2. Fabrication of monolayer of lipids as membrane models

are involved in regulating biological activities such as fertilization, host- pathogen recognition, immunity and immune response, and in cancers where changes in glycosylation are

The complex nature of biomembranes makes them challenging to study [6], see Figure 1. This complexity necessitated development of simpler model systems, which would mimic native membranes but at the same time would give control over parameters such as structure, composition, size and facilitate monitoring of molecules of interest [7]. The majority of model systems are tailored to incorporate the bilayer structure of biological membranes. These bilayer model systems can be arranged in two-dimensions on a solid support or can form threedimensional spherical structures (supported or free in solution) as in liposomes [7–9]. Liposomes may be small, large, or giant in size and can have either one (unilamellar) or multiple (multilamellar) lipid bilayers. Liposomes are the subject of intense clinical interest where they are being studied as a vehicle for drug delivery [10–12]. Alternatively, one can fabricate monolayers of biomolecules as mimetic model systems. The compositions of monolayers are chosen to mimic one of the two leaflets of a biological membrane. One can then study the changes these systems would go through when they interact with external stimuli, which could be pathogens such as bacteria or viruses, proteins, or changes in environmental factors as temperature, pressure or pH. The results of such studies may then be extrapolated to natural

The lateral organization of lipids and cholesterol in cell membranes is important for cellular functions, especially cell signaling activities. Lipid rafts [13, 14], which are aggregates of sphingolipids and cholesterols, are also known to incorporate glycolipids [15] and host key cell signaling proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins. Studies in which the lipid organization is both perturbed and also observed in living cells under culture conditions are challenging. Exposure to agents such as methyl β-cyclodextrin that perturbs the

commonly observed [5].

214 Cell Culture

biological membrane systems.

Figure 1. A representation of the complex nature of cell membranes.

Lipids are amphiphilic molecules consisting of a hydrophilic head-group and a hydrophobic tail made of one or more hydrocarbon chains that may be saturated or unsaturated.

Monolayers are assembled by depositing droplets of lipid solution onto the water surface and subsequently waiting for the solvent to evaporate. The molecules spread out while the solvent evaporates. An example of a good spreading solvent is chloroform, although not all lipids are soluble in chloroform and sometimes mixed solvents with an alcohol must be used. Once deposited on the water surface, the polar or charged head-group orients towards the water surface and the hydrophobic tail(s) aligns away from the water. The lipid molecules get spread uniformly over the water surface forming a monomolecular thick film called a Langmuir monolayer named after Irving Langmuir [20], who pioneered this technique together with Katharine B. Blodgett.

Selection of solvent is critical for uniform spreading of monolayer. An ideal solvent should be volatile, chemically inert, relatively pure and with enough solubilization power to dissolve the solutes under study. Care must also be taken to make sure that the solvents are insoluble in the subphase [21]. Chloroform, cyclohexane, benzene, hexane, and mixtures with acetone, ethanol or methanol are some commonly used solvents. Water or buffer solutions of various composition and pH are used as the subphase.

The depositions are carried out in a Langmuir-Blodgett (LB) trough, depicted in Figure 2 where some of the main monolayer techniques are also schematically depicted. The basic

Figure 2. Schematics of Langmuir-Blodgett trough along with some associated measurement techniques. (1) Trough with subphase and deposited monolayer, (2) side view of barrier, (3) surface pressure transducer with Wilhelmy plate, (4) dipping system with a solid support, (5) microscopic measurements (BAM, fluorescence), (6) spectroscopic measurements, and (7) AFM image of transferred monolayer on solid support.

thoughtful considerations were taken to address thermodynamic limitations and attempts were made to correlate the experimental evidence with the proposed model, there were some anomalies hinting at the presence of regions in the membrane where the lipids would behave differently, i.e. were different in composition and/or phase. To explain these discrepancies a new hypothesis was proposed which suggested that certain lipids within the cell membrane have unique properties which would allow them to self-associate and form segregated regions which were named "lipid rafts". Originally it was proposed that these rafts were made of sphingolipids and cholesterol and functioned as platforms for trafficking proteins; however, it was later found

Figure 3. Structures of (a) D-erythyro-sphingosine, (b) N-dodecanoyl-β-D-galactosylceramide, (c) ganglioside GM1, and

Monolayers of Carbohydrate-Containing Lipids at the Water-Air Interface

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Glycolipids are an important group of raft forming lipids. Their ability to aggregate together to form microdomains indicates their involvement in various cellular activities. Glycolipids can broadly be divided into two major categories-glycosphingolipids (GSLs) and glycoglycerolipids, the first one being widely present in animal cells and the latter in plant and microbial cells with an exception of sulfated glycoglycerolipids called seminolipids which are found in mammalian testis [26]. The major difference between these two classes of glycolipids is their lipid moiety. While GSLs have ceramide as their lipid component, which is made of an aminoalcohol base (sphingoid base) and fatty acid joined by an amide bond, glycoglycerolipids have diacylglycerol as their lipid component. The sugar units are attached to GSLs through glycosidic linkage to hydroxyl groups at the C-1 carbon of the ceramide. In glycoglycerolipids the glycosylation occurs at the C-3 hydroxyl group of glycerol, see Figure 3. From here on we attempt to understand what structural features of these glycosylated lipids gives

As mentioned above, GSLs have ceramide as their lipid moiety. Ceramides can have a variety of structures depending upon the sphingoid bases and the fatty acid combinations. This variability in the structure of ceramide adds diversity to GSLs and further diversity is

that there was more to lipid rafts than trafficking of proteins [25].

them their unique property to cluster and form microdomains.

3.1. Glycosphingolipids

(d) a glycoglycerolipid.

components of the LB trough are a Teflon trough which holds the subphase, a barrier which helps compress the spread monolayer to a targeted area or surface pressure at specified compression rates, a surface pressure transducer for measurement of surface pressure and a dipper which helps in transferring the monolayer film onto a solid substrate. Some details on the mechanism of transfer will be discussed later in the chapter. The trough can be accessorized with temperature, pH and surface potential sensors. It can also be coupled with optical and spectroscopic instruments such as a fluorescence microscope, a Brewster angle microscope or an infrared spectrometer which help in visualization and characterization of the monolayers.
