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

鞘磷脂，主要是C24和C16，仅存在于哺乳动物细胞质膜的外叶中。奇怪的是，这种双层不对称如何影响鞘磷脂-胆固醇相互作用，特别是膜微域的组织，人们知之甚少。在目前的发现期(2013-2018年)，我们能够确定体内最丰富的鞘磷脂之一，C24鞘磷脂，与胆固醇独特地相互作用，并通过产生胆固醇不对称来控制质膜的横向组织。*对于下一次发现拨款，我们建议继续这一研究方向，但扩大范围以纳入更先进的生物物理分析，并了解具有以下特定目的的生物学意义。*1.使用同源和异源FRET来了解活细胞质膜中的纳米和亚微米结构域。*我们已经将这些蛋白质用于异源FRET分析(在YFP和CFP之间)，以检测活细胞质膜中的亚微米结构域。我们从这些研究中得出的结论是，质膜上几乎没有亚微米结构域，这可能是由于C24鞘磷脂和因此20/80胆固醇在质膜中的分配所致。然而，已知质膜形成纳米结构域(~50A2)，可以检测到同源FRET(在YFP之间)。我们将使用HOMO-FREFT来确定C24SM和胆固醇分配是否影响纳米结构域。*2.发展荧光寿命技术来表征活细胞质膜中的结构域结构。*反式对羟基苯甲酸(TPA)的荧光寿命对膜的顺序很敏感，可以通过显示多个寿命自发地检测多个膜结构域，范围从1-100 ns。这种探头的寿命非常适合于检测结构域，无论其大小如何。我们将使用形成光学可分辨区域的GUV来验证该方法，然后将其应用于LUV和活细胞的质膜。*3.鉴定CerS2对活细胞质膜结构域结构的影响。*CerS2与人类疾病有关，分子机制未知。我们观察到鞘磷脂酰链长度可以改变活细胞的模型膜和质膜中的胆固醇分配和膜域。有趣的是，CerS2功能的丧失会导致C24鞘脂的减少，质膜中的C16鞘脂取代了C24鞘脂。我们将使用上述技术来表征CerS2在哺乳动物细胞中胆固醇分配和微域形成中的作用。

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.