EP164 coordinates arf6- and Rab-dependent membrane traffic in epithelial cells

描述（由申请人提供）：上皮细胞需要调节囊泡运输，以维持其动态肌动蛋白细胞骨架和极性。虽然已经研究了许多信号通路，但细胞如何协调膜运输和细胞骨架组织的调节仍不清楚。我们实验室最近的工作表明，微绒毛蛋白EPI 64对Arf 6和一种未知的Rab蛋白都具有调节功能。EPI 64的TBC结构域直接结合活性Arf 6-GTP，并且EPI 64的过表达诱导肌动蛋白包被的空泡的积累，这可能是由经由PI 4P 5 K活化的PIP 2的过度产生驱动的。仍然结合Arf 6-GTP的GAP缺陷型突变体EPI64_R160A的表达不能诱导空泡，表明空泡积累需要GAP活性。另外，通过EPI 64表达的这些空泡的出现通过Rab 8a的共表达而消除。这些结果表明，EPI 64协调Arf 6活性与Rab蛋白，可能Rab 8，以一种新的方式来调节膜交通和细胞骨架组织。本研究的目的是鉴定与EPI 64 GAP活性相关的Rab蛋白，并阐明EPI 64如何参与这些运输途径。第一个目标是鉴定哪些Rab蛋白受EPI 64过表达的影响。由于EPI 64的过表达导致细胞中肌动蛋白包被的空泡的形成，因此这可以用作筛选能够压倒EPI 64差距活性的Rab蛋白的测定，并且因此是再循环途径的相关调节剂。在目标2中，将通过在下拉试验中使用效应子测量细胞中的Rab-GTP水平来研究与EPI 64潜在相关的Rab蛋白的生物化学。将使用纯化的蛋白质定量测量EPI 64对Rab蛋白质的GAP活性，所述Rab蛋白质被发现在Rab-GTP水平中具有EPI 64依赖性变化。目标3将集中于确定当这些膜运输途径受到干扰时在细胞中形成的空泡的形成和结构。高分辨率免疫荧光活细胞成像将用于确定肌动蛋白包被的空泡形成期间GFP标记的分子的时间和空间共定位。这将通过透射电子显微镜进行补充，以在不同时间点对空泡的形成和结构进行成像。相关性：在形成肠或肾脏内部结构衬里的上皮细胞中保持独特的极性对于营养物质的吸收和分布至关重要。破坏这些细胞的细胞内运输网络可能导致营养不良，癌症和最终死亡。

DESCRIPTION (provided by applicant): Epithelial cells require regulated vesicle trafficking in order to maintain their dynamic actin cytoskeleten and polarity. Although many signaling pathways have been studied, how a cell coordinates the regulation of membrane-trafficking and cytoskeletal organization remains unclear. Recent work from our laboratory has revealed that a microvillar protein, EPI64, has regulatory functions for both Arf6 and an unknown Rab protein. EPI64's TBC domain binds directly to active Arf6-GTP and EPI64's over- expression induces an accumulation of actin-coated vacuoles likely driven by the over production of PIP2 via PI4P5K activation. Expression of a GAP-defective mutant that still binds to Arf6-GTP, EPI64_R160A, fails to induce vacuoles, indicating that GAP activity is required for vacuole accumulation. Additionally, the appearance of these vacuoles by EPI64 expression is eliminated by the co-expression of Rab8a. These results suggest that EPI64 coordinates Arf6 activity with a Rab protein, possibly Rab8, in a novel manner to regulate both membrane traffic and cytoskeletal organization. The goal of this research is to identify the relevant Rab protein(s) for EPI64's GAP activity, and to elucidate how EPI64 participates in these trafficking pathways. The first aim will be to identify which Rab proteins are affected by the overexpression of EPI64. Since overexpression of EPI64 results in the formation of actin-coated vacuoles in the cell, this can be used as an assay to screen for Rab proteins that are able to overwhelm the GAP activity of EPI64, and therefore be the relevant regulator of the recycling pathway. The biochemistry of Rab proteins potentially relevant to EPI64 will be studied in Aim 2 by measuring Rab-GTP levels in the cell using effectors in a pull-down assay. GAP activity of EPI64 on the Rab proteins found to have EPI64 dependent changes in Rab-GTP levels will be quantitatively measured using purified proteins. Aim 3 will focus on determining the formation and structure of the vacuoles that form in the cell when these membrane-trafficking pathways are perturbed. High resolution immunofluorescence live-cell imaging will be used to determine the temporal and spatial co- localization of GFP-tagged molecules during actin-coated vacuole formation. This will be complemented by transmission electron microscopy to image the formation and structure of vacuoles at various time points. Relevance: Maintaining a distinct polarity in epithelial cells that form the lining of internal structures in the intestine or kidney is vital for the uptake and distribution of nutrients. Disrupting the intracellular trafficking networks of these cells can lead to malnutrition, cancer, and eventual death.