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 15 N-enriched ions from metabolically labeled 15 N-sphingolipids in the plasma membrane, using highresolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids-both in living cells and during fixation of living cellsexclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15 N-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.SIMS | stable isotope

Background: Although cholesterol abundance affects cell function, its distribution within the plasma membrane is not established.Results: Cholesterol is uniformly distributed throughout the plasma membrane and is not enriched within sphingolipid domains.Conclusion: Sphingolipid organization in the plasma membrane is not dictated by direct cholesterol-sphingolipid interactions.Significance: Cholesterol abundance affects sphingolipid organization in the plasma membrane via an indirect mechanism.

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The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.

Characterization of the distributions of specific proteins and lipids within cellular membranes is currently a major challenge. Advances in secondary ion mass spectrometry (SIMS) now enable the distributions of isotopically labeled lipids within cellular or model membranes to be imaged with chemical specificity and high (≥50 nm) lateral resolution. Here, methods to image the distributions of sphingolipids within the membranes of intact cells with a Cameca NanoSIMS are described. For NanoSIMS detection, the incorporation of distinct stable isotopes into the lipid species of interest is essential. Metabolic labeling, cell preservation, imaging conditions, and data analysis are critical factors. The methods and principles described here can be extended to studying other membrane lipids or cholesterol.