G-protein Coupled Receptor Mediated Directional Sensing

G蛋白偶联受体介导的定向传感

• 批准号: 6987079
• 负责人: Tian Jin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6987079
• 关键词: G protein; biological signal transduction; calcium flux; calcium indicator; cell migration; cell surface receptors; chemokine receptor; chemotaxis; confocal scanning microscopy; cyclic AMP; fluorescence resonance energy transfer; intermolecular interaction; membrane structure; protein localization; single cell analysis; tissue /cell culture

Progress has been made in live cell imaging techniques. The recent advances in fluorescence microscopy and development of new fluorescent probes make imaging a powerful technique for studying signal transduction inside single living cells with high spatial and temporal resolution. Using a new generation of Laser Scanning Confocal Microscope (LSM 510 META), we are applying fluorescence resonance energy transfer (FRET) microscopic imaging methods and data analyses to monitor dynamic interactions between two proteins at the subcellular level. In addition, we are developing methods to visualize chemical gradients and changes of intracellular Ca2+ and cAMP levels. Combining imaging, spectroscopy and quantitative analyses, we have been able to quantitatively measure biochemical reactions in single living cells. To elucidate the molecular mechanisms of directional sensing, it is essential to determine spatial activities of G protein coupled receptors (GPCRs) at the subcellular level. Toward that end, we have applied live cell imaging of FRET to monitor the association and dissociation of the GPCRs G-alpha and G-beta-gamma subunits. It has been difficult to obtain reliable FRET images in living cells with the conventional systems based on filter stets and bandpass acquisition systems because of overlapping emission spectra of FRET pairs. We are using the Laser Scanning Microscope 510 META, which acquires spectral image readouts of multiple fluorescence signals simultaneously. Coupled with a liner unmixing function, the LSM 510 META allows us to separate image signals according to the fingerprint spectra of each individual protein. G-alpha2 and G-beta-gamma subunits of the D. discoideum G proteins were tagged with ECFP and EYFP respectively. Activation of GPCRs triggered dissociations between G-alpha and G-beta-gamma have been directly visualized and quantitatively determined on the membrane of single living cells. Applying live cell FRET imaging, we investigated the distribution of an IL-8 chemokine receptor, CXCR1, on the membrane of living cells. Lipid rafts were suggested to increase the efficiency and specificity of signal transduction by facilitating interactions between proteins and by preventing inappropriate crosstalk between pathways. We investigated the association between CXCR1 and lipid rafts in response to IL-8 stimuli in single cells. We established a stable cell line HEK 293 expressing a recombinant protein containing CXCR1 and cyan fluorescent protein (CFP) named as CR1F4. In CR1F4 cells, CXCR1-CFP localized in the membrane. Ca2+ elevation triggered by IL-8 stimuli was visualized in CR1F4 cells labeled with Fluo-4, a calcium indicator, illustrating that CFP tagged CXCR1 retains its function. Depletion of cholesterol with methyl-beta-cyclodextrin, disrupting the raft microdomains, eliminated IL-8 triggered Ca2+ elevation. The replenishment of cholesterol recovered the Ca2+ response. The results indicate that the function of CXCR1 depends on the integrity of raft microdomains. DiIC16 and Fast DiI specifically label raft-like and non-raft microdomains of plasma membrane, respectively. FRET was employed to detect the interaction between the CXCR1-CFP (donor) and either DiIC16 or Fast DiI (acceptors). The FRET images in living cells were obtained by visualizing the increase in intensity of CFP upon photobleaching either DiIC16 or Fast DiI. When CR1F4 cells were cultured in media containing IL-8, FRET occurred between CFP tagged CXCR1 and DiIC16 labeled raft microdomains. Little FRET signal between CXCR1-CFP and Fast DiI was visualized. After the cells were starved in serum-free media that lacks IL-8, the FRET between CXCR1-CFP and DiIC16 decreased, while the FRET between CXCR1-CFP and Fast DiI increased. Our results suggest that the CXCR1 receptor translocates from non-raft microdomain to raft microdomains upon activation by IL-8.

活细胞成像技术已经取得了进展。近年来，随着荧光显微技术的发展和新型荧光探针的发展，成像技术成为研究单个活细胞内高时空分辨率信号转导的有力手段。使用新一代激光扫描共聚焦显微镜(LSM 510META)，我们正在应用荧光共振能量转移(FRET)显微成像方法和数据分析来监测亚细胞水平上两种蛋白质之间的动态相互作用。此外，我们正在开发可视化细胞内钙离子和cAMP水平的化学梯度和变化的方法。结合成像、光谱和定量分析，我们已经能够定量测量单个活细胞的生化反应。 为了阐明定向感知的分子机制，在亚细胞水平上确定G蛋白偶联受体(GPCRs)的空间活性是必不可少的。为此，我们应用FRET的活细胞成像来监测GPCRs G-α和G-β-伽马亚单位的关联和解离。由于FRET对发射光谱的重叠，传统的基于滤光片和带通采集系统的FRET成像系统很难在活细胞中获得可靠的FRET图像。我们使用的是510META激光扫描显微镜，它可以同时获取多个荧光信号的光谱图像读数。与线性分解功能相结合，LSM 510 META允许我们根据每个单独蛋白质的指纹光谱来分离图像信号。盘状芽孢杆菌G蛋白的G-α2和G-β-γ亚基分别用ECFP和EYFP标记。GPCRs的激活触发了G-α和G-β-γ之间的解离，在单个活细胞的细胞膜上已经被直接可视化和定量测定。 应用活细胞FRET成像，我们研究了IL-8趋化因子受体CXCR1在活细胞膜上的分布。脂筏被认为通过促进蛋白质之间的相互作用和防止不适当的通路之间的串扰来提高信号转导的效率和特异性。我们在单个细胞中研究了CXCR1和脂筏对IL-8刺激的反应的相关性。我们建立了一个稳定表达CXCR1和青色荧光蛋白(CFP)重组蛋白的细胞系HEK 293，命名为CR1F4。在CR1F4细胞中，CXCR1-CFP定位于细胞膜。在钙指示剂Fluo-4标记的CR1F4细胞中，观察到由IL-8刺激引起的Ca~(2+)升高，说明CFP标记的CXCR1保留了其功能。甲基-β-环糊精耗尽胆固醇，破坏RAFT微区，消除IL-8引发的钙升高。胆固醇的补充恢复了钙离子的反应。结果表明，CXCR1的功能依赖于RAFT微区的完整性。DiIC16和Fast DII分别特异性地标记质膜的RAFT样微区和非RAFT微区。FRET检测CXCR1-CFP(供体)与DiIC16或Fast DII(受体)的相互作用。活细胞内的FRET图像是通过可视化光漂白DiIC16或Fast DII后CFP强度的增加而获得的。当CR1F4细胞在含有IL-8的培养液中培养时，CFP标记的CXCR1和DiIC16标记的RAFT微区之间发生了FRET。CXCR1-CFP与Fast DII之间几乎没有FRET信号。在缺乏IL-8的无血清培养液中饥饿后，CXCR1-CFP与DiIC16之间的FRET降低，而CXCR1-CFP与Fast DII之间的FRET增加。我们的结果表明，当IL-8激活时，CXCR1受体从非RAFT微区移位到RAFT微区。