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.

在实时细胞成像技术中取得了进展。荧光显微镜和新荧光探针的开发的最新进展使成像成为研究具有高空间和暂时分辨率的单个活细胞内信号转导的强大技术。使用新一代的激光扫描共聚焦显微镜（LSM 510 meta），我们正在应用荧光共振能量传递（FRET）显微镜成像方法和数据分析来监测亚细胞水平的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化学梯度和细胞内CA2+和cAMP水平的变化的方法。结合成像，光谱和定量分析，我们能够在单个活细胞中定量测量生化反应。 为了阐明方向传感的分子机制，必须确定在亚细胞水平上G蛋白偶联受体（GPCR）的空间活性。为此，我们应用了FRET的活细胞成像来监视GPCRS G-Alpha和G-Beta-Gamma亚基的关联和解离。由于FRET对重叠的发射光谱，具有基于滤波器的传统系统和带通通通系统的常规系统中的活细胞中很难获得可靠的FRET图像。我们正在使用激光扫描显微镜510 meta，该元元元同时获得了多个荧光信号的光谱图像读数。结合衬里的Umixing函数，LSM 510 META使我们可以根据每个蛋白质的指纹光谱分离图像信号。 D. discoideum G蛋白的G-Alpha2和G-Beta-Gamma亚基分别用ECFP和EYFP标记。 GPCR的激活触发了G-Alpha和G-Beta-Gamma之间的解离，已直接可视化并在单个活细胞的膜上进行定量确定。 应用活细胞FRET成像，我们研究了在活细胞膜上IL-8趋化因子受体CXCR1的分布。建议通过促进蛋白质之间的相互作用和防止途径之间的不适当的串扰来提高信号转导的效率和特异性。我们研究了单个细胞中IL-8刺激的CXCR1与脂质筏之间的关联。我们建立了一个稳定的细胞系HEK 293，该HEK 293表达含有CXCR1和青色荧光蛋白（CFP）的重组蛋白，称为CR1F4。在CR1F4细胞中，CXCR1-CFP位于膜中。由IL-8刺激触发的Ca2+升高在用Fluo-4标记的CR1F4细胞中可视化，该细胞是钙指示剂Fluo-4，这说明CFP标记为CXCR1的CFP保留其功能。用甲基-Beta-Cyclodextrin消耗胆固醇，破坏了筏微区，消除了IL-8触发的Ca2+升高。胆固醇的补充回收了Ca2+反应。结果表明，CXCR1的功能取决于筏微区的完整性。 DIIC16和Fast DII分别特异性标记了质膜的筏样和非羊皮微域。使用FRET来检测CXCR1-CFP（供体）和DIIC16或快速DII（受体）之间的相互作用。通过可视化DIIC16或快速DII的光漂白时，活细胞中的FRET图像是通过可视化CFP强度的增加而获得的。当CR1F4细胞在含有IL-8的培养基中培养时，在CFP标记的CXCR1和DIIC16标记的筏微区域之间发生了FRET。可视化CXCR1-CFP和Fast DII之间的几乎没有FRET信号。在缺乏IL-8的无血清培养基中饿死细胞后，CXCR1-CFP和DIIC16之间的FRET减少，而CXCR1-CFP和Fast DII之间的FRET增加了。我们的结果表明，在IL-8激活后，CXCR1受体从非生殖微区域转移到筏微区。