A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Abstract

apoptosis, and other critical cellular processes during normal cell function and disease ( 1-4 ). Insight into sphingolipid biosynthesis, transport, and subcellular distribution has been acquired by observing fl uorescent sphingolipid analogs within living cells ( 5 ). Sphingolipid derivatives that contain a fl uorophore-labeled N -acyl fatty acid are often used to investigate dynamic processes that involve acylated sphingolipids ( 5 ). Fatty acids that contain a polyene fl uorophore are especially attractive for this purpose because the structure and behavior of polyene-containing lipids are very similar to the native lipid ( 6 ). However, to study the bioactive, unacylated sphingolipids, sphingosine and sphingosine-1-phosphate, the fl uorophore must be incorporated into the sphingosine backbone. Studies have confi rmed that such fl uorescent sphingosine analogs can be metabolized to more complex fl uorescently labeled sphingolipids in living cells ( 7-10 ). Despite their potential utility, only a limited number of fl uorophores, such as pyrene, borondipyrromethene (BODIPY), and nitrobenzo-2-oxa-1,3-diazole, have been incorporated into the sphingosine backbone ( 7-9 ).To increase the utility of fl uorescent sphingosine analogs for investigating sphingolipid dynamics in living cells, derivatives with a wider range of fl uorescence properties must be developed. Lacking in particular is a bioactive fl uorescent sphingosine that has neither an excitation maximum that is in the UV range nor emission that interferes with detecting green fl uorescent protein (GFP), the most common genetically encoded fl uorescent protein label ( 11 ). A probe with these properties is expected to Abbreviations: GFP, green fl uorescent protein; BODIPY, borondipyrromethene; rt, room temperature; ER, endoplasmic reticulum. 1 R. Kim and K. Lou contributed equally to this work.

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

“…We have shown that SGPL1 disruption is easily done in a cell line of choice by CRISPR-associated RNA-guided endonuclease Cas9 in a matter of weeks and propose that this should be easily transferable to other cells lines in which functionalized sphingoid bases are used [ 15 , 19 , 20 ], such as cell lines characterized by altered sphingolipid homeostasis. In summary, SGPL1 knockout cell lines provide the basis to studying protein-sphingolipid interaction in physiological and pathophysiological states of cells.…”

“…This is also true for any other sphingoid base containing a modification (e.g. a fluorescent dye) in its backbone [ 19 , 20 ].…”

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

“…These fluorescent sphingolipid analogs are advantageous because they afford more flexibility in terms of fluorophore selection, and they can be employed for live cell imaging. Fluorescent sphingolipid precursors that permit observing the lipid distribution that results from biosynthesis and trafficking have also been developed (Peters et al, 2007; Kim et al, 2013). The main drawback to this approach is that the relatively large and chemically distinct fluorophore may alter the interactions between the labeled sphingolipid and other membrane components, which can change the lipid distribution in the membrane (Devaux et al, 2002; Maier et al, 2002; Shaw et al, 2006).…”

Quantifying Fluorescently Labeled Ceramide Levels in Human Sarcoma Cell Lines in Response to a Sphingomyelin Synthase Inhibitor

“…As such, more researchers are changing to fluorescently labeled small molecules within basic sciences [19,20]. Not only is fluorescently labeled ceramide commercially available, but several of the sphingolipids are being produced with fluorescent tags to facilitate safer and sensitive detection of these lipid molecules to implement in biochemical experiments [21,22,23].…”