Imaging Transporter Protein Dynamics in Living Cells

活细胞中转运蛋白动态成像

基本信息

批准号：
9134729
负责人：
Tim Chifong Lei
金额：
$ 15.77万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-20 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9134729
关键词：
Address Advisory Committees Aftercare Agonist Apical Atherosclerosis Binding Biochemical Biochemistry Biological Sciences Blood Vessels California Cardiovascular Diseases Carrier Proteins Cause of Death Cells Cellular biology Cholesterol Cholesterol Homeostasis Chronic Kidney Failure Clinical Confocal Microscopy Diabetes Mellitus Diffusion Disease Energy Transfer Engineering Environment Epithelial Cells Fluorescence Fluorescence Resonance Energy Transfer Glucose Transporter Glucosephosphates Goals Grant Homeostasis Hour Human body Image Imaging Techniques In Vitro Inflammation Intestines Journals Kidney Laboratories Lasers Lateral Life Light Link Lipids Liver X Receptor Measurement Membrane Membrane Lipids Membrane Microdomains Mentors Microscopy Molecular Nuclear Hormone Receptors Nuclear Receptors Optics PDZ protein Pathogenesis Pathway interactions Physiological Physiology Plasma Play Protein Dynamics Proteins Reading Regulation Regulatory Pathway Research Resolution Role SLC2A1 gene Signal Pathway Sodium Spectrum Analysis Techniques Tertiary Protein Structure Testing Total Internal Reflection Fluorescent Training Tubular formation Universities Vascular calcification Visit base blood glucose regulation brush border membrane career design genetic regulatory protein improved independency inorganic phosphate interest laboratory experience meetings microscopic imaging molecular dynamics molecular imaging novel physical science prevent protein biomarkers protein protein interaction protein transport sodium-hydrogen exchanger regulatory factor spatiotemporal symposium trafficking treatment strategy

项目摘要

DESCRIPTION (provided by applicant): As an optical engineer and laser spectroscopist, my long-term career goal is to become a productive biophysicist with the expertise to apply advanced optical techniques to the study biomedical problems. Based on my previous physical science training and current research interests, my research focuses are 1) applying advanced microscopy techniques to understand novel protein-protein, protein-lipid interactions, trafficking and signaling pathways which are important in elucidating the pathogeneses and developing new treatments for phosphate and glucose disorders that are common in chronic kidney disease and in diabetes, and 2) developing advanced microscopy and imaging techniques that are especially designed to study protein interactions, protein signaling pathways, trafficking and lipi nanodomain (lipid raft) interactions in living cells. An advisory committee will be formed including six world-renowned experts both in bioscience and advanced microscopy techniques to provide me abundant support and technical advice during the entire training period. A five-year training plan includes a three-years mentored research period and a two-years independent research period designed to 1) build a broad background in cell biology, renal and intestinal physiology; and 2) apply and develop new advanced microscopy techniques that are strongly relevant to study protein-protein, protein-lipid interactions and protein signaling pathways. The bioscience training strategy includes a) didactic training (5 courses, 25 credit hours) in cell biology, b) journal reading in renal physiology, c) biochemistry and cell biology laboratory training and d) bioscience conference and meeting attendance. The advanced microscopy training strategy includes a two month per year visit to the Laboratory for Fluorescence Dynamics at the University of California, Irvine to develop advanced microscopy techniques and to apply the latest microscopy techniques to bioscience studies. The type II sodium gradient-dependent phosphate (NaPi) cotransport proteins are the molecules responsible for reabsorption of phosphate (Pi) both in the kidney and in the gut. However, the regulatory pathways of renal and intestinal Pi transport are still largely unknown. A better understanding of the dynamic regulation of the NaPi cotransporters, their interacting regulatory proteins, and the local lipid microdomain (lipid raft) environment is critical to finding new treatment strategies for various diseases, including chronic kidney disease, atherosclerosis and vascular clarification, which are associated with the imbalance of plasma Pi concentration. Recently, we have demonstrated that the cholesterol-sensing nuclear hormone receptor, Liver X Receptor (LXR) plays an active role in Pi regulation. The renal and intestinal NaPi transporter activity and abundance are significantly reduced after treatment with LXR agonists. In this proposal, we intend to study the NaPi protein dynamics, NaPi-PDZ domain proteins, and NaPi- lipid interactions under baseline conditions and following treatments with LXR agonists in living renal and intestinal cells using advanced fluorescence dynamics microscopy techniques. These techniques include fluorescence cross-correlation spectroscopy (FCCS), cross-correlation raster image correlation spectroscopy (ccRICS), number & brightness (N&B) analysis, fluorescence resonance energy transfer (FRET), lifetime imaging, and molecular tracking (MT) techniques. These techniques allow for direct measurements of NaPi protein dynamics, NaPi-PDZ domain proteins, and NaPi-lipid interactions in living cells that can shed significant light on the regulaory pathways of Pi homeostasis. In addition, during the last two years of independent research period, sodium-gradient dependent glucose transporters (SGLT) will be studied to understand protein-protein interactions with PDZ domain proteins and protein-lipid interactions in glucose regulation using the above mentioned advanced microscopy techniques to establish my research independency.

描述（由申请人提供）：作为一名光学工程师和激光光谱学家，我的长期职业目标是成为一名富有成效的生物物理学家，拥有应用先进光学技术来研究生物医学问题的专业知识。基于我之前的物理科学训练和当前的研究兴趣，我的研究重点是 1) 应用先进的显微镜技术来了解新型蛋白质-蛋白质、蛋白质-脂质相互作用、运输和信号通路，这对于阐明疾病的发病机制和开发新的治疗方法非常重要慢性肾病和糖尿病中常见的磷酸盐和葡萄糖紊乱，2) 开发先进的显微镜和成像技术，专门用于研究活细胞中的蛋白质相互作用、蛋白质信号传导途径、运输和脂质纳米结构域（脂筏）相互作用。将成立一个由六位世界知名的生物科学和先进显微镜技术专家组成的顾问委员会，在整个培训期间为我提供充分的支持和技术建议。五年培训计划包括三年指导研究期和两年独立研究期，旨在 1) 建立细胞生物学、肾脏和肠道生理学的广泛背景； 2）应用和开发与研究蛋白质-蛋白质、蛋白质-脂质相互作用和蛋白质信号传导途径密切相关的新的先进显微镜技术。生物科学培训策略包括 a) 细胞生物学教学培训（5 门课程，25 个学分），b) 肾脏生理学期刊阅读，c) 生物化学和细胞生物学实验室培训，以及 d) 参加生物科学会议。高级显微镜培训策略包括每年两个月访问加州大学欧文分校荧光动力学实验室，以开发先进的显微镜技术并将最新的显微镜技术应用于生物科学研究。 II 型钠梯度依赖性磷酸盐 (NaPi) 共转运蛋白是负责肾脏和肠道中磷酸盐 (Pi) 重吸收的分子。然而，肾脏和肠道Pi转运的调节途径仍然很大程度上未知。更好地了解 NaPi 协同转运蛋白的动态调节、其相互作用的调节蛋白和局部脂质微结构域（脂筏）环境对于寻找各种疾病的新治疗策略至关重要，包括慢性肾病、动脉粥样硬化和血管澄清，这些疾病与血浆Pi浓度失衡有关。最近，我们证明胆固醇敏感核激素受体肝脏 X 受体 (LXR) 在 Pi 调节中发挥积极作用。用 LXR 激动剂治疗后，肾脏和肠道 NaPi 转运蛋白活性和丰度显着降低。在本提案中，我们打算使用先进的荧光动力学显微镜技术，研究基线条件下以及活肾和肠细胞中 LXR 激动剂处理后的 NaPi 蛋白动力学、NaPi-PDZ 结构域蛋白和 NaPi-脂质相互作用。这些技术包括荧光互相关光谱 (FCCS)、互相关光栅图像相关光谱 (ccRICS)、数量和亮度 (N&B) 分析、荧光共振能量转移 (FRET)、寿命成像和分子追踪 (MT) 技术。这些技术可以直接测量活细胞中的 NaPi 蛋白动力学、NaPi-PDZ 结构域蛋白和 NaPi-脂质相互作用，从而为 Pi 稳态的调节途径提供重要线索。此外，在最后两年的独立研究期间，将使用上述先进的显微镜技术研究钠梯度依赖性葡萄糖转运蛋白（SGLT），以了解蛋白质与PDZ结构域蛋白的相互作用以及葡萄糖调节中的蛋白质-脂质相互作用建立我的研究独立性。