FLUORESCENGE METHODS TO STUDY MEMBRANE ORGANIZATION 335, different glycosylphosphatidylinositol-anchored green fluorescent proteins (GPI-GFPs) by acceptor emission, between different lipid-anchored proteins attached to the inner leaflet of the plasma membrane, and between different GPI-anchored proteins and the sphingolipid GM1 labeled with CTB by increased donor intensity after acceptor bleaching and homotransfer between GPI-anchored proteins detected by fluorescence anisotropy. The results of these studies are seemingly contradictory with some con- cluding that raft proteins are randomly distributed and others suggesting that they are clustered. A recent study by Sharma et al. combined theoretical modeling with both homo- and heterotransfer FRET between GPl-anchored proteins detected by time-resolved imaging [22]. They concluded that the majority of GPI-anchored pro- teins are present as a monomer with a minor fraction (20-40%) being localized in small, cholesterol-dependent domains, Differences in membrane anchorage, cell type, and activation status may account for some of the observed discrepancies between studies. However, the important contribution of these FRET studies to our view of cell membrane organization should not be underestimated. Studies of this type lead to the consensus that lipid rafts are small (10-200 nm) and highly dynamic. 16.2.5 Total Internal Reflection Fluorescence Microscopy The fluorescence approaches discussed so far all suffer from a lack of axial resolution; therefore any illumination spot or pixel contains signals from the plasma membrane as well as the cytoplasm. ‘The use of total internal reflection fluorescence microscopy (TIRFM) partially overcomes this limitation. The excitation light in TIRFM is directed to the glass—sample interface at an angle that is greater than the critical angle and thus reflected back by the glass and is unable to penetrate the sample. This creates an evanescent field, that is, a nonpropagating, exponentially decaying wave of the same wavelength as the reflected light that is used to excite fluorophores. Because of the exponential decay of the evanescent field, only fluorophores within ~100 nm of the glass interface are excited. Hence, if cells are plated onto glass surfaces, the fluo- rophores in the cell body are not excited, resulting in an excellent signal-to-noise ratio of membrane-associated fluorescence. Because of the enhanced contrast and unprece- dented axial resolution, studies of membrane organization combine the use of TIRFM with single-particle tracking. 16.2.6 Single-Particle Tracking Single-particle tacking (SPT) is the ability to record the trajectories of individual molecules within the plane of the membrane. The molecule of interest is labeled with a probe, such 2s a fluorescent moiety, bead, or gold particle, and the location of the probe, defined by the centroid of the imaged diffraction pattern, is recorded in time, By plotting the position of the probe from all time points in a single graph, the trajectory of the probe's movement can be visualized. The mean-square displacement (MSD) versus time provides a measure of the average distance the tagged molecule travels in a given time window and can illustrate nonrandom modes of motion, such as confinement. Analysis of SPT trajectories and MSD graphs gives information about diffusion coeffi- cients, particle confinement, and the size of confinement areas, for example, regions in which the observed particle resides for a brief period of time and the residence times, or lifetimes, in confinement areas. One of the great challenges of SPT is to ensure that336 MEMBRANE ORGANIZATION just a single molecule is observed at a time. In other words, the probe attached to the molecule of interest has to be strictly monovalent, In addition, the size of the bead or particle can also influence the movement of the molecules. The relative small size of fiuorophores compared to beads and particles offers a distinct advantage. SPT has thus been applied to fluorescent lipid analogues, GFP fusion proteins, and monovalent Fab antibody fragments conjugated to fluorescent dyes and is referred to as single molecu- lar fluorescence tracking (SMFT). SPT and SMET have the ability to monitor complex movements of proteins or lipids, one molecule at a time, revealing details often missed using other approaches such as FRAP or ICS. For example, temporal confinement to a membrane domain for a short period of time can only be directly observed with SPT. Herein also lies the disadvantage—because only a few molecules are observed in each experiments, this approach is time consuming. The group led by Kusumi has revolutionized SPT by introducing sampling frequencies of 40,000 Hz equivalent to a spatial resolution of ~20 nm [18]. Tracking a large range of lipids and proteins, the group identified confinement areas of 30-230 nm with residence times of 1~25 ms for phospholipids and 3 ms~1 s for transmembrane proteins, Within each confinement area, molecules could diffuse freely whereas long-range diffusion was significantly lower because of the transition barriers that dictated confinement. These compartments were found to be dependent on the actin membrane skeleton but not on the cholesterol levels in the membrane. These observations led Kusumi to propose the picket-fence model of membrane organization (see above) Clustering of proteins, for example, with multivalent particles, greatly reduces the hop rate. Clusters of approximately six CD59 molecules, a GPI-anchored receptor, undergo frequent but transient immobilization [27]. Using simultaneous single-molecule tracking of two proteins, the authors demonstrated that CD59 clusters recruit other proteins via both protein-protein and lipid-lipid (raft) interactions and thus induce signaling. The temporal arrest of the cluster is possibly mediated by transient anchorage to the actin cytoskeleton mediated by the recruited kinases. Previously, Jacobson and co-workers observed transient confinement zones (TCZs) also using single-particle tracking [12]. TCZs are distinctly different from the corrals defined by the picket-fence model [18] and resemble more closely the transient immobilization of CD59 clusiers. SPT data have made an important contribution to our understanding of membrane organization, as it is the only technique to date able to identify the complex diffusion of proteins and lipids and the temporal variations in diffusion, It allows membrane biol- ogists to distinguish between membrane domains in native, unstimulated membranes and activated membranes that contained ligated receptors and crosslinked proteins. As discussed in Section 16.4.3, protein clusters can induce the formation of membrane domains that haye distinct biochemical and biophysical properties. 16.3 MODEL MEMBRANES The proposal of the existence of lipid-based membrane domains, particularly lipid rafts in biological membranes, has triggered a dramatic rise in biophysical studies explor- ing lateral heterogeneity in synthetic membranes, Artificial membranes can be created as supported bilayers and monolayers that form on flat surfaces such as glass slides. To alleviate the effect of the support on lipid behavior, complementary studies areREFERENCES 347 the membrane at the activation sites condensed to form highly ordered domains—a hallmark of lipid raft accumulation, This receptor-mediated membrane condensation required Lek kinase activation as well as LAT expression and phosphorylation. 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