Endocytic transport in cultured renal epithelia

内吞膜运输对肾功能的许多方面至关重要。离子、溶质和蛋白质的载体运输是由转运体和受体的极化表达调节的，这是由内吞插入和回收的速率决定的。转运蛋白的质膜表达经常受到信号受体的调节，而信号受体的表达同样受到内吞作用的调节。由于许多疾病，如胰岛素依赖型糖尿病、囊性纤维化和利德尔综合征都与内吞膜运输的破坏有关，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生物学和细胞生物学研究已经阐明了许多内吞转运步骤的分子调控。然而，在极化上皮中还没有进行类似的研究，很明显，对成纤维细胞的研究并不适用于上皮，上皮的内吞途径是由上皮特异性蛋白和在上皮更复杂的膜途径中获得新功能的无所不在的蛋白调节的。为了更好地理解上皮内吞噬的分子调控，我们建议研究已知参与膜再循环的蛋白的上皮功能，包括上皮细胞和成纤维细胞共有的Rab4a、Rab11a和Rme-1，以及上皮细胞特有的Rab17和Rab25。我们还将通过描述当极性分选被破坏时内体的蛋白质结构如何改变来确定调节内体极性分选的蛋白质。内核体蛋白分析将使用亚细胞分离、二维凝胶电泳和质谱进行分析。转染蛋白的细胞效应将通过结合使用表达突变型和野生型蛋白的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的稳定细胞系的生化分析来评估。这里提供的内吞作用分子机制的解剖将阐明调节膜循环、胞吞作用、转运到反式高尔基网络和质膜极性的过程。在此过程中，他们将为理解疾病提供框架，并开发针对转运蛋白紊乱、免疫紊乱、毒素介导的病理和极性紊乱（如多囊肾病）的治疗方法。

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.