C the lipid composition of the low density insoluble

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C. The lipid composition of the low-density, insoluble form of PLAP is just what you would expect for a lipid raft, which is enriched in sphingolipids and cholesterol and depleted for phospholipids (other than sphingomyelin). Indeed, it was experiments such as this one that established the composi- tion of lipid rafts. It remains controversial whether these experiments give information about microdomains in membranes or are artifactual results due to the method of extraction (ice-cold Triton X-100). D. The surface area of the smallest vesicles (100 nm diameter) is a little over 30,000 nm 2 [4 ¥ 3.14 ¥ (50 2 )], whereas that of a lipid raft (70 nm diameter) is a little under 4000 nm 2 [3.14 ¥ (35 2 )]. Although the presence of PLAP in the vesicles would be expected if they were derived from lipid rafts, the differ- ence in surface area is troubling because it suggests that multiple lipid rafts may have coalesced during treatment with ice-cold Triton X-100. That pos- sibility is hard to distinguish from the coalescence of well-dispersed lipids and PLAP during treatment with Triton X-100. It is this possibility of artifact that has spurred attempts to demonstrate lipid rafts directly in the plasma membranes of living cells. Reference: Brown DA & Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544. 10–31 A. For randomly dispersed receptors (see Figure 10–6A), the polarization of the fluorescent light will depend critically on the concentration of the receptors in the membrane. At high density (pixels with high-intensity fluorescence) there will be efficient FRET (high-intensity fluorescence detected by the per- pendicular filter), giving rise to a low value for polarization of the fluores- cence [( I par I perp )/( I par + I perp )]. By contrast, at low density the overall inten- sity will be lower, but FRET will be much less efficient since molecules are on average farther away from one another. As a result, what fluorescence there is will be more polarized. For receptors that are confined to microdomains such as lipid rafts (see Figure 10–6B), the overall fluorescence intensity will decrease with decreas- ing density of the rafts, which is determined just by chance distribution of rafts relative to the very small window (a pixel) being examined. The polar- ization of the fluorescence, however, will be independent of concentration. At high density and at low density of rafts, the receptors in microdomains will always be equally close to their neighbors; thus, a constant proportion of the absorbed light energy will be transferred by FRET. As a result, recep- tors in microdomains will give the same low value for polarization of fluo- rescence, regardless of the total fluorescence intensity in a pixel.
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