Visualizing lipid rafts in activated T cells

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, are a discrete compartment within the plasma membrane that is co-enriched with GPI-anchored proteins and other, often lipid-modified, proteins. Many of the proteins that reside in GEM domains function in cell signaling. Consequently, it has been postulated that GEM domains represent a signaling compartment within cell membranes. Importantly, recent studies have substantiated this hypothesis, including the observation that GEM domains are enriched in membrane caps and immune synapses in stimulated T cells. Based on this observation, it was hypothesized that GEM domains could serve as a vehicle for targeting molecules to the T cell receptor (TcR) during cell stimulation. To test this hypothesis, green fluorescent protein (GFP) was targeted to GEM domains by fusing it to the membrane-anchoring domain of p56^lck (amino acids 1 through 10) (L10-GFP). The construct was expressed in D10 T cells, and transfected cells were stimulated using conalbumin-pulsed CH27 cells. Using confocal microscopy, it was determined that GFP became enriched in the contact zone between the D10 and CH27 cells. Thus, targeting GFP to GEM domains effectively targeted it to immune synapses. Since L10-GFP lacks the CD4-binding domain of Lck, it is concluded that GEM-association alone is sufficient for targeting signaling proteins such as Lck to the site of TcR engagement.

In a second set of experiments, L10-GFP was expressed in CH27 cells that were used for antigen presentation. Intriguingly, it was found that L10-GFP became enriched in the B cell membrane at the immune synapse. Furthermore, targeting of GFP to the non-GEM membrane compartment using the membrane-anchoring signal of pp60^c-Src showed that enrichment at the immune synapse was specific for the GEM compartment of the plasma membrane. Future experiments will determine the role of aggregation of GEM domains in the B cell membrane during antigen presentation by assaying the effect of disrupting GEM domains on T cell stimulation.