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.

The ability to self-renew and differentiate into multiple types of blood and immune cells renders hematopoietic stem and progenitor cells (HSPCs) valuable for clinical treatment of hematopoietic pathologies and as models of stem cell differentiation for tissue engineering applications. To study directed HSC differentiation and identify the conditions that recreate the native bone marrow environment, combinatorial biomaterials that exhibit lateral variations in chemical and mechanical properties are employed. New experimental approaches are needed to facilitate correlating cell differentiation stage with location in the culture system. We demonstrate that multivariate analysis of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data can be used to identify the differentiation state of individual hematopoietic cells (HCs) isolated from mouse bone marrow. Here, we identify primary HCs from three distinct stages of B cell lymphopoiesis at the single cell level: HSPCs, common lymphoid progenitors, and mature B cells. The differentiation state of individual HCs in a test set could be identified with a partial least squares discriminant analysis (PLS-DA) model that was constructed with calibration spectra from HCs of known differentiation status. The lowest error of identification was obtained when the intra-population spectral variation between the cells in the calibration and test sets was minimized. This approach complements the traditional methods that are used to identify HC differentiation stage. Further, the ability to gather mass spectrometry data from single HSCs cultured on graded biomaterial substrates may provide significant new insight into how HSPCs respond to extrinsic cues as well as the molecular changes that occur during cell differentiation.

Lipid monolayers of L-α-dipalmitoylphosphatidylcholine (DPPC) are used to pattern substrates using the Langmuir-Blodgett (LB) technique. Lipid monolayers are deposited onto a freshly cleaved mica surface or glass capillary under conditions that lead to distinct patterns in the film. Exposure of the supported monolayer to ethyl 2-cyanoacrylate fumes leads to preferential polymerization in the more hydrated regions of the patterned monolayer. This method enables surfaces to be micropatterned where the lateral features are controlled by the structure present in the underlying LB film and the vertical feature size is controlled by the length of the fuming process. Atomic force microscopy (AFM) measurements confirm that the original structure in the LB film is preserved following fuming and that the lateral and vertical feature sizes can be controlled from nanometers to microns. This method, therefore, provides a rapid and versatile approach for micropatterning both flat and curved surfaces on a variety of substrates.

The plasma membrane is a selectively permeable lipid bilayer that separates cells from their surroundings. Numerous different lipid species, cholesterol, and a variety of different proteins form the plasma membranes of mammalian cells. One class of lipids, the sphingolipids, and their metabolites serve both as structural components in the plasma membranes of mammalian cells, and as bioactive signaling molecules that modulate fundamental cellular processes. Though segregation of the sphingolipids into distinct membrane domains is likely essential for cellular function, the sphingolipid distribution within the plasma membrane and the mechanisms that regulate it are poorly understood.To overcome these disadvantages, we pioneered the use of high-resolution SIMS, performed with a Cameca NanoSIMS 50, for directly imaging metabolically incorporated, stable isotope-labeled lipids in actual cell membranes. The NanoSIMS 50 is a state-of-the-art magnetic sector secondary ion mass spectrometer that can image the elemental and isotopic composition at the surface (top 5 nm) of a sample with high sensitivity and as high as 50-nm-lateral resolution. In order to visualize the cholesterol and sphingolipids in the plasma membrane, we used metabolic labeling with 15 N-sphingolipid precursors ( 15 N-sphingosine and 15 N-sphinganine) and 18O-cholesterol to selectively incorporate distinct stable isotopes, 15 N and 18 O, into the sphingolipids and cholesterol, respectively, in living mouse fibroblast cells. Then we chemically fixed the cells with a method that does not alter membrane lipid distribution [1,2]. Well-preserved cells with normal morphologies are identified by imaging with low voltage secondary electron microscopy (SEM). To increase secondary ion yields, the cell samples are coated with a thin (3-nm) iridium layer that does not alter the lipid distribution on the cells. Then we used a Cameca NanoSIMS 50 instrument to map the lipid-specific isotope enrichments on their surfaces better than 100-nm-lateral resolution.Using this approach, we have previously shown that the 15 N-sphingolipids are enriched within distinct domains in the plasma membranes of fibroblast cells [2]. Here we report how we have used this approach to probe the mechanisms responsible for this sphingolipid organization. We investigated whether the sphingolipid domains are dependent on cholesterol-sphingolipid interactions, or the diffusion barriers that are established by the cytoskeleton and its associated membrane proteins by using SIMS to image the effects of cholesterol depletion and actin depolymerisation on 15 N-sphingolipid distribution in the plasma membrane [2,3]. We also assessed whether these 15 N-sphingolipid domains are co-localized with hemagglutinin, a specific membrane protein that is thought to have an affinity for sphingolipid-enriched membrane domains. Our results indicate that the sphingolipid organizations in