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

EP164 协调上皮细胞中 arf6 和 Rab 依赖性膜运输

• 批准号: 7871350
• 负责人: David E Hokanson
• 金额: $ 5.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7871350
• 关键词: Actins; Affect; Apical; Appearance; Back; Binding; Biochemistry; Biological Assay; Cell membrane; Cell surface; Cells; Cessation of life; Clathrin; Complement; Cytoskeletal Modeling; Data; Defect; Electron Microscopy; Endocytosis; Endosomes; Epithelial Cells; F-Actin; Goals; Image; Immunofluorescence Immunologic; In Vitro; Intestines; Kidney; Laboratories; Laboratory Finding; Lead; Life; Ligands; Major Histocompatibility Complex; Malignant Neoplasms; Malnutrition; Measures; Mediating; Membrane; Membrane Protein Traffic; Modeling; Monomeric GTP-Binding Proteins; Mutation; Nutrient; Pathway interactions; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Production; Proteins; Recycling; Regulation; Research; Resolution; Signal Pathway; Signaling Molecule; Small Interfering RNA; Sorting - Cell Movement; Structure; Surface; Testing; Tetanus Helper Peptide; Time; Transmission Electron Microscopy; Tubular formation; Vacuole; Vesicle; Work; apical membrane; base; cellular imaging; cellular microvillus; design; ezrin; in vivo; knock-down; mutant; novel; overexpression; protein complex; research study; trafficking; uptake

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.

描述（由申请人提供）：上皮细胞需要受调节的囊泡运输以维持其动态肌动蛋白细胞骨架和极性。尽管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 EPI64 对 Arf6 和未知的 Rab 蛋白均具有调节功能。 EPI64 的 TBC 结构域直接与活性 Arf6-GTP 结合，并且 EPI64 的过度表达诱导肌动蛋白包被的液泡的积累，这可能是由 PI4P5K 激活导致 PIP2 过度产生所致。仍然与 Arf6-GTP 结合的 GAP 缺陷突变体 EPI64_R160A 的表达无法诱导液泡，表明液泡积累需要 GAP 活性。此外，EPI64 表达导致的这些液泡的出现被 Rab8a 的共表达所消除。这些结果表明，EPI64 以一种新的方式协调 Arf6 活性与 Rab 蛋白（可能是 Rab8），以调节膜运输和细胞骨架组织。本研究的目的是确定 EPI64 GAP 活性的相关 Rab 蛋白，并阐明 EPI64 如何参与这些贩运途径。第一个目标是确定哪些 Rab 蛋白受到 EPI64 过度表达的影响。由于 EPI64 的过度表达会导致细胞中形成肌动蛋白包被的液泡，因此可以将其用作筛选能够压倒 EPI64 GAP 活性的 Rab 蛋白的测定方法，从而成为回收途径的相关调节剂。在目标 2 中，将通过使用下拉测定中的效应器测量细胞中的 Rab-GTP 水平来研究与 EPI64 潜在相关的 Rab 蛋白的生物化学。发现 Rab-GTP 水平具有 EPI64 依赖性变化的 Rab 蛋白上的 EPI64 GAP 活性将使用纯化的蛋白进行定量测量。目标 3 将重点确定当这些膜运输途径受到干扰时细胞中形成的液泡的形成和结构。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液泡形成过程中 GFP 标记分子的时间和空间共定位。这将通过透射电子显微镜来补充，以对不同时间点的液泡的形成和结构进行成像。相关性：形成肠道或肾脏内部结构衬里的上皮细胞保持明显的极性对于营养物质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导致营养不良、癌症，甚至最终死亡。