Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of ¹⁵N-enriched ions from metabolically labeled ¹⁵N- sphingolipids in the plasmamembrane, using highresolution imagingmass spectrometry.Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involvingthe imaging offluorescent sphingolipids-both in living cells and during fixation of living cells-exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous ¹⁵N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and longrange organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.