Trafficking of NaPilla in brush border microvilli

在刷状缘微绒毛中贩运 NaPilla

• 批准号: 7160289
• 负责人: JUDITH T., BLAINE
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7160289
• 关键词: biological signal transduction; biological transport; brush border membrane; cell component structure /function; cell membrane; cellular polarity; cytoskeleton; fluorescence microscopy; fluorescence recovery after photobleaching; hormone regulation /control mechanism; kidney cell; membrane activity; membrane transport proteins; molecular /cellular imaging; parathyroid hormones; phosphates; postdoctoral investigator; protein structure function; renal tubular transport; spectrometry; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Disorders of inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems. Aging, diabetes mellitus, malignancy, renal failure, alcoholism, transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. The evidence to date indicates that the majority of renal tubular Pi regulation by dietary and hormonal factors is mediated by the type Ha renal sodium phosphate cotransporter (NaPilla). These factors alter NaPilla surface abundance and thus activity by altering the cotransporter's insertion into and retrieval from brush border membrane (BBM) microvilli. Relatively little is known, however, about the precise events governing NaPilla trafficking to and from the cell membrane. These studies have been limited by the inability to visualize protein trafficking in microvilli with high resolution in real time. Our lab has developed a novel application of total internal reflection fluorescence microscopy (TIR-FM) which allows visualization of trafficking events in brush border microvilli in real time. The proposed studies will use TIR-FM microscopy coupled with fluorescence recovery after photobleaching (FRAP) and image correlation spectroscopy (ICS) techniques to examine how alterations in extracellular Pi and PTH modulate NaPilla trafficking in BBM microvilli. These studies will be performed in opposssum kidney (OK) cells, a well-established model of the proximal tubule. NaPilla trafficking is also belived to be regulated by PDZ proteins (scaffolding proteins that link the cotransporter to the cytoskeleton) and by the cytoskeleton itself. The proposed work will investigate modulation of NaPilla trafficking in BBM microvilli in response to alterations in extracellular Pi or PTH by PDZ proteins and by the cytoskeleton. This work will use dynamic imaging techniques to study physiologic processes at the single protein level in real time. These techniques, besides being widely applicable to other processes, will allow insights into the renal regulation of Pi homeostasis which is crucial for normal cellular function. This knowledge may ultimately lead to the development of novel therapies to treat phosphate imbalance.

描述（由申请人提供）：无机磷酸盐 (Pi) 浓度紊乱和 Pi 重吸收受损是常见的临床问题。众所周知，衰老、糖尿病、恶性肿瘤、肾功能衰竭、酗酒、移植、艾滋病和几种治疗药物主要通过影响肾小管Pi转运而引起或与低磷血症或高磷血症相关。肾脏在调节 Pi 稳态中起着至关重要的作用。迄今为止的证据表明饮食和激素因素对肾小管Pi的调节大部分是由Ha型肾磷酸钠协同转运蛋白(NaPilla)介导的。这些因素通过改变协同转运蛋白插入刷状缘膜 (BBM) 微绒毛和从刷状缘膜 (BBM) 微绒毛中回收来改变 NaPilla 表面丰度和活性。然而，关于控制 NaPilla 进出细胞膜的精确事件，人们知之甚少。这些研究因无法以高分辨率实时可视化微绒毛中的蛋白质运输而受到限制。我们的实验室开发了一种全内反射荧光显微镜（TIR-FM）的新颖应用，可以实时可视化刷状缘微绒毛中的贩运事件。拟议的研究将使用 TIR-FM 显微镜结合光漂白后荧光恢复 (FRAP) 和图像相关光谱 (ICS) 技术来研究细胞外 Pi 和 PTH 的变化如何调节 BBM 微绒毛中的 NaPilla 运输。这些研究将在负鼠肾 (OK) 细胞中进行，这是一种完善的近端小管模型。 NaPilla 运输也被认为受到 PDZ 蛋白（将协同转运蛋白与细胞骨架连接起来的支架蛋白）和细胞骨架本身的调节。拟议的工作将研究 BBM 微绒毛中 NaPilla 运输的调节，以响应 PDZ 蛋白和细胞骨架对细胞外 Pi 或 PTH 的改变。这项工作将利用动态成像技术实时研究单一蛋白质水平的生理过程。这些技术除了广泛适用于其他过程之外，还将有助于深入了解对正常细胞功能至关重要的 Pi 稳态的肾脏调节。这些知识可能最终导致治疗磷酸盐失衡的新疗法的开发。