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.

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein’s activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests of whether specific proteins co-localize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein co-localization with specific lipid species.

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.

Principal component analysis (PCA) of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data enables differentiating structurally similar molecules according to linear combinations of multiple peaks in their spectra. However, in order to use PCA to correctly identify variations in lipid composition between samples, the discrimination achieved must be based on chemical differences that are related to the lipid species, and not sample-associated contamination. Here, we identify the positive-ion TOF-SIMS peaks that are related to phosphatidylcholine lipid headgroups and tail groups by PCA of spectra acquired from lipid isotopologs. We demonstrate that restricting PCA to a contaminant-free lipid-related peak set reduces the variability in the spectra acquired from lipid samples that is due to contaminants, which enhanced differentiating different lipid standards, but adversely affected the contrast in PC scores images of phase-separated lipid membranes. We also show that PCA of a restricted data set consisting of the peaks related to lipids and amino acids increases the likelihood that the discrimination of TOF-SIMS data acquired from intact cells is based on differences in the lipids and proteins on the cell surface, and not sample-specific contamination without compromising sample discrimination. We expect that the lipid-related peak database established herein will facilitate interpreting the TOF-SIMS data and PCA results from studies of both model and cellular membranes, and enhance identifying the origins of the peaks that contribute to discriminating different types of cells.