Transient loss of plasma membrane asymmetry in mammalian cells: mechanisms and function

项目摘要/摘要 真核细胞质膜的一个主要特征是各种脂质的不对称分布 类型横跨两个膜的小叶。维持这种跨双层组织需要投资 重要的能量来源，暗示了小叶间脂类分离的重要功能作用。一位杰出的 例如带电的脂质，如磷脂酰丝氨酸(PS)：通常仅限于细胞质PM小叶 对于健康的细胞，它们在外质表面的永久暴露标志着细胞被巨噬细胞吞噬。 这一模式最近受到了观察的挑战，因为观察到PS暂时翻转到外部PM叶 在抗原诱导的健康免疫细胞激活过程中。这一现象的目的和机制 完全未知，也不清楚这种重新分配是否是PS特有的，或者更确切地说，导致批发 两个PM小叶的脂体学和生物物理混杂。为了调查这些有趣的问题，我们 建议研究这种可逆脂质的特性、调节机制和功能作用 在免疫细胞激活过程中争先恐后。在目标1中，我们将使用先进的脂质组学和成像技术来 描述免疫细胞激活过程中膜脂组织的变化。我们假设 抗原活化诱导一过性、高度局部性和非特异性的脂质扰乱，而不是 典型的永久性PS暴露与细胞凋亡相关。在目标2中，我们将识别分子 造成这种影响的机制。使用超分辨率显微镜，我们将监测纳米级 各种脂类转运体、翻转的脂类和免疫信号级联中的关键机制的组织。 我们希望观察到这些关键分子在特定PM领域的空间隔离和限制。 最后，在目标3中，我们将通过评估PM的效果来检查脂质扰乱的功能含义 蛋白质-膜相互作用的不对称性。我们假设，激活诱导的扰乱降低了 内PM叶的电荷密度，导致电荷依赖的信号蛋白从 薄膜。为了验证这一假设，我们将使用原位荧光显微镜以及生物物理工具 在体外和硅胶中-检测含有多碱结构域的蛋白质的膜结合亲和力，包括 癌基因RAS，在对称和不对称膜中。这些目标的成功实现将 对膜组织及其结构的生理调节变化产生重要而新颖的见解 在免疫细胞激活中的作用。此外，这些研究将提供第一个详细的脂质学和生物物理学 未受干扰和功能修改的PM不对称性的表征。这一发现可能是可扩展的 在各种其他情况下对细胞的激活，因此预计将通过揭示 为理解和治疗病理学提供新的潜在靶点。

PROJECT SUMMARY / ABSTRACT A central feature of the plasma membrane (PM) of eukaryotic cells is the asymmetric distribution of various lipid types across the two membrane leaflets. Maintaining such transbilayer organization requires investment of significant energetic resources, implying an important functional role for interleaflet lipid segregation. A prominent example are charged lipids like phosphatidylserine (PS): normally restricted to the cytoplasmic PM leaflet in healthy cells, their permanent exposure on the exoplasmic face marks the cell for phagocytosis by macrophages. The paradigm has recently been challenged by observations that PS temporarily flips to the outer PM leaflet during antigen-induced activation in healthy immune cells. The purpose and mechanisms of this phenomenon are wholly unknown, nor is it clear whether this redistribution is specific for PS, or rather leads to wholesale lipidomic and biophysical scrambling of the two PM leaflets. To investigate these intriguing questions, we propose to study the characteristics, regulatory mechanisms, and functional roles of this reversible lipid scrambling during immune cell activation. In Aim 1, we will use advanced lipidomics and imaging techniques to characterize the changes in membrane lipid organization during immune cell activation. We hypothesize that antigen activation induces transient, highly localized, and non-specific lipid scrambling, in contrast to the canonical permanent PS exposure associated with apoptosis. In Aim 2, we will identify the molecular mechanisms responsible for this effect. Using super-resolution microscopy, we will monitor the nanoscale organization of various lipid translocators, flipped lipids, and key machinery in the immune signaling cascade. We expect to observe spatial segregation and confinement of these key molecules into specialized PM domains. Finally, in Aim 3 we will examine the functional implications of lipid scrambling by evaluating the effect of PM asymmetry on protein-membrane interactions. We hypothesize that activation-induced scrambling reduces the charge density of the inner PM leaflet, leading to dissociation of charge-dependent signaling proteins from the membrane. To test this hypothesis, we will use fluorescence microscopy in situ—as well as biophysical tools in vitro and in silico—to examine the membrane binding affinity of polybasic-domain containing proteins, including the oncogene Ras, in symmetric and asymmetric membranes. The successful execution of these aims will produce important and novel insights into physiologically regulated changes in membrane organization and its role in immune cell activation. Moreover, these studies will provide the first detailed lipidomic and biophysical characterization of both unperturbed and functionally modified PM asymmetry. The findings may be extensible to cell activation in a variety of other contexts, and are thus expected to have far-reaching impacts by revealing new potential targeting for understanding and treating pathologies.