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

项目摘要 上皮细胞极化是一个重要的生物学过程，具有多种生理功能，包括组织 形态发生和伤口愈合。极化的一个定义特征是细胞质膜的分离 (PM)脂质和蛋白质进入顶端和基底外侧区室，其间的分子交换是 限制。几十年前，发现顶端PM富含饱和脂质、糖脂和胆固醇。 解释顶端PM的生物成因和独特组成的机制假说包括自我- 组装膜结构域（即，脂筏），特异性蛋白分选基序，和翻译后 介导蛋白质分选的修饰。然而，无论是蛋白质的详细组成，还是蛋白质的机制， 以及上皮细胞中PM结构域之间的脂质分选。基底外侧动脉的脂质分布 PM仍然没有解决，留下了关于顶侧和基底侧的分化和脂质分离的疑问 下午.蛋白质分选的一般决定因素知之甚少，过去的研究集中在 特定的蛋白质或贩运机制。重要的是，顶端PM宿主广泛的细胞外糖萼 由糖脂、糖蛋白和多糖组成，可通过糖结合蛋白交联 称为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 尚未发现，尽管大部分顶端蛋白被糖基化。最后， 极化上皮呈现高度向外弯曲的膜形状，例如纤毛或微绒毛，但还必须 同时能够形成向内的膜内陷，用于细胞信号传导和从细胞中摄取。 细胞外间隙形成这些高度弯曲的膜结构的驱动力仍然存在 不确定。我们将调查这些知识差距在膜和上皮生物学。在目标1中，我们 使用先进的脂质组学和成像技术来表征脂质组织、膜 组成和细胞极化过程中的膜特性。我们假设， 相对于基底外侧PM，PM将富含高度饱和的脂质和糖脂， 生物物理特性反映了筏富集环境。在目标2中，我们将系统地评估 跨膜蛋白顶端与基底外侧分选的结构决定因素，包括跨膜 结构域特征、蛋白筏亲和力和糖基化。我们假设这些蛋白质特征协同作用 直接将蛋白分选至顶端PM。在目标3中，我们将探讨顶端PM糖萼在驱动细胞生长中的作用。 膜弯曲我们假设凝集素介导的相互作用控制着细胞的曲率和组织。 膜。这些目标的成功实现将解决膜研究中几个很大程度上尚未解决的问题 生物学关于脂质组成，膜特性和蛋白质结构特征如何协调组织 极化细胞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.