Trifunctional lipid probes for comprehensive studies of single lipid species in living cells.
Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid-protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo-cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann-Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.|Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
Doris
0000-0002-6862-0076
D
Höglinger
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.|Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
André
A
Nadler
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Per
P
Haberkant
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.|Leibniz Institute on Aging - Fritz Lipmann Institute, 07745 Jena, Germany.
Joanna
J
Kirkpatrick
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Martina
M
Schifferer
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Frank
F
Stein
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Sebastian
S
Hauke
Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Forbes D
FD
Porter
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; schultz@embl.de.
Carsten
C
Schultz
D020013:Calcium Signaling; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis* / Q000737:chemistry; D018613:Microscopy, Confocal; D052556:Niemann-Pick Disease, Type C / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D021381:Protein Transport; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013110:Sphingosine / Q000737:chemistry* / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods
C011439:1,2-diacylglycerol; D004075:Diglycerides; D008055:Lipids; D020543:Proteome; D013110:Sphingosine
Niemann−Pick disease type C; caged lipids; diacylglycerol; lipid−protein interaction; sphingosine