Composition, sorting, and morphology of the apical plasma membrane in epithelial cells.

项目摘要 上皮细胞极化是一种基本的生物过程，具有许多生理作用，包括组织 形态发生与伤口愈合。极化的一个重要特征是细胞质膜的分离。 (PM)脂类和蛋白质进入心尖室和基底侧室，其间进行分子交换 有限制。几十年前，顶端PM被发现富含饱和脂肪、糖脂和胆固醇。 解释心尖部PM的生物发生和独特成分的机械假说包括 组装膜结构域(即脂筏)、特定的蛋白质分类基序和翻译后 调节蛋白质分选的修饰。然而，无论是蛋白质的详细组成还是作用机制 并且上皮细胞中PM结构域之间的脂质分类已经得到解决。基底外侧的脂类分布 PM仍然没有解决，留下了关于根尖和基底外侧的分化和脂质分离的疑问。 下午。蛋白质分类的一般决定因素知之甚少，过去的研究主要集中在这两个方面 特定的蛋白质或运输机器。重要的是，心尖部PM有广泛的细胞外糖萼。 由糖脂、糖蛋白和多糖组成，可通过糖结合蛋白进行交联 天然存在于细胞外空间的凝集素。这些糖萼分子在蛋白质分选中的作用 还没有被揭示，尽管顶端蛋白的主要部分是糖基化的。最后，最重要的PM 极化上皮具有高度向外弯曲的膜形状，如纤毛或微绒毛，但也必须 能够同时形成向内的膜内陷，用于细胞信号传递和从 细胞外空间。形成这些高度弯曲的膜结构的驱动力仍然存在 未定。我们将调查这些在膜和上皮生物学方面的知识差距。在目标1中，我们将 使用先进的脂质组学和成像技术来表征脂质组织、膜的变化 细胞极化过程中的组成和膜的性质。我们假设心尖部 相对于基底外侧PM，PM将富含高度饱和的脂类和糖脂，而心尖PM 反映浮筏丰富环境的生物物理特性。在目标2中，我们将对 包括跨膜在内的顶端与基侧分选的跨膜蛋白结构决定因素 结构域特征、蛋白RAFT亲和力和糖基化。我们假设这些蛋白质是协同作用的 直接对根尖PM进行蛋白质分选在目标3中，我们将探索顶端PM糖萼在驾驶中的作用 膜弯曲。我们假设凝集素介导的相互作用控制着 膜。这些目标的成功实施将解决几个在很大程度上悬而未决的问题 关于脂质组成、膜特性和蛋白质结构特征如何协调组织的生物学 极化电池PM的组成和结构

Project Summary Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane (PM) lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical PM was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical PM include self- assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between PM domains in epithelial cells have been resolved. The lipid profile of the basolateral PM remains unresolved, leaving doubts about the differentiation and lipid separation of the apical and basolateral PM. The general determinants of protein sorting are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical PM hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides, which can be crosslinked by sugar binding proteins called lectins natively present in the extracellular space. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. Finally, the apical PM of polarized epithelia takes on highly outward-curved membrane shapes such as cilia or microvilli, but must also be simultaneously capable of forming inward membrane invaginations for cell signaling and uptake from the extracellular space. The driving forces for the formation of these highly-curved membrane structures remain undetermined. We will investigate these knowledge gaps in membrane and epithelial biology. In Aim 1, we will use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We hypothesize that the apical PM will be enriched in highly saturated lipids and glycolipids relative to the basolateral PM, with apical PM biophysical properties reflecting a raft-enriched environment. In Aim 2, we will systematically evaluate the transmembrane protein structural determinants of apical versus basolateral sorting including transmembrane domain features, protein raft affinity, and glycosylation. We hypothesize that these protein features cooperatively direct protein sorting to the apical PM. In Aim 3, we will explore the role of the apical PM glycocalyx in driving membrane bending. We hypothesize that lectin-mediated interactions control the curvature and organization of membranes. Successful execution of these aims will address several largely open questions in membrane biology about how lipid composition, membrane properties, and protein structural features coordinate to organize composition and structure of the polarized cell PM.