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

长酰基链鞘脂在哺乳动物细胞质膜侧向组织中的作用

• 批准号: RGPIN-2018-04229
• 负责人: Zha, Xiaohui
• 金额: $ 1.75万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712816
• 关键词: role; long; acyl; chain; sphingolipids

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.

鞘脂，主要是C24和C16，仅存在于哺乳动物细胞质膜的外小叶中。奇怪的是，很少有人知道这种双层不对称性如何影响鞘脂-胆固醇相互作用，特别是膜微区的组织。在当前的发现资助期间（2013-2018），我们能够确定体内最丰富的鞘脂之一C24鞘磷脂与胆固醇独特地相互作用，并且通过产生胆固醇不对称性来控制质膜中的侧向组织。 对于下一个发现补助金，我们建议继续这一研究方向，但扩大范围，纳入更先进的生物物理分析，并了解具有以下特定目标的生物学意义。 1.使用家庭和hetero-FRET了解纳米和亚微米结构域的质膜活细胞。 我们已经使用这些蛋白质进行异源FRET分析（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，质膜几乎没有亚微米结构域，可能是由于C24鞘脂，因此20/80胆固醇在质膜中分配。 然而，已知质膜形成纳米结构域（~50 A2），其可以被homo-FRET（在YFP之间）检测到。 我们将使用homo-FRFT来确定C24 SM和胆固醇分配是否影响纳米结构域。 2.开发荧光寿命技术，以表征活细胞质膜中的域结构。 反式parinatic酸（tPA）的荧光寿命是敏感的膜秩序，并可以自发地检测多个膜域显示多个寿命，范围从1-100 ns。 该探针的寿命非常适合检测域，无论大小如何。 我们将使用GUV，形成光学可分辨的域，以验证该方法，然后将其应用于LUV和活细胞的质膜。 3.表征CerS 2对活细胞质膜结构域的影响。 CerS 2与人类疾病有关，分子机制未知。我们观察到，鞘脂酰基链的长度可以改变胆固醇分配和膜结构域模型膜和活细胞的质膜。有趣的是，CerS 2的功能丧失导致C24鞘脂减少，其在质膜中被C16鞘脂取代。 我们将使用上述技术来表征CerS 2在哺乳动物细胞中胆固醇分配和微区形成中的作用。