Short chain ceramides disrupt immunoreceptor signaling by inhibiting segregation of Lo from Ld plasma membrane components

Lipid phase heterogeneity in plasma membranes is thought to play a key role in targeting cellular signaling, but efforts to test lipid raft and related hypotheses are limited by the spatially dynamic nature of these phase-based structures in cells and by experimental characterization tools. We suggest that perturbation of plasma membrane structure by lipid derivatives offers a general method for assessing functional roles for ordered lipid regions in membrane and cell biology. We previously reported that short chain ceramides with either C2 or C6 acyl chains inhibit antigen-stimulated Ca2+ mobilization (Gidwani et al., 2003). We now show that these short chain ceramides inhibit liquid order (Lo)-liquid disorder (Ld) phase separation in giant plasma membrane vesicles that normally occurs at low temperatures. Furthermore, they are effective inhibitors of tyrosine phosphorylation stimulated by antigen, as well as store-operated Ca2+ entry. In Jurkat T cells, C6-ceramide is also effective at inhibiting Ca2+ mobilization stimulated by either anti-TCR or thapsigargin, consistent with the view that these short chain ceramides effectively interfere with functional responses that depend on ordered lipid regions in the plasma membrane.