The cell membrane is not only a homogeneous two dimensional fluid, which Singer and Nilcolson suggested.[77] The plasma membrane of an eucaryotic cell has many dif-ferent components which can be divided into two major groups, lipids and membrane proteins. The lipid builds up the membrane matrix in which the membrane proteins are incorporated. The membrane has many different lipids which are divided into the subgroups glycerolipids, sterols and sphingolipids.[78,79] The glycerolipids as well as the sphingolipids have different head groups. Very often, a phosphate is present in the lipid head group called phopholipids, which can be either a glycerolipid or a sphingolipid. The main lipid content are the phosphoglycerolipids, which are divided into the different head groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositolphosphate (PIP) and phosphatidylserine (PS). The glyc-erolipids as well as the sphingolipids have different fatty acids.
The membrane has a huge amount of different lipids aggregated into a two di-mensional fluid. The different lipids and different proteins cluster in different ways and amounts which results in a heterogeneous membrane.[80–83] These clusters or do-mains are called lipid rafts (figure 1.4).[80] Lipid rafts are defined as "small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions."[84]Three dif-ferent lipid rafts are illustrated in figure 1.4. First, difdif-ferent proteins are activated in the lipid raft which are inactive in the non-raft membrane (figure 1.4).[85,86] It was reported that the actin network binds to a lipid raft.[87] The cell used the gly-cosphingolipids for signaling processes and communications which are enriched in such lipid rafts.[88] The last illustrated lipid raft contains the receptor lipid for STx it is Gb3 and the STx binds to it.[64] It is not clear if the lipid rafts exist first, be-fore the toxin bind, or if the lipid raft is created by the binding of the toxin. A problem is that such lipid rafts are not visualized with the newest technical equip-ment in eukaryotes.[89]Only indirect information from detergent restistant membrane (DRM),[90]fluorescence resonance energy transfer (FRET)[91,92] and fluorescence cor-relation spectroscopy (FCS)[93,94] measurements hints that such rafts exist. In bacte-rial small domains of∼40 nm in diameter were found.[95]
The heterogeneity of the lipid raft model was analyzed in model membrane systems with the three major membrane lipids of eucaryotic cells, a low melting phospholipid (POPC or DOPC), a high melting lipid (DPPC or sphingomyelin (SM)) and sterol (cholesterol (Chol)).[97,98]These model membranes undergo phase separation in a wide
Figure 1.4.: A schematic drawing of the cell membrane with three different lipid rafts.
From left to right, the first lipid raft activates a channel protein,[85] with the second one the actin is bound[87] and in the third one glycolipids are enriched and shiga toxin (STx) binds to it.[88] The cell membrane has different lipid species such as cholesterol (Chol), sphingomyelin (SM), phosphatidylcholine (PC), glycolipid as globotriaosyl ceramide (Gb3), phosphatidylinositolphosphate (PIP), phosphatidylethanolamine (PE) and phosphatidylse-rine (PS).[96]
region of the lipid compositions.[99] The lipid raft hypothesis is mimicked by such phase-separated membranes. The two phases of the raft mimic model membranes are the liquid ordered (lo) phase which is enriched in high melting lipids and sterols and mimics the raft domains and the liquid disordered (ld) phase which is enriched in low melting lipids and mimics the membrane matrix. The non-physiological lipid DOPC is preferential used instead of POPC, because the domains are larger and better visible with the fluorescent microscope.[97] The raft mimic model membranes which represent the cell membranes and the phase distribution of the STx receptor lipid will be analyzed. These data are an indication of the position from Gb3 in the plasma membrane relative to the lipid raft theory.
The measurement of the raft like lipid composition can be accomplished on dif-ferent membrane systems which can be the giant unilamellar vesicles (GUVs),[100]
adhered GUVs,[101] pore-spanning membranes (PSMs)[102] and solid supported mem-branes (SSMs)[103] (figure 1.5). GUVs and SSMs mimic the cell membrane with the same conditions for each membrane region (figure 1.5 A, D). The two different leaflets can have different surroundings. In GUVs the buffer inside can be different from the buffer outside of a GUV.[104,105]The different leaflets of the SSMs are distinguishable:
One is in contact with the surface of the support and the other leaflet is in contact with the solution. SSMs are very easy to handle and the membrane is, in contrast to the GUVs, two dimensional which is useful for microscopy studies (figure 1.5 D).
With the supported membrane, the SSMs mimic the membrane cell adhesion to a
surfaces. The increase of water between support and the membrane of SSM makes sure that the surface interaction has no influence of the properties from integral mem-brane proteins.[106] Therefore polymer-supported membranes were developed which increase the water film between the membrane and the substrates from few nm to many nm.[107] This increase of the water layer also increases the activity of proteins in the SSMs and brings it nearer to the membrane system of GUVs which mimic the membrane part of a cell which has no contact. To combine the adhesion membrane model (SSM) and the membrane model without any adhesion (GUV), the whole cellular membrane is described, because the cellular membrane has parts with ad-hesion and parts without. The cell membrane interacts not only with the surface outside of the cell but it has also adhesion properties to the inside of the cell.[108] The actin network stabilizes the plasma membrane and interacts strongly over actin bind-ing proteins with the membrane.[109] This can be mimicked with membrane systems which have different adhesion properties such as the adhered GUVs and the PSMs (figure 1.5 B and C). Both membrane systems have an adhered area as in SSMs and in both membrane systems there are lipid areas where the membrane is as free-standing as in GUVs.[102,110]These membrane systems mimic the cell membranes with different adhesion properties to a surface or to the actin network.[102]
Figure 1.5.:Different model membrane systems are giant unilamellar vesicles (GUVs) (A), adhered GUVs (B), pore-spanning membranes (PSMs) (C) and solid supported membranes (SSMs). The shown systems are schematically drawn with phase-separated membranes, which contain DOPC/sphingomyelin (SM)/cholesterol (Chol). The liquid disordered (ld) phase is enriched in DOPC and the liquid ordered (lo) phase is enriched in SM and Chol.
The substrates for the adhered GUVs, PSMs and SSMs are functionalized with two different layers. The functionalization can differ for each system.
The actin network has an influence on the phase separation in membranes. Liu et al.showed that thelo phase is co-localized with the actin network.[87] This can be a rearrangement of lipids or that the phase diagram of such a lipid mixture changes with different adhesion regions which are adhesion to the action network or not. A phase diagram of PSMs was investigated which mimics such two different adhesion
regions for one membrane. This model system should clarify if cell adhesion also has an influence on the raft building or if only the different lipid and protein components do that. These influences have then also an effect on the distribution of Gb3 in the plasma membrane. The distribution of the Gb3 was investigated with a raft mimic membrane system in which fluorescently labeled Gb3s were incorporated. This analysis was done in GUVs. The knowledge of the distribution of the Gb3 in plasma membrane or plasma mimic membrane can help to understand the binding properties of STxB to Gb3 so that different drugs against EHEC can be designed which changes the Gb3 distribution in the plasma membrane and prevent the uptake of STx into the cell.