Polyene-lipids: A new tool to image lipids

Lars Kuerschner¹, Christer S Ejsing¹, Kim Ekroos², Andrej Shevchenko¹, Kurt I Anderson¹ & Christoph Thiele¹

¹ Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.

² AstraZeneca R&D, 43183 Moelndal, Sweden.

Figure 1. A versatile procedure to introduce conjugated pentaene tags into lipids. The precursors, octatrienal (1) and decatetraenal (2), were constructed by repeated rounds of Wittig reactions. Cis- and trans-pentaene−fatty acids20, (3) and (4), respectively, were obtained by a final Wittig reaction with a triphenylphosphonium salt of a w-bromocarboxylic acid. The -9 double bond can be isomerized from cis (3) to trans (4) configuration. Fatty acids were activated and coupled to sphingosine or sphingosylphosphorylcholine to give pentaene-ceramide (5) or pentaene-SM (6), respectively. A Wittig reagent derived from a protected sn-1-O-alkylglycerol (7) couples to aldehyde (2) to give pentaene-alkylglycerol (8) after deprotection. Note that molecules (3) and (4) are the c and t 16:5-fatty acids, respectively, when N = 1 and c and t 18:5−fatty acids when N = 2. Full Figure and legend (17K)

Figure 2. Analysis of phase partitioning of fluorescent SM analogs. (a,b) For a fluorescence quenching assay, series of liposomes comprising a liquid-disordered phase only (a), or coexisting liquid-ordered and liquid-disordered phases (b) were prepared containing variable amounts of a quencher lipid, 1-palmitoyl-2-stearoyl-(12-DOXYL)-PC, and traces of the fluorescent SM analogue. The quencher lipid prefers the liquid-disordered (L-) phase. Normalized fluorescence (F/Fo)cor is plotted as a function of the quencher concentration. Data are mean range, n = 2 for open symbols, or mean s.d., n = 4 for closed symbols. Error bars smaller than symbol size were omitted. Note the difference between polyene-SMs and BODIPY-SMs in b, suggesting a preference of polyene-SMs for the liquid-ordered (raft) phase. DOPC, dioleoylphosphatidycholine. (c) Partitioning of fluorescent SM analogs into detergent-resistant membranes (DRMs) of COS7 cells. Cells were incubated with NBD-, BODIPY-, or polyene-ceramide prior to detergent solubilization with Lubrol-WX on ice. DRMs were floated on a linear sucrose gradient and fractions were analyzed for cell-derived SMs by thin-layer chromatography (TLC). Plates were photographed under UV illumination to visualize fluorescent SMs, followed by sulfuric acid charring to visualize total SMs and PCs. DRMs are found in fractions 3−5, solubilized material in fractions 6−8. Full Figure and legend (44K)

We fed 5 M concentrations of polyene−fatty acids to COS7 cells for 20 h and analyzed fluorescent metabolic products by TLC. We found that c/t16:5−fatty acids were incorporated mainly in PC, and longer ones (c/t18:5 and c/t22:5) were also incorporated into other phospholipids (Fig. 3a). Similar metabolism, apart from reduced SM labeling, was observed for radioactively-labeled natural fatty acids (Fig. 3b), demonstrating that polyene−fatty acids resemble natural fatty acids of comparable length with respect to utilization by the lipid biosynthetic enzymes.

Figure 3. Comparison of lipid metabolites of polyene-labeled or radioactive fatty acids in COS7 cells. Cells were incubated with 5 M of the respective fatty acid for 20 h. Lipids were extracted and analyzed by TLC for fluorescent (a), or radioactive (b) metabolites, that were identified by comigrating lipid standards. Abbreviations are: CE, cholesterol ester; Cer, ceramide; DG, diglyceride; FA, fatty acid; ori, origin of application; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TG, triglyceride. Full Figure and legend (11K)

Since polyene−fatty acids can be used in diverse cell types, we fed 50 M t16:5−fatty acid to kidney epithelial cells (COS7, Fig. 4a), adipocytes (3T3-L1, Fig. 4b) and muscle cells (C2C12, Fig. 4c), all of which incorporated the polyene−fatty acid into various lipids. Different labeling times for different cell lines were necessary to incorporate sufficient polyene-lipid for high-quality imaging. High labeling selectivity, supporting unequivocal interpretation of imaging data, was achieved at lower concentrations for incubation times of a few hours. COS7 cells incubated with 3 M t16:5−fatty acid for 2 h showed a prominent labeling of PC (Fig. 4d). When fed with c16:5-monoalkyl-1-glycerol, a polyene precursor for ether lipids, COS7 cells produced labeled ether lipids (Fig. 4e). Prominently-labeled classes were ether-PC and ether-PE, which are also known to contain the majority of natural ether lipids²⁵. To label sphingolipids, we fed polyene-ceramide complexed to -cyclodextrin²⁶ at final concentrations of 0.15−10 M for 2-4 h to COS7 cells, which resulted in uptake and conversion to polyene-SM (Fig. 4f). As a -cyclodextrin complex, synthetic polyene-SM was also efficiently transferred to the cells (data not shown).

Figure 4. Lipid metabolites of polyene−fatty acid in different cell types. (a−c) COS7 kidney epithelial cells (a), 3T3-L1 adipocytes (b), and C2C12 muscle cells (c) were incubated with 50 M t16:5−fatty acid for 30 min (a), 15 min (b) or 1 h (c).(d−f) Lipid metabolites of different polyene lipid precursors in COS7 cells. Cells were incubated with 3 M t16:5−fatty acid (d), c16:5-monoalkyl-1-glycerol (e) or c18:5-ceramide (f) for 2 h (d,e) or 4 h (f). Lipids were extracted and analyzed by TLC for fluorescent metabolites, which were identified by comigrating lipid standards. Abbreviations as in Figure 3. Full Figure and legend (15K)

Saturated and unsaturated fatty acids display different preferences for the SN1 and SN2 positions in phospholipids. The preferences of polyene−fatty acids were analyzed by mass spectrometry. Lipid extracts of cells labeled with either t16:5− or c16:5−fatty acids were analyzed by precursor ion scanning for t/c16:5-containing species. Major species found were PCs containing 14:0−, 16:0−, 16:1− and 18:1−fatty acids and 16:0-ether−PC (Supplementary Fig. 1 and Supplementary Table 1 online). Analysis for positional preference²⁷ showed both t16:5− and c16:5−fatty acids predominantly at SN2 positions when combined with 14:0−, 16:0− and 18:1−fatty acids. In combination with palmitoleic acid, t16:5−fatty acid was preferentially located at the SN1 position, but c16:5−fatty acid was equally distributed (Supplementary Table 1 online). Taking into account the relative amounts of labeled species, the majority of both 16:5−fatty acids was found at the SN2 position. Further fatty acid profiling showed that there was no metabolism of 16:5− to 16:4− or 18:5−fatty acids (data not shown). To detect effects of c/t16:5−fatty acids on unlabeled phospholipids, precursor ion scans for lipids containing 16:0−, 16:1−, 18:0−, 18:1−, 18:2−, 20:4− and 22:5−fatty acids were performed. No relevant differences between control cells and labeled cells were found (data not shown). Quantitative analysis revealed that labeling with 3 M fatty acid for several hours or with 50 M fatty acid for several minutes resulted in labeling of 0.5−2% of the total of the respective lipid class (data not shown), which seems unlikely to severely perturb membrane organization. Therefore, we used these concentrations for in vivo imaging of COS7 cells.

For live-cell imaging of polyene-lipids, photodestruction of the polyene fluorophore was substantially reduced by using two-photon excitation²⁸, which allowed sequential acquisition of up to 50 images. The distribution of polyene-lipids in kidney epithelial cells (COS7, Fig. 5a), adipocytes (3T3-L1, Fig. 5b) and muscle cells (C2C12, Fig. 5c) was imaged, and we observed labeling of the nuclear envelope, lipid droplets and both endoplasmic and sarcoplasmic reticula. To accurately identify labeled cellular structures, we generated fusion proteins of mRFP1²⁹ as organellar markers. Colocalization studies of polyene-lipids and these markers in living COS7 cells were performed (Fig. 6). Labeling with 3 M t16:5−fatty acid for 2 h generated mostly PC (Fig. 4d) and resulted in staining of nuclear envelope and endoplasmic reticulum (Fig. 6) plus lipid droplets and mitochondria (data not shown). Labeling inside the nucleus, shown for NBD− and BODIPY−fatty acids³⁰, was not observed. Labeling with polyene-alkylglycerol, which yielded fluorescent ether lipids (Fig. 4e), allowed their visualization in living cells for the first time. A distribution similar to that of PC was found (Fig. 6). Ether lipids did not accumulate in peroxisomes (Fig. 6), although the early steps of ether lipid biosynthesis occur there²⁵. Labeling with polyene-ceramide generated fluorescent SM (Fig. 4f), resulting in plasma membrane staining (Fig. 6) with little, if any, labeling of the Golgi complex, contrary to observations with NBD-ceramide³¹ and BODIPY-ceramide⁸.

Figure 5. Imaging of fluorescent polyene-lipids in various types of living mammalian cells. COS7 kidney epithelial cells (a), 3T3−L1 adipocytes (b) and C2C12 muscle cells (c) were grown with 50 M t16:5−fatty acid for 30 min (a), 15 min (b), or 1 h (c). During this time fluorescent polyene-lipids (see Fig. 4a−c) were generated by cellular metabolism. Lipid distribution in living cells was imaged using two-photon-excitation microscopy. Bars, 10 m. Full Figure and legend (45K)

Figure 6. Localization of different lipid classes in living COS7 cells. COS7 cells transiently expressing mRFP1 targeted to the ER (upper panels), to peroxisomes (middle panels) or to the Golgi complex (lower panels) were grown for 2 h with 3 M t16:5−fatty acid (upper panels), c16:5-monoalkyl-1-glycerol (middle panels) or c18:5-ceramide (lower panels). During this time mostly fluorescent PC (upper panels), various ether lipids (middle panels) and SM (lower panels) were generated by cellular metabolism (Fig. 4d−f). Two-photon−excited lipid distribution in living cells is shown in green, and mRFP1 signal is in red. Bars, 10 m. Full Figure and legend (19K)

	Top

We analyzed the metabolic fate of polyene lipid precursors upon application to living cells. As degradation of the polyene fluorophore via -oxidation yields only non-fluorescent products, invisible in TLC and microscopic images, our analysis only accounts for those products that give a signal in microscopic imaging. Cells readily take up fluorescent precursors and convert them to the expected products with no apparent disturbance by the tag. Polyene-ceramide is converted to polyene-SM, and fluorescent ether lipids are generated from polyene-alkylglycerol. The labeling pattern of polyene−fatty acids follows that of radiolabeled natural fatty acids of the same length: shorter fatty acids show preference for incorporation into PC, longer ones have a wider distribution. The unequal degree of SM-labeling observed for fluorescent and radioactive fatty acids points to a preference of ceramide synthase for pentaene−fatty acids. This is unexpected because ceramide synthase normally prefers saturated fatty acids and it indicates that the kink in the geometry of cis-pentaene−fatty acids is not as pronounced as for a monounsaturated fatty acid, consistent with molecular modeling data (see Supplementary Fig. 2 online). Labeling with higher concentrations of polyene−fatty acid resulted in accumulation of fluorescent intermediates of lipid biosynthesis, for example, free fatty acid and diglyceride, as well as labeled storage forms of lipids, such as triglyceride and cholesterol ester, suggesting saturation of the phospholipid biosynthesis. The results demonstrate that polyene-lipids are very similar to their natural counterparts in how they are used by the lipid biosynthetic enzymes, in contrast to the NBD− and BODIPY−fatty acids, which are poorly incorporated into cellular lipids³⁰.

	Top

BODIPY-FL-C5-ceramide, BODIPY-FL-C5-SM and NBD-C6-ceramide were from Molecular Probes. Acyl or alkyl chains of polyene-lipids are denoted as follows: cis/trans configuration of the -9 double bond (c/t), followed by the number of carbon atoms and the number of double bonds, separated by a colon. All polyenes are pentaenes featuring conjugated double bonds at -9, -7, -5, -3 and -1 positions; the latter four in trans configuration.

COS7, 3T3-L1, and C2C12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing high glucose, GlutaMAX I and pyruvate (31966; Gibco) with 10% FCS. COS7 cells were transiently transfected using Cellfectin (Invitrogen). Confluent 3T3-L1 cells were differentiated to adipocytes with 500 mM 3-isobutyl-1-methylxanthine, 10 mM dexamethasone and 5 mg/ml insulin. After 3 d, 3-isobutyl-1-methylxanthine and dexamethasone were omitted. For up to 10 d, fresh medium was applied every 2 d until 90% of cells were differentiated. Confluent C2C12 cells were differentiated to myotubes in DMEM containing low glucose, L-glutamine and pyruvate (31885; Gibco) with 2% horse serum for 3 d. Sixteen hours prior to all experiments, cells were transferred to microscopy medium, DMEM with low glucose and pyruvate, free of glutamine and phenol red (11880; Gibco) and supplemented with 2 mM L-glutamine, 5% de-lipidated⁴⁰ FCS. For microscopy cells were grown in number 1.5 glass bottom dishes (MatTek); for lipid analysis in 10 cm plates. Fatty acids or alkylglycerol at final concentration of up to 5 M were added to microscopy medium by ethanolic injection. Polyene lipids or NBD- or BODIPY-tagged lipids at higher concentration were complexed to 1% delipidated BSA (Applichem) in HBSS as described³¹. Polyene-ceramide or polyene-SM was complexed to a twofold molar excess of -cyclodextrin (Applichem), dissolved in water by slowly injecting a few microliters of the lipid stock into vigorously stirring solution. After lyophilization, the complex was redissolved in microscopy medium and used immediately.

Two-photon microscopy was performed using a Biorad Radiance 2100 MP setup attached to a Nikon Eclipse TE300 inverted microscope equipped with a Planapo 60 (1.2 NA). Pulsed excitation (pulse duration 200 fs, repetition rate 76 MHz, average power 600 mW) at 725 nm was provided by a 5 W Verdi/Mira laser (Coherent). Fluorescent images were acquired using the non-descanned detectors. Long and short wavelengths were split using a 560DCXR beamsplitter (Chroma), and the longer ones were filtered with a D630/50m bandpass filter (Chroma). Images (852 852 pixels, 104 104 m, recording time, 17 s per frame) were acquired using LaserSharp 2000 software (Biorad). Using this equipment, a lower limit of detection of pentaene lipids is at a concentration of 2 nmol/mg of cellular protein. All live cell imaging was done at 37 °C; the temperature of the specimen was controlled with a Tempcontrol 37−2 digital unit (Bachhoffer) and that of the objective, by an objective heater (Bioptechs). Images were processed using Adobe Photoshop 6.0.

	Top

1. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569−572 (1997). | Article | PubMed | ISI | ChemPort |
2. Brown, D.A. & London, E. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164, 103−114 (1998). | Article | PubMed | ISI | ChemPort |
3. Magee, T., Pirinen, N., Adler, J., Pagakis, S.N. & Parmryd, I. Lipid rafts: cell surface platforms for T cell signaling. Biol. Res. 35, 127−131 (2002). | PubMed | ISI | ChemPort |
4. Holthuis, J.C., van Meer, G. & Huitema, K. Lipid microdomains, lipid translocation and the organization of intracellular membrane transport (Review). Mol. Membr. Biol. 20, 231−241 (2003). | PubMed | ChemPort |
5. Maier, O., Oberle, V. & Hoekstra, D. Fluorescent lipid probes: some properties and applications (Review). Chem. Phys. Lipids 116, 3−18 (2002). | PubMed | ChemPort |
6. Ishitsuka, R., Yamaji-Hasegawa, A., Makino, A., Hirabayashi, Y. & Kobayashi, T. A lipid-specific toxin reveals heterogeneity of sphingomyelin-containing membranes. Biophys. J. 86, 296−307 (2004). | PubMed | ChemPort |
7. Chattopadhyay, A. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem. Phys. Lipids 53, 1−15 (1990). | PubMed | ChemPort |
8. Pagano, R.E., Martin, O.C., Kang, H.C. & Haugland, R.P. A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J. Cell Biol. 113, 1267−1279 (1991). | Article | PubMed | ISI | ChemPort |
9. Stoffel, W. & Michaelis, G. Chemical syntheses of novel fluorescent-labelled fatty acids, phosphatidylcholines and cholesterol esters. Hoppe-Seyler's Z. Physiol. Chem. 357, 7−19 (1976). | PubMed | ChemPort |
10. Somerharju, P. Pyrene-labeled lipids as tools in membrane biophysics and cell biology. Chem. Phys. Lipids 116, 57−74 (2002). | PubMed | ChemPort |
11. Antes, P., Schwarzmann, G. & Sandhoff, K. Distribution and metabolism of fluorescent sphingosines and corresponding ceramides bearing the diphenylhexatrienyl (DPH) fluorophore in cultured human fibroblasts. Eur. J. Cell Biol. 59, 27−36 (1992). | PubMed | ChemPort |
12. Pagano, R.E. & Sleight, R.G. Defining lipid transport pathways in animal cells. Science 229, 1051−1057 (1985). | PubMed | ChemPort |
13. Kaiser, R.D. & London, E. Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence quenching. Biochim. Biophys. Acta 1375, 13−22 (1998). | PubMed | ChemPort |
14. Wang, T.Y. & Silvius, J.R. Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers. Biophys. J. 79, 1478−1489 (2000). | PubMed | ISI | ChemPort |
15. Sklar, L.A., Hudson, B.S. & Simoni, R.D. Conjugated polyene fatty acids as membrane probes: preliminary characterization. Proc. Natl. Acad. Sci. USA 72, 1649−1653 (1975). | PubMed | ChemPort |
16. Lentz, B.R. Use of fluorescent probes to monitor molecular order and motions within liposome bilayers. Chem. Phys. Lipids 64, 99−116 (1993). | PubMed | ChemPort |
17. Mateo, C.R., Souto, A.A., Amat-Guerri, F. & Acuna, A.U. New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions. Biophys. J. 71, 2177−2191 (1996). | PubMed | ChemPort |
18. Quesada, E., Acuna, A.U. & Amat-Guerri, F. New transmembrane polyene bolaamphiphiles as fluorescent probes in lipid bilayers. Angew. Chem. Int. Edn. Engl. 40, 2095−2097 (2001). | Article | ChemPort |
19. Quesada, E., Acuna, A.U. & Amat-Guerri, F. Synthesis of carboxyl-tethered symmetric conjugated polyenes as fluorescent transmembrane probes of lipid bilayers. Eur. J. Org. Chem. 2003, 1308−1318 (2003). | Article |
20. Souto, A.A., Ulises Acuna, A. & Amat-Guerri, F. A general and practical synthesis of linear conjugated pentaenoic acids. Tetrahedr. Lett. 35, 5907−5910 (1994). | ChemPort |
21. London, E. & Feigenson, G.W. Fluorescence quenching in model membranes. 1. Characterization of quenching caused by a spin-labeled phospholipid. Biochemistry 20, 1932−1938 (1981). | PubMed | ChemPort |
22. Ahmed, S.N., Brown, D.A. & London, E. On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36, 10944−10953 (1997). | Article | PubMed | ISI | ChemPort |
23. Brown, D.A. & Rose, J.K. Sorting of GPI-Anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533−544 (1992). | Article | PubMed | ISI | ChemPort |
24. Roper, K., Corbeil, D. & Huttner, W.B. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat. Cell Biol. 2, 582−592 (2000). | Article | PubMed | ISI | ChemPort |
25. Nagan, N. & Zoeller, R.A. Plasmalogens: biosynthesis and functions. Prog. Lipid Res. 40, 199−229 (2001). | Article | PubMed | ChemPort |
26. Tanhuanpaa, K. & Somerharju, P. Gamma-cyclodextrins greatly enhance translocation of hydrophobic fluorescent phospholipids from vesicles to cells in culture. Importance of molecular hydrophobicity in phospholipid trafficking studies. J. Biol. Chem. 274, 35359−35366 (1999). | Article | PubMed | ChemPort |
27. Ekroos, K. et al. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J. Lipid Res. 44, 2181−2192 (2003). | PubMed | ChemPort |
28. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73−76 (1990). | PubMed | ISI | ChemPort |
29. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877−7882 (2002). | Article | PubMed | ChemPort |
30. Huang, H., Starodub, O., McIntosh, A., Kier, A.B. & Schroeder, F. Liver fatty acid-binding protein targets fatty acids to the nucleus Real time confocal and multiphoton fluorescence imaging in living cells. J. Biol. Chem. 277, 29139−29151 (2002). | Article | PubMed | ChemPort |
31. Lipsky, N.G. & Pagano, R.E. A vital stain for the Golgi apparatus. Science 228, 745−747 (1985). | PubMed | ISI | ChemPort |
32. Bielawska, A., Crane, H.M., Liotta, D., Obeid, L.M. & Hannun, Y.A. Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide. J. Biol. Chem. 268, 26226−26232 (1993). | PubMed | ISI | ChemPort |
33. Li, L., Tang, X., Taylor, K.G., DuPre, D.B. & Yappert, M.C. Conformational characterization of ceramides by nuclear magnetic resonance spectroscopy. Biophys. J. 82, 2067−2080 (2002). | PubMed | ChemPort |
34. Mukherjee, S. & Maxfield, F.R. Role of membrane organization and membrane domains in endocytic lipid trafficking. Traffic 1, 203−211 (2000). | Article | PubMed | ISI | ChemPort |
35. Brugger, B., Erben, G., Sandhoff, R., Wieland, F.T. & Lehmann, W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 94, 2339−2344 (1997). | Article | PubMed | ISI | ChemPort |
36. Sprong, H., van der Sluijs, P. & van Meer, G. How proteins move lipids and lipids move proteins. Nat. Rev. Mol. Cell Biol. 2, 504−513 (2001). | Article | PubMed | ISI | ChemPort |
37. Pagano, R.E., Sepanski, M.A. & Martin, O.C. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: Interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J. Cell Biol. 109, 2067−2079 (1989). | Article | PubMed | ISI | ChemPort |
38. Rodemer, C. et al. Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet. 12, 1881−1895 (2003). | Article | PubMed | ChemPort |
39. Tanhuanpaa, K., Virtanen, J. & Somerharju, P. Fluorescence imaging of pyrene-labeled lipids in living cells. Biochim. Biophys. Acta 1497, 308−320 (2000). | PubMed | ChemPort |
40. Thiele, C., Hannah, M.J., Fahrenholz, F. & Huttner, W.B. Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat. Cell Biol. 2, 42−49 (2000). | Article | PubMed | ISI | ChemPort |
41. Bligh, E.G. & Dyer, W.J. A rapid method for total lipid extraction and purifiaction. Can. J. Biochem. Physiol. 37, 911−917 (1959). | PubMed | ISI | ChemPort |
42. Dasgupta, S. & Hogan, E.L. Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids. J. Lipid Res. 42, 301−308 (2001). | PubMed | ChemPort |
43. Ekroos, K., Chernushevich, I.V., Simons, K. & Shevchenko, A. Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Anal. Chem. 74, 941−949 (2002). | PubMed | ChemPort |

	Top

We thank colleagues who kindly provided plasmids: P. Keller and J. White (ManII), M. Rolls (Sec61) and R.E. Tsien (mRFP1). We thank M. Gruner and A. Rudolph for NMR analysis and K. Sandhoff for helpful suggestions. We acknowledge financial support by the Deutsche Forschungsgemeinschaft (SFB-TR 13, D2).

Competing interests statement:  The authors declare that they have no competing financial interests.

	Top

Natureproducts is an online service detailing information about specific products used in this article, you can view the product descriptions, request information and compare with other similar products. The products used are listed in alphabetical order. A-Z product listing 11880 (MatTek) 31885 (MatTek) 560DCXR beamsplitter (Chroma) BODIPY-FL-C5-ceramide, BODIPY-FL-C5-SM (Molecular Probes) Canon (D60 camera) Cellfectin (Invitrogen) See more natureproducts

	Top