Size Of Lipid Rafts Research Articles

Plasma Membrane Organization

Plasma membrane organization The composition of the plasma membrane has long been modeled as a mosaic fluid (Singer and Nicolson, 1972). Studies in the last few years have identified microdomains, lipid rafts and caveolae, that constrain membrane proteins within a small region of the cellular plasmamembrane. These domains facilitate anchoring of different signaling proteins for example H-Ras that has been shown to co-localize with lipid rafts upon activation (Lommerse et al. 2005, Rotblat et al. 2004). The signaling cascade on the plasma membrane of cells is dependent on the location of different membrane proteins. Therefore it is of interest to further investigate these domains. Photo Activated Localization Microscopy (PALM) achieves sub-diffraction limited resolution by imaging single fluorophores and determining their position at high precision. By using photoactivatable fluorescent proteins the amount of fluorescing molecules can be altered by variation of the intensity of the activation beam. This way single molecule fluorescence microscopy is achieved. Dendra2 is a fluorescent protein thatwas photoconverted from a green to a red fluorescent state using a 405nm light-puls. For this research 3T3-cells have been transfected to express H-Ras-Dendra2. Using Photo Activated Localization Microscopy (PALM) the position of H-Ras was determined with a precision of 30 nm. By analyzing the trajectories of single molecules of H-Ras we showed that a significant percentage of H-Ras molecules was confined within small (∼200 nm) domains. Choleratoxin B (CtxB) is a ligand to the ganglioside GM1 which has been used as a marker for lipid rafts at the outside of the lipid membrane. Since it binds to five GM1 molecules it was suggested that CtxB enlarges the size of lipid rafts. Here we have treated cells with CtxB that indeed enlarged both the outside and inside lipid raft and therefore the confinement of H-Ras.

Docosahexaenoic Acid Modifies the Clustering and Size of Lipid Rafts and the Lateral Organization and Surface Expression of MHC Class I of EL4 Cells

An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

Single-Molecule Diffusion Reveals Similar Mobility for the Lck, H-Ras, and K-Ras Membrane Anchors

Recent evidence on the occurrence of small (5–700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these “lipid rafts”, in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.

Rafts and the evil amyloids

Lipid rafts are bad news for those with Alzheimer's disease (AD). On page 113, Ehehalt et al. show that formation of the β-amyloid peptide (Aβ), which is tightly linked to AD, depends on the raft association of one of its creators. Figure APP (green, bottom) meets β-secretase (green, top) in lipid rafts (red). The creating enzyme is β-secretase, which cleaves the amyloid precursor protein (APP) to release a product that is then processed into Aβ. Several recent lines of evidence suggest that cholesterol is somehow linked to Aβ production. For instance, high cholesterol levels are correlated with an increased likelihood of developing AD. As cholesterol is found in membrane lipid rafts, the authors investigated whether APP and β-secretase were linked with these compartments. They found that, indeed, both proteins were found in lipid rafts. Further increasing the fraction of APP and β-secretase in lipid rafts (by oligomerizing each protein) released more Aβ. The small size of lipid rafts makes it unlikely that both proteins are found within the same raft. The group demonstrates, however, that endocytosis is necessary for Aβ formation. Thus, endocytosis may lead to a clustering of rafts that puts β-secretase within striking distance of APP. The regulation and mechanism of this clustering await further studies. Decreasing cholesterol levels in cells limited Aβ secretion, but how cholesterol is involved is also not yet clear. Perhaps specific lipids are required to activate β-secretase. The authors plan to use purified secretase and APP to determine whether raft lipids are needed for processing. Prion scrapies are also formed in lipid rafts, so rafts seem to be conducive to the formation of amyloids. Potential therapeutic drugs should therefore function within raft environments if they are to be successful at preventing amyloid formation. ▪

Sphingolipid–Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.