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- Mahammad, S, et al.
(författare)
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Limited cholesterol depletion induces T cell activation and increases the plasma membrane fraction of higher order leading to clustering of signaling molecules
- 2009
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Ingår i: The 49th Annual American Society of Cell Biology Meeting, San Diego, December 2009. ; , s. 2562/M-L20-
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The plasma membrane of eukaryotic cells contains nanodomains known as lipid rafts. Cholesterol depletion is a widely used technique for studying lipid rafts and their involvement in cellular processes. Cholesterol depletion has been reported to cause both increased and abolished T cell signaling. The abolished cell signaling upon cholesterol depletion is likely to be caused by substantial cell death as demonstrated by cell viability measurements. We have investigated how cholesterol depletion alters T cell activation by analyzing Jurkat T cells upon extraction of 10, 20, 30, 40 and 50% of total cholesterol using methyl β cyclodextrin (MBCD), a protocol in which cholesterol depletion does not have any adverse effect on cell viability.Upon cholesterol depletion peripheral actin polymerization and aggregation of the lipid raft marker GM1 in the plasma membrane is observed. The aggregation of GM1 upon cholesterol depletion is dependent on signaling protein Lck. The aggregated GM1 domains colocalize with signaling proteins such as Lck and LAT. To confirm that the effects seen by cholesterol depletion using cyclodextrin are actually due to cholesterol depletion and not cyclodextrin treatment itself, control experiments having Jurkat T cells treated with MBCD-cholesterol complexes to keep the cellular cholesterol content at equilibrium. A larger fraction of ordered (lo) plasma membrane is observed upon cholesterol depletion, a study performed by using laurdan. A relative membrane order is given by normalized ratio of the two emission regions termed as general polarization (GP). GP is defined analogously to fluorescence polarization by measuring the intensities (I) between 385 and 470 nm and 480 and 508 nm. Change in the membrane order and increased peripheral actin polymerization indicates that actin polymerization is in correlation to the formation of liquid ordered (lo) domains in the plasma membrane upon cholesterol depletion. Our results conclude that limited cholesterol depletion leads to T cell activation and an increase in the amount of liquid ordered domains in the plasma membrane. This activation is followed by aggregation of GM1 enriched domains.
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