Membrane lateral phase separation

FIGURE 9.8 An illustration of the concept of lateral phase separations in a membrane. Phase separations of phosphatidylserine (green circles) can be indnced by divalent cations snch as Ca-+. 

The measurement of fluorescence lifetimes is an integral part of the anisotropy, energy transfer, and quenching experiment. Also, the fluorescence lifetime provides potentially useful information on the fluorophore environment and therefore provides useful information on membrane properties. An example is the investigation of lateral phase separations. Recently, interest in the fluorescence lifetime itself has increased due to the introduction of the lifetime distribution model as an alternative to the discrete multiexponential approach which has been prevalent in the past. 

The fluorescence lifetime is sensitive to the environment of the fluorophore, and in membranes this usually means the surrounding fatty acyl chains or the membrane protein interfacial region (see summary in Table 5.3). Generally, the lifetime of membrane-bound fluorophores is rather less sensitive to the types of subtle alterations which are encountered in membranes as compared to the fluorescence anisotropy parameters. The gel-to-liquid crystalline phase transition is a notable exception where most fluorophores show an alteration in lifetime properties. Although, again, the anisotropy (see below) is the most sensitive parameter in this regard, the fluorescence lifetime has been used with considerable success in the study of phase transitions and lateral phase separations. Fluorophores used to yield information on the... 

The use of DPH lifetimes for the analysis of phase separations and membrane properties has been described for mode) systems.n fl) In the case of both parinaric acids and DPH, one of the motivations for examining phase separation in a model lipid bilayer is the possibility that phase separations might be detectable in natural membranes. However, this technique has not been able to satisfactorily resolve lateral phase separations in natural membranes, either because they do not exist or because they are much more complex and even possibly transient in nature. Alternatively, it could be argued that if a probe could be found with the characteristics of trans-parinaric acid but perhaps with an even greater phase partitioning ability, then this approach might be reevaluated. 

Membrane conformational changes are observed on exposure to anesthetics, further supporting the importance of physical interactions that lead to perturbation of membrane macromolecules. For example, exposure of membranes to clinically relevant concentrations of anesthetics causes membranes to expand beyond a critical volume (critical volume hypothesis) associated with normal cellular function. Additionally, membrane structure becomes disorganized, so that the insertion of anesthetic molecules into the lipid membrane causes an increase in the mobility of the fatty acid chains in the phospholipid bilayer (membrane fluidization theory) or prevent the interconversion of membrane lipids from a gel to a liquid form, a process that is assumed necessary for normal neuronal function (lateral phase separation hypothesis). 

Fig. 2. Phase diagram describing lateral phase separations in the plane of bilayer membranes for binary mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoyl-phosphatidylcholine (DPPC). The two-phase region (F+S) represents an equilibrium between a homogeneous fluid solution F (La phase) and a solid solution phase S presumably having monoclinic symmetry (P(J. phase) in multilayers. This phase diagram is discussed in Refs. 19, 18, 4. The phase diagram was derived from studies of spin-label binding to the membranes.

Wu, S. H. W. and McConell, H. M. Lateral phase separations and perpendicular transport in membranes. Biochemical and Biophysical Research Communications 55 4S4, 1973. 

Shimshick, E. J. et al. Lateral phase separations in membranes. Journal of Supramolecular Structure 2 285-295,1973. 

Shimshick EJ, McConnel HM. Lateral phase separation in phospholipid membranes. Biochemistry 1973 12 2351-2360. 

The simple model does allow an entry point into the study of self-assembly of multicomponent lipid systems, lateral phase separation (clustering), membrane asymmetry, and in particular how these relate to curvature through packing. These form a central class of problems in membrane biology. 

Phase separation is observed when mixtures of phospholipids are incorporated into the membrane where the single components possess certain differences in their structure. In this case the phase separation is indicated in the thermogram by two or more signals. Structural characteristics which could lead to phase separation are differences in chain length and different degrees of saturation. Mixtures of dipalmitoylphosphatidic acid (DPPA) and diphosphatidylcholine (DPPC) or of distearoyllecithin with dimyristoyllecithin, for example, show the phenomena of phase separation. The reason for this is a lateral phase separation caused by domain formation. 

Lateral Phase Separation in Phospholipid Membranes Caused by Lateral Diffusion of Lipid Chains... 

Lateral phase separation in phospholipid membranes caused by lateral diffusion of lipid chain is detected by the time dependence of the local concentration which is estimated from the spin-spin exchange interaction. 

Although the Fluid Mosaic Model assumes a homogeneous distribution of lipid throughout the bilayer, lateral phase separation can be observed in model membranes composed of mixtures of lipids (Figure 2b). The first indication that similar effects occurred in vivo was the discovery of cholesterol-rich and detergent-resistant fractions of cell membranes, dubbed lipid rafts. These rafts, described... 

The fourth reported method, specific to peroxidative damage to membrane components, is via stabilisation of the membrane bilayer. It has been demonstrated that changes in membrane fluidity and lateral phase separation can markedly affect the rate of lipid oxidation [4, 18,19]. 

A wide variety of shape transformations of fluid membranes has been extensively studied theoretically in the past two decades using a bending elasticity model proposed by Canham and Helfrich [1]. The model has succeeded in explaining equilibrium shapes of the erythrocyte. However, much attention has recently been paid to shape deformations induced by internal degrees of freedom of membranes. For example, the bending elasticity model cannot explain the deformation from the biconcave shape of the erythrocyte to the crenated one (echinocytosis) [2, 3]. It is pointed out [3] that a local asymmetry in the composition between two halves of the bilayer plays an important role in the crenated shape. It has been observed [4] that a lateral phase separation occurs on an artificial two-component membrane where domains prefer local curvatures depending on the composition. In order to study the shape deformation accompanied by the intramembrane phase separation, we consider a two-component membrane as the simplest case of real biomembranes composed of several kinds of amphiphiles. 

When membranes fuse, the so-called stalk hypothesis suggests that the intermediate hemifusion state (Fig. 6.4c) comprises a structure in which proximal monolayers layers are connected by a bent stalk and the distal layers are pulled towards each other, thus forming a dimple (see also Fig. 6.5) The stalk model has been supported by theoretical and experimental observations. The fusion of model membranes appears to occur via the same series of fusion intermediates as those in vivo, although the approach of membranes is not Rab/SNARE mediated but is driven by reduced bilayer repulsion forces arising from hydration, electrostatic interactions, thermal fluctuations (Helfrich interaction) or osmotic stress. Membrane fusion is also promoted by defects introduced into the membrane by lateral phase separation (for example of lipid rafts, see above), high spontaneous membrane curvature, or addition of macromolecules or proteins into the membrane. 

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