Understanding the function of recycling endosomes

了解回收内体的功能

• 批准号: RGPIN-2021-02889
• 负责人: Lacy, Paige
• 金额: $ 3.06万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760822
• 关键词: Understanding; function; recycling; endosomes

My research program is focused on the role of membrane-bound recycling endosomes (REs) in protein trafficking and secretion. REs are specialized secretory compartments that were originally characterized for their ability to recycle cell surface receptors through endocytic and exocytic pathways. Recently, REs were found to have a role in intracellular protein trafficking and secretion beyond recycling receptors to cell surfaces. Studies have shown that REs are implicated in the secretion of proteins to the extracellular milieu. However, the function of REs in trafficking and secretion of proteins remains poorly understood. In this proposal, we aim to study the specific role of REs in contributing to the trafficking and release of cell-derived mediators using epithelial cells. Epithelial cells have robust protein trafficking systems that serve as perfect models for studying RE trafficking, and the secretion of proteins is well defined in these cells. They are also polarized cells, capable of directional release of proteins through apical and basolateral surfaces. Therefore, epithelial cells provide an ideal system for understanding protein trafficking through REs. Objectives The long-term objective is to determine the function of REs in protein trafficking and secretion in epithelial cells. There is very little known about the role of REs in mediator release from epithelial cells, and yet there is strong evidence for their existence in these cells. The specific short-term objectives are to: 1. Specifically label REs in epithelial cells and study their redistribution in response to specific stimuli (e.g. Toll-like, tyrosine kinase, and G-protein coupled receptors). 2. Determine mechanisms of RE-dependent secretory pathways in epithelial cells by application of gene deletion technology to effectively remove this compartment. 3. Characterize the role of rapidly and slowly recycled compartments of REs in mediator trafficking in epithelial cells. Specifically, we propose to resolve the requirements for REs in the release of proteins and mediators from epithelial cells. REs will be characterized for their role in mediator trafficking by the application of gene deletion technology. We will also delve more deeply into defining and characterizing RE subcompartments, known as rapidly and slowly recycled compartments of REs, in protein trafficking in epithelial cells. Using high resolution imaging, gene deletion technology (CRISPR and si/shRNA) and ultrastructural studies, we will map the fine distribution of RE-associated proteins during epithelial cell secretion of mediators. Outcomes My research program will reveal novel membrane trafficking compartments required for mediator trafficking in epithelial cells, important cells in the maintenance of barrier function. Understanding the function of REs in epithelial cells will lead to the elucidation of novel membrane trafficking pathways for REs that serve an essential function in protein secretion.

我的研究项目主要集中在膜结合再循环内体（RE）在蛋白质运输和分泌中的作用。RE是特化的分泌隔室，其最初的特征在于其通过内吞和外吞途径回收细胞表面受体的能力。最近，发现RE在细胞内蛋白质运输和分泌中的作用超出了将受体再循环到细胞表面。研究表明，RE与蛋白质分泌到细胞外环境有关。然而，RE在蛋白质运输和分泌中的功能仍然知之甚少。在这项提案中，我们的目标是研究的具体作用，RE在促进贩运和释放的细胞源性介质使用上皮细胞。上皮细胞具有强大的蛋白质运输系统，可作为研究RE运输的完美模型，并且蛋白质的分泌在这些细胞中被很好地定义。它们也是极化细胞，能够通过顶端和基底侧表面定向释放蛋白质。因此，上皮细胞提供了一个理想的系统，通过REs. Objectives了解蛋白质贩运的长期目标是要确定的功能RE在上皮细胞中的蛋白质贩运和分泌。关于RE在上皮细胞释放介质中的作用知之甚少，但有强有力的证据表明它们存在于这些细胞中。具体的短期目标是：1。特异性标记上皮细胞中的RE，并研究它们对特定刺激（例如Toll样受体、酪氨酸激酶受体和G蛋白偶联受体）的再分布。2.通过应用基因缺失技术有效去除上皮细胞中的RE依赖性分泌途径，确定其机制。 3.表征RE的快速和缓慢再循环隔室在上皮细胞中的介质运输中的作用。具体来说，我们建议解决的要求RE在释放蛋白质和介质从上皮细胞。RE将通过基因缺失技术的应用表征其在介体运输中的作用。我们还将更深入地研究定义和表征RE亚室，称为RE的快速和缓慢回收室，在上皮细胞中的蛋白质运输。利用高分辨率成像、基因缺失技术（CRISPR和si/shRNA）和超微结构研究，我们将绘制上皮细胞分泌介质过程中RE相关蛋白的精细分布。 结果我的研究计划将揭示新的膜运输隔间所需的介质运输上皮细胞，重要的细胞在维护屏障功能。了解RE在上皮细胞中的功能将导致阐明RE在蛋白质分泌中起重要作用的新的膜运输途径。