A Long Wavelength Fluorescent Sphingosine As a Tool to Visualize Sphingolipid Transport and Location in Living Cells

Sphingolipids are cell membrane components that also play critical roles in multiple biological events, including cell growth and apoptosis signaling. Information on  dynamic sphingolipid processes, such as trafficking, sorting, and organization in membranes, has been acquired by visualizing fluorescent sphingolipid analogs in living cells.  Investigations of the transport and subcellular distributions of sphingomyelin, ceramide and gangliosides have been facilitated by the commercial availability of fluorescent sphingolipid analogs in which the fluorophore is attached to the N-acyl fatty acid side chain. To study the subcellular locations and functions of biologically important, unacylated sphingolipid metabolites, such as sphingosine and sphingosine-1-phosphate, the fluorophore must instead be incorporated into the sphingoid backbone.  Despite the utility of such derivatives, only a small number of fluorescent sphingolipid analogs have been reported, and the fluorescence emissions of most of these analogs overlap with that of the most common genetically encoded fluorescent protein label, green fluorescent protein (GFP).  Development of a fluorescent sphingosine analog that emits at a longer wavelength is desirable to permit visualizing the fluorescent sphingolipid analog in parallel with GFP-labeled proteins.  Here, we report the use of a new fluorescent sphingolipid precursor in which a long wavelength borondipyrromethene fluorophore, BODIPY 540, is incorporated into the sphingosine backbone.  The BODIPY 540 sphingosine analog and its fluorescent metabolites were detected in the organelles where sphingolipid metabolism occurs, and could be imaged in parallel with GFP-labeled proteins within living cells. Thin layer chromatography and mass spectrometry analysis confirmed that mammalian cells  readily metabolized the BODIPY 540 sphingosine into multiple fluorescent sphingolipids. We expect that this BODIPY 540 sphingosine analog  will be a useful tool to study dynamic sphingolipid events and may also facilitate the detection of abnormal sphingolipid regulation.