Single-particle tracking studies, which involves observing the motion of a single molecule tagged with nanometer-sized colloidal gold or fluorescent dye , has been used to elucidate the fine structure of biological membranes (Fig. 3.4). Sheets and coworkers used video-enhanced bright field microscopy on 40-nm colloidal gold labeled to antibodies against Thy-1 or cholera toxin B (CtxB) subunit to study the movement of Thy-1 or GM1 respectively, in C3H 10T1/2 fibroblasts . They divided the observed motion of molecules into four categories: fast diffusion; slow diffusion; confined diffusion; and stationary at 6.6 s time scale (200 frames at video rate of 30 frames per second). While significantly higher fraction of Thy-1 and GM1 showed confined diffusion (37% and 35%, respectively), only 16% of fluorescein phosphatidylethanolamine (fl-PE; phospholipid analogue) molecules displayed confined diffusion. Moreover, the treatment of cells with a glycosphingo-lipid inhibitor (which reduced the glycosphingolipid level by ~40%) reduced the fraction of Thy-1 (28%) molecules undergoing confined diffusion. This indicates a role of glycosphingolipids in the reduced mobility of Thy-1. At this timescale of 6.6 s, Thy-1 and GM1 were found in transient confinement zones (TCZs) averaging 325 to 370 nm in diameter. At the 60 s observation window, Thy-1 and GM1 were confined to TCZs for 7-9 s, with an average diameter of between 260 and 330 nm. Interestingly, the extraction with cold Triton X-100 did not affect either fraction of molecules showing confined diffusion or diameter of confined region. The authors concluded that these confined reasons might be in-vivo correlates of detergent-insoluble complexes. The same group conducted a further characterization  and found that the lipid analogues 1,2-dio-leoyl-sn-glycero-3-phosphoe-thanolamine-fluorescein (FL-DOPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoe-thanolamine-fluorescein (FL-DPPE) spent significantly less time in TCZs compared to Thy-1 and GM1. For Thy-1, the confinement times were significantly reduced upon cholesterol depletion. The authors investigated the mobility of Thy-1 within the TCZs at a higher time resolution, by capturing data using a high-speed
Fig. 3.4 Single-molecule tracking of membrane molecules suggests a model of a plasma membrane that resembles a partitioned fluid supported by a cytoskeleton mesh. High-speed tracking of single molecules in cell membranes reveals an unexpected behavior of diffusing particles which suggests the plasma membrane is a fluid that is partitioned by a membrane cytoskeleton fence. Using this technique, Kusumi et al. identified three types of lipidic structures that formed in the plasma membrane . Type (a) is prevalent in the absence of extracellular stimulation; these are small (perhaps consisting of several molecules) and unstable (the lifetimes may be less than 1 ms), and may be the type that monomeric GPI-anchored proteins associate with . Type (b) may appear when receptor molecules form oligomers upon liganding or crosslinking. The receptors may be GPI-anchored receptors or transmembrane receptors with some affinity to cholesterol and saturated alkyl chains. Oligomer-ized receptors may then induce small but stable rafts around them, perhaps due to the slight reduction in the thermal motion around the cluster and the subsequent assembly of cholesterol. Given the rather stable oligomerization of the receptor molecules, the type (b) raft may be stable for minutes, although the associated raft-constituent molecules may be exchanged frequently between the raft and the bulk domains. Type (c) structures may be formed around these receptor rafts. (Image reprinted with permission from .)
digital video camera (500 frames per second for up to 10 s). The diffusion coefficient within TCZs was reduced by a factor of ~2 compared to the one outside of the TCZs. In another study, the diffusion of GPI and transmembrane-anchored forms of neural cell adhesion molecule (NCAM) was studied in NIH-3T3 fibroblasts . Surprisingly, a similar confinement was shown for GPI as well as the transmembrane forms of NCAMs.
Schutz et al. used the single-particle tracking of lipid labeled with fluorescent dye, a method referred to as "single dye tracing" . In this approach, dilute quantities of Cy5-labeled, saturated lipid probe 1,2-dimyristol-sn-glycero-3-phos-phoethanolamine (DMPE-Cy5) and a mono-unsaturated lipid probe DOPE-Cy5 were introduced into human coronary artery smooth muscle (HASM) cells. The single dye tracing data indicated rapid and confined diffusion for DMPE-Cy5 with a long residence time and a confinement area in the order of ~600-700 nm. By contrast, DOPE-Cy5 displayed a relatively unconfined diffusion within the membrane.
Pralle et al. examined the viscous drag of GPI and transmembrane-anchored proteins in regions much smaller than TCZs (as defined by video-based single-particle tracking experiments ). These authors performed high-resolution particle tracking with a bead (labeled with antibody as well as a fluorophore) held by laser trap and bound to membrane protein, and then observed the amplitude of fluctuations of a 2-mm bead in a laser trap of defined spring constant. The amplitude of fluctuations in turn may be related to the local viscosity by the Saffman-Delbruk model of diffusion in two dimensions . The authors reported that raft proteins (by cold detergent-insolubility criterion) such as hemagglutinin (HA), PLAP or chimeric YFPGLGPI (a GPI-anchored protein containing the signal sequence of lactase-phlorizin hydrolase (LPH) fused to yellow fluorescent protein; YFP) experienced an approximately three-fold higher viscous drag than non-raft LYFPGT46 (YFP ectodomain fused to the transmembrane domain of the LDL receptor). Cholesterol depletion resulted in a significant decrease in the viscous drag of HA and GPI-APs, whereas viscous drag of LYFPGT46 was unchanged after cholesterol depletion. By applying the Saffman-Delbruck model for diffusion in biological membranes, Pralle et al. estimated the size of rafts as being ~26 ± 13 nm. However, the application of this theory to membrane diffusion in an heterogeneous environment is fraught with complications, and even if applicable the extremely insensitive relationship between particle size and diffusion coefficient [Diffusion coefficient ~ ln (1/R)] makes the estimation of size inaccurate.
Kusumi et al.  presented a comprehensive review of the various factors that might influence the diffusion of a protein in the plasma membrane (Fig. 3.4). These authors suggested that there might be two different types of raft in the plasma membrane: (1) steady-state rafts or "reserve rafts" that exist at all times; and (2) "clustered rafts", which are more stable long-lived structures that might be created when processes such as signaling are initiated. Kusumi et al. also defined all possible physical interactions that take place between lipids and proteins that would give rise to segregation and, most importantly, associated lifetimes to each of these processes that are critical when designing any type of experiment to detect such structures. For many years Kusumi's group has focused its efforts on understanding the diffusion of proteins in membranes, by using single-particle tracking . This technology allows single particles to be tracked at a time resolution of 25 ms, whereupon most proteins and lipids are seen not to undergo free unrestricted diffusion but rather to diffuse freely into small compartments between 30 and 250 nm in size that are bounded by cytoskeletal "fences". The proteins and lipids cross these fences at a frequency ranging from 1 to 25 ms, depending on the cell type . Such a phenomenon is called "hop diffusion", and the frequency with which molecules hop from one compartment to the other is called the "rate of hop diffusion". Both theoretical models and experiments appear to coincide in this understanding of the structure of the cell membrane where the plasma membrane is actively partitioned by the cytoskeleton that it rests on into membrane skeleton pickets and fences.
When such experiments were performed on GPI-anchored proteins, the latter were found to have a rate of hop diffusion that was indistinguishable from that of phospholipids . Surface scanning resistance (SSR) measurements are performed by coating a bead with antibodies to a receptor on the cell surface, and this bead is held by a laser trap while the stage is scanned in two dimensions . The resistance felt by the bead is reflective of the barriers to diffusion present on the cell surface, and can be used to determine the linkages of the receptor to the cytoskeleton. SSR measurements performed on Qa-2 (GPI-anchored MHC class I) revealed that, using a low concentration of antibodies on the bead, the barriers encountered by the bead were similar to that observed by a monomeric GPI-AP . When a higher concentration of antibody was used, it resulted in cross-linking of Qa-2 and the barriers observed were indicative of an association with a transmembrane protein that has linkage with the actin cytoskeleton. This result was surprising, and questioned most existing models of rafts. The authors interpreted their results by saying that these were the representatives of reserve rafts that consisted of a very small number of GPI-anchored proteins having a hop diffusion rate of 25 ms on average. If the fraction of molecules that resided in aggregated species was small, then in single-particle measurements the probability of labeling those species is also lowered and it becomes difficult to obtain statistically significant data on those events.
As indicated above, by using single-particle tracking, albeit at a lower time resolution, Jacobson and coworkers reported a phenomenon for raft-like molecules. They found that GPI-anchored proteins - but not other transmembrane proteins -underwent slow diffusion in small zones that were 200 nm in size [36,38, 86], called TCZs. Kusumi and coworkers clarified that these TCZs could not be compared to the membrane compartments that they observed because the time resolution of the two techniques was very different . It is also possible that due to the inability to have single antibody per gold particle, Jacobson and co-workers might have observed diffusion of small clustered molecules. The issue of multi-valency of the tagging particle remains a matter of great concern in the correct interpretation of results from these techniques .
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