Lateral Lipid Organization in Membranes

Several processes have been proposed for producing non-homogeneous inplane organization of different lipid species in membranes (Binder et al. 2003). Some of these processes suggest self-organization due to equilibration of the membrane system towards thermodynamically favorable states, without a necessary major participation of specific lipid-lipid interactions. An example of this type of process is the phase separation that spontaneously occurs in bilayers made from a mixture of lipids with significantly different gel-to-fluid transition temperatures (Tm), where segregation of phases occurs at temperatures between the Tm's of the different lipids (Fig. 5.1b) (Bagatolli and Gratton 2000; 2000a,b).

Fig. 5.1. Gel/fluid-like phase coexistence in phospholipid monolayers and bilayers. (a) Liquid-condensed/liquid-expanded regions coexist in interfacial films of dipalmitoylphosphatidyl-choline (DPPC) compressed to 11 mN/m, at 25°C. Condensed domains were visualized either by exclusion of 1 mol% of the fluorescent probe NBD-PC in the epifluorescence images (scale bar, 25 p.m), or by their greater thickness visualized with AFM of films transferred onto mica supports (scale bar, 10 p.m). (b) Gel/liquid crystalline phase coexistence in giant unilamellar vesicles (GUVs) made of an equimolar mixture of dilauroylphosphatidylcholine (DLPC) and DPPC at 25°C in the presence of 0.5 mol% fluorescent probe rhodamine-dipalmitoylphos-phatidylethanolamine (Rh-DPPE) (Bagatolli and Gratton 2000a)

Fig. 5.1. Gel/fluid-like phase coexistence in phospholipid monolayers and bilayers. (a) Liquid-condensed/liquid-expanded regions coexist in interfacial films of dipalmitoylphosphatidyl-choline (DPPC) compressed to 11 mN/m, at 25°C. Condensed domains were visualized either by exclusion of 1 mol% of the fluorescent probe NBD-PC in the epifluorescence images (scale bar, 25 p.m), or by their greater thickness visualized with AFM of films transferred onto mica supports (scale bar, 10 p.m). (b) Gel/liquid crystalline phase coexistence in giant unilamellar vesicles (GUVs) made of an equimolar mixture of dilauroylphosphatidylcholine (DLPC) and DPPC at 25°C in the presence of 0.5 mol% fluorescent probe rhodamine-dipalmitoylphos-phatidylethanolamine (Rh-DPPE) (Bagatolli and Gratton 2000a)

The molecules of the component with the higher Tm nucleate and segregate into highly packed ordered regions excluding the more disordered molecules of the lipid with lower Tm, which still have chains with a high number of spontaneous trans-gauche isomerizations, at these intermediate temperatures. Phase separation can be envisaged as a typical entropically driven phenomenon. The process can be easily modeled in interfacial monolayers where the packing state of the lipids can be controlled by compressing or expanding the films in a surface balance (Fig. 5.1a) (Nag and Keough 1993; Mohwald et al. 1995). Single phospholipid monolayers compressed to pressures into the liquid-expanded to liquid-condensed transition plateau show coexistence of condensed highly packed regions with disordered loosely packed areas. Films made from a mixture of two lipids with significantly different Tm's show two-dimensional phase segregation at a wide range of lateral pressures, including those thought to be equivalent to the ones occurring in cellular membranes (around 30 mN/m). Phase separation has also been observed in membrane bilayers made of phospholipids with very different chain lengths, especially, although not exclusively, at temperatures inducing gel-like solid phases (Fig. 5.1b). This should also be considered an entropy-driven process because the phase separation minimizes the amount of hydrophobic segments, arising from the hydrophobic mismatch of the different lipids, which are potentially exposed to the polar solvent.

Alternatively, certain lipid complexes could be established by selective or preferential molecular interactions that also induce lateral organization of lipids in processes that may be considered not only entropic, but also enthalpically driven. For instance, this would be the case for the lipid-lipid interactions that induce the formation of the sphingolipid/cholesterol complexes thought to be the basis for the organization of rafts (Veiga et al. 2001; Collado et al. 2005). In this case, hydrogen bonding between the amide of the sphingomyelin skeleton and the hydroxyl of cholesterol would supply an important stabilizing energy. Formation of other lipid condensates, such as the phosphatidylcholine/cholesterol condensed complexes proposed by McConnell (McConnell and Vrljic 2003; Mc-Connell 2005), would still have a mainly entropic origin. This demonstrates that entropic and enthalpic contributions for membrane domain triggering cannot be easily separated. Simple ternary mixtures containing variable proportions of saturated and unsaturated phospholipid species and cholesterol show that two fluid phases exist in both interfacial monolayers and bilayers (Fig. 5.2) (Dietrich et al. 2001; Veatch and Keller 2002; Veatch and Keller 2003).

The saturated lipid molecules tend to pack with cholesterol and form what is called a liquid-ordered (lo) phase. The lipid acyl chains are mostly extended and tightly packed against the planar structure of the sterol forming an "ordered phase", while the unsaturated lipid molecules maintain a high degree of transla-tional mobility forming a "fluid phase". The ability of the sphingomyelin backbone to form a hydrogen bond with the cholesterol molecule increases the affinity between these two components, favoring segregation of the sphingolipid/cholesterol complexes from unsaturated lipid-enriched regions in membranes. These complexes are extraordinarily resistant to detergent solubilization (Shogomori and Brown 2003; Chamberlain 2004). Other processes can also produce lateral organization via a major enthalpic contribution, for instance, the segregation that occurs in mixtures of charged and non-charged lipids in the presence of proper counter-charged molecules (Russ et al. 2003; Mbamala et al. 2005).

PC/SM/Chol 25:45:30 DOPC/DPPCS/Chol 30:30:40

Fig.5.2. Fluid/fluid-like phase coexistence in lipid monolayers and bilayers. (a) Fluid-or-dered/fiuid-disordered coexistence in interfacial films made of egg yolk phosphatidylcholine (PC)/bovine brain sphingomyelin (SM)/Cholesterol (Choi) (25:45:30, w/w/w) compressed to 17 mN/m at 25°C. (b) Liquid-ordered/liquid-disordered phase coexistence in GUVs made of the ternary mixture dioieoyiphosphatidylcholine (DOPC)/DPPC/Chol (30:30:40, w/w/w) in the presence of 0.5 mol% BODIPY-PC and 0.5 mol% DilC18. Scale bar in the left panel represents 25 l_im and image in the right panel also have 25 |_im width

PC/SM/Chol 25:45:30 DOPC/DPPCS/Chol 30:30:40

Fig.5.2. Fluid/fluid-like phase coexistence in lipid monolayers and bilayers. (a) Fluid-or-dered/fiuid-disordered coexistence in interfacial films made of egg yolk phosphatidylcholine (PC)/bovine brain sphingomyelin (SM)/Cholesterol (Choi) (25:45:30, w/w/w) compressed to 17 mN/m at 25°C. (b) Liquid-ordered/liquid-disordered phase coexistence in GUVs made of the ternary mixture dioieoyiphosphatidylcholine (DOPC)/DPPC/Chol (30:30:40, w/w/w) in the presence of 0.5 mol% BODIPY-PC and 0.5 mol% DilC18. Scale bar in the left panel represents 25 l_im and image in the right panel also have 25 |_im width

Simplistic molecular mechanisms for producing lateral sorting of lipids in membranes are often invoked to explain the nature of membrane domains. However, the compositional complexity of the real membranes could sustain simultaneous processes, perhaps synergistically potentiated or mutually influenced, that promote membrane lateral organization to different extents and lead to the coexistence of many different types of lipid domains. In this context, the notion of rafts, as is often used in the literature, can be misleading.

Lateral separation of domains or phases in lipid membranes due to some of these different processes has been mostly documented in simple model systems such as vesicles or interfacial monolayers made of structurally different lipids. Under the appropriate environmental conditions, spontaneous self-organized micrometer-sized domains can be visualized in model bilayers or monolayers (Binder et al. 2003). The study of these models demonstrates that pure lipid systems may have defined intrinsic properties for lateral self-organization, providing a structural scaffold that is probably very sensitive to compositional complexity. Microscopic techniques such as epifluorescence or Brewster angle microscopy are suitable to detect and analyze lateral domains in the microscopic size range, while atomic or scanning force microscopy (AFM, SFM) has produced pictures showing lipid domains from micrometer to hundreds of nanometer size. Other techniques, such as fluorescence resonance energy transfer (FRET), fluorescence quenching (Silvius 2003; de Almeida et al. 2005), or more recently, fluorescence correlation spectroscopy (FCS) (Kahya et al. 2004) have provided indirect evidence for the existence of membrane domains in the size range of a few nanometers. However, little is known about the occurrence oflipid self-organizing processes in the context of compositionally complex cellular membranes. Recent studies have shown that native membranes from the pulmonary surfactant system maintain a stable

Fig. 5.3. Presence of condensed regions In pulmonary surfactant monolayers and bilayers. (a) Dark condensed domains In interfacial monolayers made from an organic extract of porcine pulmonary surfactant, containing all the lipid components plus the hydrophobic proteins SP-B and SP-C with 1 mol% NBD-PC added and compressed to 35 mN/m at 25°C. (b) Coexistence of fluid-ordered (red) and fluid-disordered (yellow) regions In GUVs made from organic extracted material from porcine pulmonary surfactant doped with 0.5 mol% BODIPY-PC and 0.5 mol% DIIC18. Scale bars In left panels represent 25 ^m and Images In right panels also have 25 ^m width

Fig. 5.3. Presence of condensed regions In pulmonary surfactant monolayers and bilayers. (a) Dark condensed domains In interfacial monolayers made from an organic extract of porcine pulmonary surfactant, containing all the lipid components plus the hydrophobic proteins SP-B and SP-C with 1 mol% NBD-PC added and compressed to 35 mN/m at 25°C. (b) Coexistence of fluid-ordered (red) and fluid-disordered (yellow) regions In GUVs made from organic extracted material from porcine pulmonary surfactant doped with 0.5 mol% BODIPY-PC and 0.5 mol% DIIC18. Scale bars In left panels represent 25 ^m and Images In right panels also have 25 ^m width coexistence of two fluid phases in the form of micrometer-sized fluid disordered domains immersed in a cholesterol-enriched fluid-ordered background (Fig. 5.3) (Bernardino de la Serna et al. 2004).

Liposomes made from the full lipid fraction extracted from certain membranes also show the coexistence of ordered and disordered regions (Dietrich et al. 2001; Nag et al. 2002). These experiments indicate that the lipid fraction of membranes plays a major role in determining their lateral structure. Considerable effort is being directed toward the detection of membrane domains in the membranes of whole cells. Several studies have shown the existence of large micrometer sized domains in certain cell membranes (Gousset et al. 2002; Gaus et al. 2003). Other studies suggest that domains in intact cell membranes could be very small and highly dynamic, thus making their detection and morphological characterization a technical challenge (Helms and Zurzolo 2004; Kusumi et al. 2004; Simons and Vaz 2004). It maybe difficult to distinguish between the presence of real "domains" with a defined lifetime and what could be considered transient lipid fluctuations caught in a given instant (Nielsen et al. 2000).

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