TRANSFORMATIVE LIPID EXCHANGE APPROACHES TO STUDY MEMBRANE ORGANIZATION

我们的目标是通过实验室取得的突破来了解生物膜的结构/功能 环糊精催化脂对膜磷脂和鞘磷脂组成的调控 交换。脂交换通常是有效的(80%-100%)，并允许制备脂泡 在脂质不对称性方面与自然膜非常相似，在脂质方面的差异 内层和外层脂单分子层(叶)的组成。实验室现在已经将这种方法扩展到 控制哺乳动物细胞质膜外叶的脂质成分(不损害 它们)，并且不干扰膜类固醇。该方法正被应用于解决长期存在的问题 膜的组织和功能问题。我们在1994年提出了仍然有效的工作模式： 由于神经鞘脂脂-胆固醇富集液的有序分离而在细胞中形成膜结构域 (LO)不饱和富脂液体无序结构域。在上一次授权期内，使用类脂 交换制备不对称膜，我们发现每个小叶中的脂质结构是如何控制的 当存在LO结构域的整体诱导或抑制时。对于生理上相关的脂类，我们 发现LO结构域的形成因脂质不对称性的丧失而大大增强，并证实了这一点 在天然质膜囊泡中也是如此。这一点很重要，因为一过性的脂肪损失 非对称性发生在信号转导过程中。在下一个授权期内，我们建议检验这一假设 LO结构域的形成是通过使用红细胞使完整细胞中的脂质不对称性丧失而引起的， 它们有许多优点，包括只有一个膜，已知的脂质不对称，已知的 方法原位控制脂质不对称性，便于脂质交换。然后我们将延长 对代谢活性细胞的研究，以了解脂质不对称性的丧失是否稳定了LO结构域 免疫RBL-2H3细胞中抗体触发的信号转导及LOG的重要性 通过使用促进或抑制LO结构域形成的脂类交换结构域。在上一次授权期内 我们还使用脂质交换来证明形成LO结构域的能力是必要的和充分的 支持胰岛素受体功能的脂质。我们提出了在LO域中定位的假设， 它们是厚的，减少胰岛素受体的跨膜螺旋倾斜，激活自磷酸化。 我们将通过定义激活前后的受体结构域定位以及通过 延长或缩短其跨膜片段的突变，以查看预测的活性变化是否 观察到的。我们还建议测试一种新的竞争分析来定义受体结构域的定位。 最后，我们建议将脂交换扩展到膜包膜病毒。一系列的血脂将是 与SARS CoV-2尖峰蛋白或VSV-G蛋白交换到假病毒的包膜中，了解如何 病毒脂/结构域调节细胞的摄取。这些研究可能有助于改进疫苗的设计。

We aim to understand biomembrane structure/function with the aid of a lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. Lipid exchange is generally efficient (80-100%), and permits preparation of lipid vesicles mimicking natural membranes closely in terms of lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets). The lab has now extended this method to control lipid composition of plasma membrane outer leaflets in mammalian cells (without harming them), and without disturbing membrane sterol. The method is being applied to solve long-standing issues of membrane organization and function. We proposed in 1994 what remains the working model: that membrane domains form in cells due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. In the last grant period, using lipid exchange to prepare asymmetric membranes, we found how lipid structure in each leaflet controls when there is overall induction or suppression of Lo domains. With physiologically-relevant lipids, we found that Lo domain formation is greatly enhanced by loss of lipid asymmetry, and confirmed this is also true in natural plasma membrane vesicles. This is important because transient loss of lipid asymmetry occurs during signal transduction. In the next grant period we propose to test the hypothesis that Lo domain formation is induced by loss of lipid asymmetry in intact cells by using red blood cells, which have a number of advantages including only a single membrane, known lipid asymmetry, known methods to control lipid asymmetry in situ, and facile use for lipid exchange. We will then extend studies to metabolically-active cells to see if loss of lipid asymmetry stabilizes Lo domains during antibody-triggered signal transduction in immune RBL-2H3 cells, and defining the importance of Lo domains by exchange using lipids that enhance or inhibit Lo domain formation. In the last grant period we also used lipid exchange to show the ability to form Lo domains is necessary and sufficient for a lipid to support insulin receptor function. We proposed the hypothesis that localization in Lo domain, which are thick, decreases insulin receptor transmembrane helix tilt, activating autophosphorylation. We will test this hypothesis by defining receptor domain localization before and after activation, and by mutations that lengthen or shorten its transmembrane segment, to see if predicted activity changes are observed. We also propose to test a novel competition assay for defining receptor domain localization. Finally, we propose to extend lipid exchange to membrane enveloped viruses. A range of lipids will be exchanged into the envelope of pseudovirus with SARS CoV-2 spike or VSV-G proteins to see how viral lipids/domains modulate cellular uptake. These studies may help design of improved vaccines.