Lipocalin-dependent glycocalyx organization and tube protection

项目摘要 一种富含脂质和糖的顶端（管腔）细胞外基质（aECM）形成并保护着胃管， 我们的身体例如，肺表面活性物质有助于狭窄的气道保持开放， 保护血管系统中的毛细血管。对这种管腔基质的损伤可导致管塌陷或破裂。 泄漏，导致疾病。尽管管腔aECM的重要性，我们只有一个初步的 了解它的多层组织，它是如何组装的，以及如何适当的脂质含量是 确保。专门的溶酶体相关细胞器（LRO）储存，分泌和回收aECM脂质，但 控制脂质转运至LRO和aECM以及在LRO和aECM之间转运的机制尚不清楚。也不清楚 饮食和代谢变化如何影响aECM脂质，以及这可能如何影响管 完整不幸的是，管腔aECM的脆弱性质使得在大多数情况下难以可视化和研究。 系统.我们将利用透明模式生物C。来研究两个 aECM中脂质转运蛋白-脂质运载蛋白和清道夫受体B（SCARB）蛋白的保守家族 组织和管保护。实时成像和电子显微镜都使我们能够可视化管腔 aECM前所未有的细节。正向和反向遗传学方法使我们能够识别和操纵 基质中的相关糖蛋白和脂质。C.前表皮线虫 aECM和哺乳动物糖萼之间以及C.线虫和哺乳动物LRO的研究表明， 系统将揭示aECM贩运和组装的广泛相关原理。目标1将检验假设 两种脂质运载蛋白顺序地将磷脂酰胆碱（PC）转运到LRO和aECM。我们将使用 成像、遗传和生物化学方法来鉴定脂质运载蛋白的细胞和aECM区室， 和货物，测试对aECM的代谢影响，并将脂质运载蛋白与脂质运输的特定步骤联系起来， aECM总成。目的2将检验SCARB与人LIMP-2（溶酶体积分）相关的假设 膜蛋白2）通过对LRO中PC代谢的影响对抗脂质运载蛋白。我们将结合联合收割机成像和 遗传学方法来鉴定相关的SCAV-2区室和货物，并测试PC水平升高是否可以 解释SCAV-2缺失如何抑制脂质运载蛋白突变体致死性。我们还将克隆其他抑制基因座， 确定SCAV-2合作伙伴。总之，这些目标将揭示管腔aECM组装的基本原理， 组织，并阐明特定脂质转运蛋白家族的功能和之间的关系， 在人类生物学和疾病中的不同作用。

Project Summary A lipid and sugar-rich apical (luminal) extracellular matrix (aECM) shapes and protects the narrowest tubes in our bodies. For example, lung surfactant helps narrow airways remain open, and the endothelial glycocalyx protects capillaries in the vascular system. Damage to such luminal matrices can cause tube collapse or leakage, contributing to disease. Despite the importance of luminal aECM, we have only a rudimentary understanding of its multi-layered organization, how it is assembled, and how appropriate lipid content is ensured. Specialized lysosome-related organelles (LROs) store, secrete and recycle aECM lipids, but mechanisms controlling lipid transport to and between LROs and aECM are poorly defined. It is also unclear how dietary and metabolic changes affect aECM lipids, and how this might contribute to effects on tube integrity. Unfortunately, the fragile nature of the luminal aECM makes it difficult to visualize and study in most systems. We will take advantage of the transparent model organism C. elegans to study opposing roles of two conserved families of lipid transporters - lipocalins and Scavenger Receptor B (SCARB) proteins - in aECM organization and tube protection. Both live imaging and electron microscopy allow us to visualize luminal aECM in unprecedented detail. Forward and reverse genetic approaches allow us to identify and manipulate relevant glycolproteins and lipids in the matrix. Molecular similarities between the C. elegans pre-cuticular aECM and the mammalian glycocalyx, and between C. elegans and mammalian LROs, suggest that this system will reveal broadly relevant principles of aECM trafficking and assembly. Aim 1 will test the hypothesis that two lipocalins act sequentially to transport phosphatidylcholine (PC) to LROs and aECM. We will use imaging, genetic, and biochemical approaches to identify lipocalins' cellular and aECM compartments, partners and cargoes, test metabolic influences on aECM, and tie lipocalins to specific steps of lipid trafficking and aECM assembly. Aim 2 will test the hypothesis that a SCARB related to human LIMP-2 (lysosomal integral membrane protein 2) opposes lipocalins via effects on PC metabolism in LROs. We will combine imaging and genetic approaches to identify relevant SCAV-2 compartments and cargos, and test if elevated PC levels can explain how SCAV-2 loss suppresses lipocalin mutant lethality. We will also clone other suppressor loci to identify SCAV-2 partners. Together, these Aims will reveal basic principles of luminal aECM assembly and organization, and elucidate the functions of, and relationships between, specific lipid transporter families that have previously been studied for separate roles in human biology and disease.