Uncovering the routes of distribution and metabolism of extracellular sphingosine 1-phosphate in vivo and in vitro by imaging and analyzing fluorescently-labelled sphingolipids

• Antragsteller: Professor Dr. Markus Gräler, Ph.D.
• Fachliche Zuordnung: Zellbiologie
• Förderung: Förderung von 2009 bis 2013
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 92820999

Sphingosine 1-phosphate (S1P) in blood is stored by erythrocytes, and it is inducibly released into plasma, where it acts as an extracellular stimulus for S1P-receptors. Based on preliminary data we hypothesize that blood serves as a major source for S1P, which is distributed to lymphoid and peripheral tissues and lymph. In order to define the pathways that are involved in the distribution of S1P, fluorescently-labelled sphingolipids will be used to track the release and distribution of S1P in vivo and in vitro. The release and distribution of S1P from erythrocytes into plasma and surrounding cells and tissues will be monitored in tissue sections by confocal laser scanning microscopy and in co-culture experiments by flow cytometry. Mice deficient for the sphingosine-phosphorylating enzyme sphingosine kinase 2 and for the S1P-degrading enzyme S1P-lyase will be used as recipient mice to determine the role of de- and re-phosphorylation for tissue distribution of blood-borne S1P, and for determining the routes and origin of accumulating S1P in tissues of S1P-lyase deficient mice. To uncover the metabolic fate of extracellular S1P, fluorescently-labelled sphingolipid metabolites will be identified and analyzed using fluorescence detection in high performance liquid chromatography (HPLC) combined with tripple-quadrupole mass spectrometry (Q-Trap). The subcellular localization of fluorescently-labelled sphingolipids will be analyzed by confocal laser scanning microscopy in vitro using tissue cell lines and primary cells. We hypothesize that differences in subcellular sphingolipid localization separate incorporated extracellular and endogenous sphingolipids, and determine their fate whether they are secreted, stored, transported, or degraded.

• DFG-Verfahren: Sachbeihilfen