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Direct Chemical Evidence for Sphingolipid Domains in the Plasma

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Direct Chemical Evidence for Sphingolipid Domains in the Plasma Direct chemical evidence for sphingolipid domains PNAS PLUS in the plasma membranes of fibroblasts Jessica F. Frisza,1, Kaiyan Loua,1, Haley A. Klitzinga, William P. Hanafina, Vladimir Lizunovb, Robert L. Wilsona, Kevin J. Carpenterc, Raehyun Kima, Ian D. Hutcheonc, Joshua Zimmerbergb, Peter K. Weberc, and Mary L. Krafta,2 aSchool of Chemical Sciences, University of Illinois, Urbana, IL 61801; bProgram in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and cGlenn T. Seaborg Institute, Lawrence Livermore National Laboratory, Livermore, CA 94551 Edited* by Thomas S. Reese, National Institutes of Health, Bethesda, MD, and approved December 28, 2012 (received for review September 23, 2012) Sphingolipids play important roles in plasma membrane structure To visualize the sphingolipids within the plasma membrane, we and cell signaling. However, their lateral distribution in the plasma isotopically labeled them and then mapped their distributions in membrane is poorly understood. Here we quantitatively analyzed the plasma membrane, using a secondary ion mass spectrometry the sphingolipid organization on the entire dorsal surface of intact (SIMS) instrument (Cameca NanoSIMS 50) with 50-nm lateral cells by mapping the distribution of 15N-enriched ions from metabol- resolution and an analysis depth of <5 nm at the cell surface (14– ically labeled 15N-sphingolipids in the plasma membrane, using high- 16); this analysis depth is less than the thickness of the plasma resolution imaging mass spectrometry. Many types of control experi- membrane. Previous reports establish that NanoSIMS imaging ments (internal, positive, negative, and fixation temperature), along does not alter biomolecule distribution within subcellular com- with parallel experiments involving the imaging of fluorescent sphin- partments or lipid membranes (14, 17–19). Thus, this approach golipids—both in living cells and during fixation of living cells— enabled us to quantitatively determine the sphingolipid organi- exclude potential artifacts. Micrometer-scale sphingolipid patches zation. Sphingolipids were segregated in ∼200-nm diameter mi- consisting of numerous 15N-sphingolipid microdomains with mean crodomains that nonrandomly clustered into sphingolipid-rich diameters of ∼200 nm are always present in the plasma membrane. plasma membrane patches with micrometer-scale dimensions. Depletion of 30% of the cellular cholesterol did not eliminate the The effects of cholesterol depletion and cytoskeleton disruption CHEMISTRY sphingolipid domains, but did reduce their abundance and long- on this sphingolipid organization indicate these sphingolipid range organization in the plasma membrane. In contrast, disruption assemblages are not lipid rafts and thus should at present be re- of the cytoskeleton eliminated the sphingolipid domains. These re- ferred to as sphingolipid domains. sults indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched Results plasma membrane domain that depends upon cortical actin. Metabolic Labeling Achieved a High Level of Molecule-Specific 13C- and 15N-Incorporation. We metabolically labeled Clone 15 cells, SIMS | stable isotope using uniformly 13C-labeled fatty acids and the 15N-sphingolipid precursors, 15N-sphingosine and 15N-sphinganine, such that the CELL BIOLOGY 15 patial organization of the lipids and proteins in the plasma resulting N-sphingolipids (i.e., sphingomyelin, ceramide, and Smembranes of eukaryotic cells is critical to coordinating lipid glycosphingolipids) reached a steady-state distribution in the plasma membrane without 15N-incorporation into proteins (20). signaling and numerous other processes that occur at the cell 13 surface (1). Thus, the hypothesis that lipids self-organize in com- The uniformly C-labeled fatty acids varied in chain length and positionally and functionally distinct plasma membrane domains degree of saturation to promote their incorporation into all sphingolipid and glycerolipid species, presumably producing an has been investigated for decades (2). Microdomains with a variety 13 of lipid compositions and dependencies on lipid and protein even distribution of C-lipids in the cell membrane. This global interactions may exist in the plasma membrane (3–5), but the most labeling of most lipids with carbon-13 allowed evaluation of compelling lipid microdomain is the lipid raft because such plasma membrane integrity and detection of spatially dependent variations in analysis sensitivity or artifactual lipid clustering. domains self-assemble in synthetic membranes (6, 7). Lipid rafts N are defined as dynamic and small (10- to 200-nm) plasma mem- Approximately 80% of the -palmitoyl sphingomyelin, the most abundant cellular sphingolipid we detected, contained one 15N- brane domains that are enriched with cholesterol and sphingoli- 13 ∼ pids (6, 7). Despite intense research, the distributions of most lipid isotope, and C16-palmitic acid was incorporated into 80% of the N-palmitoyl sphingomyelin (Fig. S1). Little (<2%) 15N-en- species, including cholesterol and sphingolipids, in cellular mem- richment of phosphatidylethanolamine due to 15N-sphingolipid branes are still difficult to determine (7). The ability to directly catabolism was detected (SI Methods), so 15N-incorporation into visualize specific lipid species without the use of potentially per- nonsphingolipid species was negligible. Using previously reported turbing tags would help to address outstanding questions regarding methods (21), we determined that ∼60% of the fatty acids in the distinct lipid domains in the plasma membrane (7–9). 13 cellular glycerolipids were uniformly C-labeled. The cells were Here we use a high-resolution chemical imaging approach to address the question: How are sphingolipids distributed in the plasma membranes of intact cells? We focused on sphingolipids i Author contributions: I.D.H., J.Z., P.K.W., and M.L.K. designed research; J.F.F., K.L., H.A.K., because ( ) they are postulated to be segregated within composi- W.P.H., V.L., K.J.C., R.K., P.K.W., and M.L.K. performed research; K.L., R.L.W., R.K., and J.Z. tionally distinct lipid domains, such as lipid rafts (10–12), and (ii) contributed new reagents/analytic tools; J.F.F., H.A.K., V.L., R.L.W., and M.L.K. analyzed sphingolipid metabolites are signaling molecules that regulate cell data; and J.F.F., J.Z., P.K.W., and M.L.K. wrote the paper. survival and proliferation (13). We studied a transfected mouse The authors declare no conflict of interest. fibroblast cell line that stably expresses influenza hemagglutinin *This Direct Submission article had a prearranged editor. (Clone 15 cell line) because the hemagglutinin clusters in their 1J.F.F. and K.L. contributed equally to this work. plasma membranes are thought to be associated with sphingolipid 2To whom correspondence should be addressed. E-mail: [email protected]. and cholesterol domains (10–12). Thus, we hoped to increase the See Author Summary on page 2701 (volume 110, number 8). probability of detecting sphingolipid-enriched plasma membrane This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. domains by using the Clone 15 cell line. 1073/pnas.1216585110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1216585110 PNAS | Published online January 28, 2013 | E613–E622 Downloaded by guest on September 26, 2021 prepared for SIMS analysis by chemical fixation in glutaraldehyde, ological samples (26), the sputtering depth (<2 nm) was much followed by osmication, a procedure known to immobilize lipids less than the plasma membrane’s thickness (7.5 nm) (27), en- because osmium tetroxide acts as a multivalent cross-linker (22). suring that a minimum number of secondary ions were collected This procedure produces a membrane whose organization is re- from the underlying cytoplasm. tained during the inevitable drying process needed for introduction Secondary electron images of the Clone 15 fibroblast cells were into the vacuum of the NanoSIMS analysis chamber (22). Well- collected simultaneously to the secondary ions to allow the isotopic preserved cells with intact microextensions and normal morpholo- data to be related to cell location and morphology (Fig. 1 and Fig. gies were identified by low-voltage scanning electron microscopy S3). The high levels of 15N-isotope incorporation into the cellular (SEM) imaging and subsequently analyzed with NanoSIMS. sphingomyelin allowed the location and density of sphingolipids in the membrane to be mapped with a high signal-to-noise ratio. NanoSIMS Images Show Sphingolipid-Enriched Domains in the Plasma Local elevations in 15N-enrichment were dispersed within a matrix Membrane. The samples were coated with a thin (3-nm) iridium of lower 15N-enrichment on the surfaces of the Clone 15 cells layer that prevents charging during NanoSIMS analysis; prior (Fig. 2 and Fig. S3). Control experiments excluded the possibilities work establishes these metal coatings do not alter the lipid dis- that these local elevations in 15N-enrichment were artifacts in- tribution at the cell surface (23). To visualize the distributions of duced by cell topography (Fig. S2), the iridium coating (Fig. S4), 13C-lipids and 15N-sphingolipids in the plasma membrane, the or isotope-labeled material adsorbed to the cell (Fig. S5). Con- − − − − 12C1H , 13C1H , 12C14N , and 12C15N secondary ions were si- sequently, these local
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