Endocytic transport in cultured renal epithelia

培养的肾上皮细胞的内吞转运

• 批准号: 6883966
• 负责人: KENNETH W DUNN
• 金额: $ 25.55万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6883966
• 关键词: cellular polarity; confocal scanning microscopy; endocytosis; epithelium; gel electrophoresis; intracellular transport; mass spectrometry; membrane transport proteins; protein structure function; renal tubular transport; renal tubule; tissue /cell culture; transfection

Endocytic membrane transport is critical to many facets of renal function. Vectorial transport of ions, solutes and proteins is regulated by the polarized expression of transporters and receptors, which is determined by rates of endocytic insertion and retrieval. The plasma membrane expression of transporters is frequently regulated by signaling receptors, whose expression is likewise regulated by endocytosis. As many diseases, such as insulin-dependent diabetes, cystic fibrosis and Liddle's syndrome are associated with disruptions of endocytic membrane transport, it is clearly important to understand the molecular mechanisms regulating endocytosis. Combined molecular and cell biological studies of fibroblasts have elucidate the molecular regulation of many endocytic transport steps. However, comparable studies have not been conducted in polarized epithelial and it is clear that the studies of fibroblasts do not apply to epithelia, whose endocytic pathways are regulated by epithelial-specific proteins, and by ubiquitous proteins that acquire new functions within the more complex membrane pathways of epithelia. To better understand molecular regulations of epithelial endocytosis we propose to address the epithelial function of proteins known to participate in membrane recycling, both those common to epithelia and fibroblasts, Rab4a, Rab11a and Rme-1, as well as those specific to epithelial cells, Rab17 and Rab25. We will also identify proteins that regulate polar sorting in endosomes by characterizing how the protein constitution of endosomes is altered when polar sorting is disrupted. Endosome protein analysis will be assayed using subcellular fractionation, 2-dimensional gel electrophoresis and mass spectroscopy. The cellular effects of transfected proteins will be evaluated through the combined use of quantitative confocal microscopy of polarized cells expressing GFP chimeras of mutant and wildtype proteins and biochemical analysis of stable cell lines expressing transfected proteins. The dissection of the molecular mechanisms of endocytosis provided here will illuminate the processes that regulate membrane recycling, transcytosis, transport to the trans-Golgi network and plasma membrane polarity. In so doing, they will provide the framework for understanding diseases, and developing therapeutics for transporter disorders, immune disorders, toxin-mediated pathologies and polarity disorders, such as polycystic kidney disease.

内吞膜转运对肾功能的许多方面至关重要。离子、溶质和蛋白质的载体转运受转运蛋白和受体的极化表达调节，其由内吞插入和回收速率决定。转运蛋白的质膜表达通常受信号受体调节，信号受体的表达同样受内吞作用调节。由于许多疾病，如胰岛素依赖型糖尿病，囊性纤维化和Liddle综合征与内吞膜转运的破坏有关，因此了解调节内吞作用的分子机制显然很重要。结合成纤维细胞的分子和细胞生物学研究阐明了许多内吞转运步骤的分子调控。然而，在极化上皮细胞中尚未进行类似的研究，很明显，成纤维细胞的研究不适用于上皮细胞，其内吞途径受上皮细胞特异性蛋白质和在上皮细胞更复杂的膜途径中获得新功能的普遍存在的蛋白质的调节。为了更好地了解上皮内吞作用的分子调节，我们建议解决已知参与膜回收的蛋白质的上皮功能，包括上皮和成纤维细胞常见的Rab 4a、Rab 11 a和Rme-1，以及上皮细胞特异性的蛋白质Rab 17和Rab 25。我们还将通过表征当极性分选被破坏时内体的蛋白质组成如何改变来鉴定调节内体中极性分选的蛋白质。将使用亚细胞分级分离、二维凝胶电泳和质谱法分析内体蛋白。将通过结合使用表达突变体和野生型蛋白质的GFP嵌合体的极化细胞的定量共聚焦显微镜和表达转染蛋白质的稳定细胞系的生化分析来评价转染蛋白质的细胞效应。本文对内吞作用的分子机制进行了剖析，阐明了调节膜再循环、胞吞转运、转运至高尔基体网络和质膜极性的过程。 在这样做的过程中，他们将为理解疾病提供框架，并为转运蛋白紊乱、免疫紊乱、毒素介导的病理和极性紊乱（如多囊肾病）开发治疗方法。