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Anatomical distribution of lipids in human brain by imaging mass spectrometry
Abstracts / Chemistry and Physics of Lipids 163S (2010) S1–S18
influenced the ability of the ceramides to stabilize SM-rich domains, and to displace sterol from the domains. References Megha, et al., 2007. BBA 1768. Nybond, et al., 2005. BBA 1718.
doi:10.1016/j.chemphyslip.2010.05.009 SO2 Coexistence of three structurally and kinetically distinct lipid micrometric domains at the plasma membrane L. D’Auria 1,∗ , P. Van Der Smissen 1 , F. Bruyneel 2 , P.J. Courtoy 1 , D. Tyteca 1 1 CELL Unit, Université catholique de Louvain and de Duve Institute, Brussels, Belgium 2 CHOM Unit, Université catholique de Louvain, Louvain-la-Neuve, Belgium
Beside well-accepted nanometric rafts, micrometric lipid compartmentation at the plasma membrane (PM) of living cells is disputed. We have recently shown that fluorescent analogs of Lo sphingomyelin (SM) label sharp submicrometric domains on living erythrocytes and CHO cells. We here report that insertion in the same cells of Ld -lipid analogs, i.e. various phosphatidylcholine (PC) or a lactosylceramide with a non-natural stereochemistry (L-tLacCer), also led to the formation of sharp submicrometric domains at ≤30 ◦ C but larger regions with fuzzy boundaries at 37 ◦ C. Insertion of Lo -glycosphingolipid analogs with natural stereochemistry (D-e-GSLs) led to the opposite, with large surface regions at ≤30 ◦ C and micrometric domains at 37 ◦ C. The coexistence of three types of domains of increasing order, respectively enriched in PC/L-t-LacCer, SM and GSLs and covering ∼70% of the CHO cell surface, was confirmed by (i) controlled perturbations of endogenous GSLs/SM, (ii) double-labeling imaging together with a GPI-anchored protein and (iii) fluorescence recovery after photobleaching (FRAP). Indeed, in contrast to GSL analogs, Ld -PC and L-t-LacCer analogs showed at 30 ◦ C a severe restriction to their lateral diffusion over long-range, but not short-range, consistent with sharp boundaries. Combined GSLs and SM depletion by fumonisin B1 relaxed this restriction, which was restored by repletion either with the GSL GM3 or a shortchain non-fluorescent SM, indicating that both GSLs and SM restrict Ld -analogs. We propose that endogenous PM lipids are organized at the micrometric size into non-overlapping islands enriched in PC and SM enclosed among a continuous GSL phase at ≤30 ◦ C, and the Ld -phase becoming continuous at 37 ◦ C. doi:10.1016/j.chemphyslip.2010.05.010 SO3 Anatomical distribution of lipids in human brain by imaging mass spectrometry Antonio Veloso 1,∗ , Estíbaliz González de San Román 2 , Egoitz Astigarraga 1 , Gabriel Barreda-Gome 2 , Iván Manuel 2 , María T. ˜ Ochoa 3 , Olatz Fresnedo 3 , Jose A. Fernandez 1 , Giralt 2 , Begona Rafael Rodríguez-Puertas 2 1
Departamento de Química-Física, Universidad del País Vasco, Spain Departamento de Farmacología, Universidad del País Vasco, Spain 3 Departamento de Fisiología, Universidad del País Vasco, Spain 2
The human brain’s lipid gray matter contains 36–40% lipid, white matter 49–66%, and myelin 78–81%. In addition to the structural role of the lipids in the cellular membranes and in the energetic
S3
metabolism, their relevance as signalling molecules in the central nervous system is increasing our understanding of the brain physiology. The aim of this study is to analyse the lipid composition and anatomical distribution of the different lipid species in human brain. We have applied the MALDI-TOF mass spectrometry technique on slices, followed by a lipidomic analysis in different postmortem tissue samples of human brain. An external calibration was used to achieve a mass accuracy of ∼10 ppm in the spectra, allowing us to propose identification for a number of lipid species (m/z from 500 to 2000). The spectra recorded directly from tissue slices (imaging) show an excellent s/n ratio, both, in reflectron positive and negative modes. Furthermore, they retain the information about the anatomical distribution of the molecular species present in autopsied frozen tissue. The comparison between the spectra of striatum, hippocampus and frontal cortex recorded directly from the tissue, reflects differences in lipid composition between the three brain areas. doi:10.1016/j.chemphyslip.2010.05.011 SO4 Lipidomic profile of mutants in GPI anchor biosynthesis Ursula Loizides-Mangold 1,∗ , Victor Nesatyy 2 , Fabrice David 2 , Isabelle Riezman 1 , Howard Riezman 1 1
Department of Biochemistry, University of Geneva, CH-1211 Geneva, Switzerland 2 Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Faculty of Life Sciences, Lausanne, Switzerland Here, we describe a mass spectrometry-based approach to detect and quantify molecular lipid species from a complex biological sample using crude lipid extracts. We applied tandem mass spectrometry coupled with multiple reaction monitoring (MRM) to analyze over 800 phospho- and sphingolipids. Synthetic lipids with fatty acid compositions that are not naturally occurring were used as internal standards. In addition, we were able to study the sterol composition of the same lipid extract by GC–MS. Following this approach, we analyzed the lipid profile of a series of Chinese hamster ovary (CHO) cells that have a defect in glycosylphosphatidylinositol (GPI) anchor biosynthesis. In eukaryotes, anchoring of proteins to the extracellular surface of the plasma membrane via the GPI anchor is essential for cellular function and development. Importantly, the trafficking of GPI anchored proteins is affected by alterations in sphingolipids and sterols. Using our lipidomics approach, we observed an overall downregulation of monoglycosylated ceramides in the majority of GPI anchor mutants. In contrast, total sphingomyelin levels increased in the same set of mutants. In addition, we observed an increase in certain phospholipid species that are potential precursor molecules for GPI anchor biosynthesis. We then focused on the mutant PIG-F, which has a defect in the late phase of GPI anchor biosynthesis and cannot synthesize a full-length GPI anchor. PIG-F deficient cells were complemented with the wild-type PIG-F gene and the observed effects on sphingolipids and GPI anchor lipid precursors were reversed back to wild-type in the complemented cells.
Acknowledgement This project was supported by the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation. doi:10.1016/j.chemphyslip.2010.05.012
influenced the ability of the ceramides to stabilize SM-rich domains, and to displace sterol from the domains. References Megha, et al., 2007. BBA 1768. Nybond, et al., 2005. BBA 1718.
doi:10.1016/j.chemphyslip.2010.05.009 SO2 Coexistence of three structurally and kinetically distinct lipid micrometric domains at the plasma membrane L. D’Auria 1,∗ , P. Van Der Smissen 1 , F. Bruyneel 2 , P.J. Courtoy 1 , D. Tyteca 1 1 CELL Unit, Université catholique de Louvain and de Duve Institute, Brussels, Belgium 2 CHOM Unit, Université catholique de Louvain, Louvain-la-Neuve, Belgium
Beside well-accepted nanometric rafts, micrometric lipid compartmentation at the plasma membrane (PM) of living cells is disputed. We have recently shown that fluorescent analogs of Lo sphingomyelin (SM) label sharp submicrometric domains on living erythrocytes and CHO cells. We here report that insertion in the same cells of Ld -lipid analogs, i.e. various phosphatidylcholine (PC) or a lactosylceramide with a non-natural stereochemistry (L-tLacCer), also led to the formation of sharp submicrometric domains at ≤30 ◦ C but larger regions with fuzzy boundaries at 37 ◦ C. Insertion of Lo -glycosphingolipid analogs with natural stereochemistry (D-e-GSLs) led to the opposite, with large surface regions at ≤30 ◦ C and micrometric domains at 37 ◦ C. The coexistence of three types of domains of increasing order, respectively enriched in PC/L-t-LacCer, SM and GSLs and covering ∼70% of the CHO cell surface, was confirmed by (i) controlled perturbations of endogenous GSLs/SM, (ii) double-labeling imaging together with a GPI-anchored protein and (iii) fluorescence recovery after photobleaching (FRAP). Indeed, in contrast to GSL analogs, Ld -PC and L-t-LacCer analogs showed at 30 ◦ C a severe restriction to their lateral diffusion over long-range, but not short-range, consistent with sharp boundaries. Combined GSLs and SM depletion by fumonisin B1 relaxed this restriction, which was restored by repletion either with the GSL GM3 or a shortchain non-fluorescent SM, indicating that both GSLs and SM restrict Ld -analogs. We propose that endogenous PM lipids are organized at the micrometric size into non-overlapping islands enriched in PC and SM enclosed among a continuous GSL phase at ≤30 ◦ C, and the Ld -phase becoming continuous at 37 ◦ C. doi:10.1016/j.chemphyslip.2010.05.010 SO3 Anatomical distribution of lipids in human brain by imaging mass spectrometry Antonio Veloso 1,∗ , Estíbaliz González de San Román 2 , Egoitz Astigarraga 1 , Gabriel Barreda-Gome 2 , Iván Manuel 2 , María T. ˜ Ochoa 3 , Olatz Fresnedo 3 , Jose A. Fernandez 1 , Giralt 2 , Begona Rafael Rodríguez-Puertas 2 1
Departamento de Química-Física, Universidad del País Vasco, Spain Departamento de Farmacología, Universidad del País Vasco, Spain 3 Departamento de Fisiología, Universidad del País Vasco, Spain 2
The human brain’s lipid gray matter contains 36–40% lipid, white matter 49–66%, and myelin 78–81%. In addition to the structural role of the lipids in the cellular membranes and in the energetic
S3
metabolism, their relevance as signalling molecules in the central nervous system is increasing our understanding of the brain physiology. The aim of this study is to analyse the lipid composition and anatomical distribution of the different lipid species in human brain. We have applied the MALDI-TOF mass spectrometry technique on slices, followed by a lipidomic analysis in different postmortem tissue samples of human brain. An external calibration was used to achieve a mass accuracy of ∼10 ppm in the spectra, allowing us to propose identification for a number of lipid species (m/z from 500 to 2000). The spectra recorded directly from tissue slices (imaging) show an excellent s/n ratio, both, in reflectron positive and negative modes. Furthermore, they retain the information about the anatomical distribution of the molecular species present in autopsied frozen tissue. The comparison between the spectra of striatum, hippocampus and frontal cortex recorded directly from the tissue, reflects differences in lipid composition between the three brain areas. doi:10.1016/j.chemphyslip.2010.05.011 SO4 Lipidomic profile of mutants in GPI anchor biosynthesis Ursula Loizides-Mangold 1,∗ , Victor Nesatyy 2 , Fabrice David 2 , Isabelle Riezman 1 , Howard Riezman 1 1
Department of Biochemistry, University of Geneva, CH-1211 Geneva, Switzerland 2 Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Faculty of Life Sciences, Lausanne, Switzerland Here, we describe a mass spectrometry-based approach to detect and quantify molecular lipid species from a complex biological sample using crude lipid extracts. We applied tandem mass spectrometry coupled with multiple reaction monitoring (MRM) to analyze over 800 phospho- and sphingolipids. Synthetic lipids with fatty acid compositions that are not naturally occurring were used as internal standards. In addition, we were able to study the sterol composition of the same lipid extract by GC–MS. Following this approach, we analyzed the lipid profile of a series of Chinese hamster ovary (CHO) cells that have a defect in glycosylphosphatidylinositol (GPI) anchor biosynthesis. In eukaryotes, anchoring of proteins to the extracellular surface of the plasma membrane via the GPI anchor is essential for cellular function and development. Importantly, the trafficking of GPI anchored proteins is affected by alterations in sphingolipids and sterols. Using our lipidomics approach, we observed an overall downregulation of monoglycosylated ceramides in the majority of GPI anchor mutants. In contrast, total sphingomyelin levels increased in the same set of mutants. In addition, we observed an increase in certain phospholipid species that are potential precursor molecules for GPI anchor biosynthesis. We then focused on the mutant PIG-F, which has a defect in the late phase of GPI anchor biosynthesis and cannot synthesize a full-length GPI anchor. PIG-F deficient cells were complemented with the wild-type PIG-F gene and the observed effects on sphingolipids and GPI anchor lipid precursors were reversed back to wild-type in the complemented cells.
Acknowledgement This project was supported by the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation. doi:10.1016/j.chemphyslip.2010.05.012