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

描述(由申请人提供)：上皮细胞需要有调控的囊泡运输，以维持其动态的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然不清楚。我们实验室最近的工作表明，微绒毛蛋白EPI64对Arf6和一个未知的Rab蛋白都具有调节功能。EPI64‘S TBC结构域直接与活性Arf6-GTP结合，EPI64’S过表达诱导肌动蛋白包裹的空泡积累，可能是由PI4P5K激活过量产生PIP2驱动的。表达仍然与Arf6-GTP结合的GAP缺陷突变体EPI64_R160A不能诱导液泡，表明GAP活性是液泡积累所必需的。此外，由于EPI64的表达，这些空泡的出现被Rab8a的共表达所消除。这些结果表明，EPI64以一种新的方式与Rab蛋白(可能是Rab8)协调Arf6的活性，以调节膜运输和细胞骨架组织。本研究的目的是确定EPI64 S GAP活性的相关Rab蛋白(S)，并阐明EPI64是如何参与这些转运途径的。第一个目标是确定哪些Rab蛋白受到EPI64过表达的影响。由于EPI64的过表达导致细胞内形成肌动蛋白包裹的空泡，这可以作为一种检测方法来筛选能够压倒EPI64间隙活性的Rab蛋白，从而成为循环途径的相关调节因子。潜在与EPI64相关的Rab蛋白的生物化学将在Aim 2中通过使用下拉试验中的效应器测量细胞中的Rab-GTP水平来研究。在Rab-GTP水平中发现EPI64依赖于EPI64的变化的Rab蛋白上的EPI64的GAP活性将使用纯化的蛋白进行定量测量。目标3将集中于确定当这些膜运输途径被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被用来确定在肌动蛋白包裹的空泡形成过程中GFP标记分子的时间和空间共定位。这将得到透射电子显微镜的补充，以成像不同时间点的空泡的形成和结构。相关性：维持构成肠道或肾脏内部结构衬里的上皮细胞的明显极性对营养的吸收和分配至关重要。破坏这些细胞的细胞内运输网络可能会导致营养不良、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.