T lymphocytes or T cells are involved primarily in controlling cell-mediated immune reactions, having the ability to recognize specific peptide antigens through the receptors on their cell surface. The association of various lipids and proteins, such as T cell receptors (TCR), in the cell lipid bilayer is important in the signaling pathway of the T cell. Aggregating the TCR complexes is necessary for activation of the cell, which causes the complexes to associate with specialized parts of the plasma membrane. These regions have been labeled as detergent resistant membranes (DRM), as they remain intact after being treated with a mild detergent. The DRM are rich in glycolipids, cholesterol, and signaling proteins that are thought to act as rafting devices that allow this signaling to occur. These lipid rafts are also hypothesized to associate with cytoskeletal proteins during activation.
In this project, the lipids in Jurkat T cells will be analyzed. First all the lipids will be extracted from whole cells with organic solvents (methanol: chloroform). The different classes of lipids will be separated and then analyzed by TLC. To activate the T cells an anti-TCR antibody will be used to aggregate the TCR complexes. A mild detergent will then be added to break up the lipids in the membranes of the activated and regular cells. We will then separate the soluble and involuble fractions, extract lipids from them, and analyze by TLC. The lipid classes and proportions will be determined for the soluble and insoluble cell extracts for both the regular and activated cells to see if raft components become associated with the insoluble part after activation. A sucrose density gradient will be performed to further separate the components, and a TLC will be used to analyze the lipid components of each fraction.