The role of long acyl chain sphingolipids in plasma membrane lateral organizations of mammalian cells

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Sphingolipids, primarily C24 and C16, reside exclusively in the outer leaflet of mammalian cell plasma membrane. Curiously, little is known how this bilayer asymmetry influences sphingolipid-cholesterol interactions and, particularly, the organization of membrane microdomains. During the current Discovery grant period (2013-2018), we are able to establish that one of the most abundant sphingolipids in vivo, C24 sphingomyelin, uniquely interacts with cholesterol and is central to governing the lateral organization in the plasma membrane by generating cholesterol asymmetry.For next the Discovery grant, we propose to continue this direction of research, but broaden the scope to incorporate more advanced biophysical analysis and also to understand the biological significance with following specific aims.1. Use home- and hetero-FRET to understand nano- and submicron-domains in the plasma membrane of live cells.We have used these proteins for hetero-FRET analysis (between YFP and CFP) to detect sub-micron domains in the plasma membrane of live cells. Our conclusion from these studies was that plasma membranes have little submicron domains, likely due to C24 sphingolipids and hence 20/80 cholesterol partitioning in the plasma membrane. However, the plasma membrane is known to form nano-domains (~50 A2), which can be detected homo-FRET (between YFPs). We will use homo-FREFT to determine if C24 SM and cholesterol partitioning affect nano-domains. 2. Develop fluorescence lifetime techniques to characterize domain structures in the plasma membrane of live cells.The fluorescence lifetime of trans-parinatic acid (tPA) is sensitive to membrane order and can spontaneously detect multiple membrane domains by displaying multiple lifetime, ranging from 1-100 ns. The life time of this probe is a well-suited to detect domains, regardless of size. We will use GUVs, which form optically resolvable domains, to validate the method and then apply it to LUVs and the plasma membrane of live cells. 3. Characterize the impact of CerS2 on the domain structures in the plasma membrane of live cells.CerS2 is linked to human diseases with unknown molecular mechanisms. We observed that sphingolipid acyl chain length can alter cholesterol partitioning and membrane domains in both model membranes and the plasma membrane of live cells. Interestingly, loss-of-function of CerS2 causes a reduction of C24 sphingolipids, which are replaced by C16 sphingolipids in the plasma membrane. We will use the techniques described above to characterize the role of CerS2 in cholesterol partition and microdomain formation in mammalian cells.