Endocytic transport in cultured renal epithelia

内细胞膜转运对肾功能的许多方面都是至关重要的。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，而转运体和受体的极化表达是由胞内插入和提取的速率决定的。转运蛋白的质膜表达通常受信号受体的调节，信号受体的表达同样受内吞作用的调节。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细胞膜运输中断有关，因此了解调控内吞作用的分子机制显然很重要。成纤维细胞的分子生物学和细胞生物学相结合的研究阐明了许多细胞内转运步骤的分子调控。然而，在极化上皮细胞中还没有进行类似的研究，很明显，成纤维细胞的研究不适用于上皮细胞，因为上皮细胞的内吞途径受上皮特有的蛋白质调节，也不适用于在更复杂的上皮膜通路中获得新功能的普遍存在的蛋白质。为了更好地了解上皮细胞内吞作用的分子调控，我们建议研究已知的参与膜循环的蛋白质的上皮功能，包括上皮细胞和成纤维细胞共同的蛋白质Rab4a、Rab11a和RME-1，以及上皮细胞特有的蛋白质Rab17和Rab25。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是如何改变的，来识别调节内体极性分选的蛋白质。内体蛋白分析将使用亚细胞分离、二维凝胶电泳和质谱仪进行分析。通过表达突变型和野生型蛋白GFP嵌合体的极化细胞的定量共聚焦显微镜和稳定表达转基因蛋白的细胞系的生化分析相结合的方法来评估转基因蛋白的细胞效应。这里提供的内吞作用分子机制的剖析将阐明调节膜循环、跨细胞作用、向跨高尔基网络的运输和质膜极性的过程。通过这样做，他们将提供了解疾病的框架，并开发针对转运蛋白障碍、免疫障碍、毒素介导的病理和极性障碍的治疗方法，如多囊肾病。

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.