthetic lipid monolayers, for example using DPPC (1,2-dipalmitoylsn-glycerol-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), or

complex mixtures such as the so-called canonical mixture (containing POPC, sphingomyelin and

cholesterol), were employed to mimic cellular membranes as well as lung surfactants17¨C26. Even

though Langmuir monolayers are not well suited to address transmembrane processes, their

versatility allows the investigation of lipid¡¯ structure and organisation of single membrane leaflets

at the air/water interface, by exploiting a plethora of interfacial in situ techniques27¨C29, thus offering

the possibility of untangling different questions of biophysical interest. Here, Langmuir

monolayers composed of natural lipids were studied by surface pressure-area isotherms, Brewster

angle microscopy (BAM) and neutron reflectometry (NR). The latter technique is especially suited

for the study of biological interfaces. Indeed, neutrons are non-destructive, thus allowing structural

characterisation with sub-nanometric resolution in physiological conditions and temperatures.

Moreover, neutrons interact very differently with hydrogen and deuterium nuclei, therefore,

through isotopic substitution, it is possible to highlight structural and chemical differences in

specific regions of interest such as, for example, identifying the presence of water molecules and

the position and orientation of hydrophobic tails and polar headgroups.27

3

The combined experimental approach used in this work shed light on the interfacial structure

of in vitro model systems of the plasma and ERGIC membranes, with emphasis on the structural

effects associated with the incorporation of cholesterol in the membranes. Although natural lipids

were previously used in the form of bi- and multilayers to mimic eukaryotic cell membranes, which

were then characterized by neutron reflectometry30,31 and diffraction32,33, to the best of our

knowledge, this is the first report describing the use of natural lipids extracted from yeast and

studied in the form of monolayers at the air/water interface, in order to mimic biological relevant

membranes, and to provide direct structural evidences about the role of cholesterol. The possibility

of tuning and controlling the composition and surface density of phospholipids makes Langmuir

films effective models for mimicking single leaflets of cell membranes under different

environmental conditions. Such in vitro lipid model systems could potentially be exploited to study

the interaction of cell membranes with external stimuli, such as viral proteins34 or antimicrobial

peptides35¨C37.

2. Experimental section

2.1. Natural lipid extraction and purification

Phospholipids were extracted from perdeuterated and hydrogenous Pichia pastoris biomasses.

Harvested cells were suspended into 10 mL deionised water and lysed by probe sonication on an

ice bath for 3¡Á5 min with 30 s intervals, 25% duty cycle. The resulting cell lysate was poured into

boiling ethanol containing 1% butylated hydroxytoluene (BHT) followed by vigorous stirring to

denature lipases. The total lipid mixtures were then extracted according to the method proposed

by Folch et al.38, followed by evaporation of the organic phase under a N2 stream and their final

reconstitution in CHCl3. Purification of the various classes of phospholipid mixtures containing

molecular species of mixed acyl chain lengths was achieved through sequential purifications steps,

first by passing the lipid extracts through an amino-bonded solid-phase extraction column followed

by a diol-modified silica stationary phase column coupled to a High-performance Liquid

Chromatography-Evaporative light scattering detector (HPLC-ELSD) (Agilent 1260, United

Kingdom)

system.

The

mobile

phase

employed

was

a

gradient

of

solvent

A

(CHCl3/CH3OH/NH4OH, 80:20.5:0.5 v/v) and solvent B (CHCl3/CH3OH/H2O/NH4OH,

60:35:5.5:0.5 v/v)39. TLC analysis was carried out on a High-Performance Thin-Layer

Chromatography (HP-TLC) system (CAMAG, Muttenz, Switzerland) to assess the identity and

4

purity of each of the purified classes. Fatty acid compositions of such purified mixtures were

measured by Gas Chromatography-Flame Ionization Detection (GC-FID), see Table S1.

2.2. Other lipids and reagents

Cholesterol (purity¡Ý99.0%) and natural extracted sphingomyelin (egg, chicken, purity

¡Ý99.0%) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Deuterated cholesterol

was obtained from the National Deuteration Facility (NDF) in ANSTO (Australia). Ultra-pure

water was generated by passing deionized water through a Milli-Q unit (total organic content=4

ppb; resistivity=18 M¦¸¡¤cm, Milli-Q, Merck KGaA, Darmstadt, Germany). D2O (99.9% of

isotopical purity) was purchased from Sigma Aldrich and used as received. HEPES-NaCl buffer

(5 mM HEPES, 150 mM NaCl, pH=7) was used for the experiments. HEPES (in solution, 1 M in

H2O, and powder, purity 99.5%), and sodium chloride (purity¡Ý99.0%) were purchased from Sigma

Aldrich.

2.3. Lipid mixture solutions preparation

Lipid stock solutions were prepared in chloroform stabilized with ethanol (purity 99.8%;

Sigma Aldrich, St. Louis, MO, USA), and stored at -20¡ãC. Solutions with the desired composition

(shown in Figure 1A and Table S2) of 0.2 mg.mL-1 were then prepared and used to perform all

the experiments. The relative percentage of 16 and 18 carbon chains of each lipid could be

estimated from their molar ratios and acyl chain composition (see Table S3).

2.4. Surface pressure (¦°)-area (A) isotherm

The surface pressure (¦°)-area per molecule (A) isotherms were measured using a Langmuir

trough (KIBRON, Helsinki, Finland) with a maximum area of 166.4 cm2 coupled to a force balance

fitted with a contact probe. The trough was carefully cleaned with Decon90, ethanol and Milli-Q

water before filling it with 120 mL of HEPES-NaCl buffer. Subsequently, the lipid solutions were

spread on the clean subphase using a Hamilton micro-syringe with a precision of ¡À1 ?L. After the

chloroform was evaporated for about 20 min, the variation of surface pressure during compression

was recorded using a Wilhelmy plate made of filter paper as contact probe and applying a barrier

speed of 8 cm2¡¤min?1. The temperature of the subphase was maintained at 21.0¡À0.1 ¡ãC. The trough

is placed inside a polyethylene box to control the atmosphere of the system. The temperature inside

the box was controlled by passing thermostated water through the jacket of the trough. The

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