eins are present in all parts of the membrane, but some integral membrane For in
ID: 205901 • Letter: E
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eins are present in all parts of the membrane, but some integral membrane For instance, the Prot proteins seem to be associated with lipid rafts in particular cell types. protein, linker for activation of T-cells (LAT) is associated with rafts in cera populations. This change in lipid:protein ratio leads to a during ultracentrifugation at 280,000 x g following disruption of the ce detergents. In th in change in the mobility of lipid rafts ese experiments, lipid rafts are located in more buoyant fractions (higher erence in density compared to other membrane the centrifuge tube) indicating a diff regions (located to the bottom of the tube) In the followi lipid raft formation. They applied col D (CD; ultracentrifugation as described above. Fractions were numbered from 1 centrifuge tube) to 12 (bottom of the centrifuge tube). A western blot was performed to ng experiments, investigators looked at the role of cytoskeletal proteins in chicin (Colch; a microtubule inhibitor) or cytochalsin- an actin filament inhibitor) to T-cells and then isolated lipids using detergent and (top of the he presence of LAT in separated lipid fractions. LAT is a protein "marker" for lipid control (untreated cells), LAT is found in buoyant fractions, raft formation in T-cells. In the which is indicative of lipid raft formation. A) Control LAT 4 5 6 7 89 10 11 12 B) Colch 4 5 6 7 8 9 10 11 12 C) CD LAT Modified from Head et al. (2006)J Biol Chem 4 5 6 7 89 10 11 12Explanation / Answer
Answer 5). The hypothesis being tested here is role of cytoskeleton proteins in formation of lipid rafts. LAT is used as lipid raft marker protein in this set of experiment.
Answer 6). In this experiment lipid rafts are located in more buoyant fraction in centrifugation tube, and in control experiment these fraction are in lane 4-5. After treatment with colch or CD, The LAT is not present in same fraction in western blots. This indicate that LAT is no longer associated with more buoyant fractions.
Answer 7). The disruption of cytoskeleton inhibits lipid raft formation as indicated in western blots conclude that
Answer 8). a) Apply cytoskeletal inhibitors and look for the presence of LAT in buoyant fractions that would clarify that LAT localization to lipid rafts depend upon intact cytoskeletal network.
b) Apply cytoskeletal inhibitors and look for the presence of another raft marker in buoyant fractions which would clarify that Lipid raft formation depends upon intact cytoskeletal network.
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