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。荧光显微镜的最新进展和新荧光探针的开发使成像成为研究单个活细胞内信号转导的强大技术，具有高的空间和时间分辨率。使用新一代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转移（FRET）显微成像方法和数据分析，以监测两种蛋白质之间的动态相互作用在亚细胞水平。此外，我们正在开发可视化化学梯度和细胞内Ca 2+和cAMP水平变化的方法。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中的生化反应。 为了阐明定向传感的分子机制，有必要在亚细胞水平上确定G蛋白偶联受体（GPCRs）的空间活性。为此，我们应用FRET的活细胞成像来监测GPCR G-α和G-β-γ亚基的缔合和解离。由于荧光共振能量转移对的发射光谱相互重叠，传统的基于滤光片和带通采集系统的荧光共振能量转移系统很难在活细胞中获得可靠的荧光共振能量转移图像。我们使用的是激光扫描显微镜510 Meta，它可以同时获取多个荧光信号的光谱图像读数。结合线性解混功能，LSM 510 Meta允许我们根据每个蛋白质的指纹光谱分离图像信号。G-α 2和G-β-γ亚基。分别用ECFP和EYFP标记discoideum G蛋白。GPCR的激活触发G-α和G-β-γ之间的解离已经在单个活细胞的膜上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CXCR 1在活细胞膜上的分布。脂筏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。我们研究了单细胞中CXCR 1和脂筏对IL-8刺激的反应之间的关系。我们建立了一个稳定的细胞系HEK 293表达的重组蛋白CXCR 1和青色荧光蛋白（CFP）命名为CR 1F 4。在CR 1F 4细胞中，CXCR 1-CFP定位于膜。在用Fluo-4（钙指示剂）标记的CR 1F 4细胞中观察到由IL-8刺激触发的Ca 2+升高，这说明CFP标记的CXCR 1保留了其功能。用甲基-β-环糊精消耗胆固醇，破坏筏微结构域，消除IL-8触发的Ca 2+升高。补充胆固醇可恢复Ca 2+反应。结果表明，CXCR 1的功能依赖于筏微区的完整性。DiIC 16和Fast DiI分别特异性标记质膜的筏状和非筏状微区。采用FRET检测CXCR 1-CFP（供体）与DiIC 16或Fast DiI（受体）之间的相互作用。活细胞中的FRET图像是通过观察DiIC 16或Fast DiI光漂白后CFP强度的增加而获得的。当CR 1F 4细胞在含有IL-8的培养基中培养时，在CFP标记的CXCR 1和DiIC 16标记的筏微结构域之间发生FRET。CXCR 1-CFP和Fast DiI之间的FRET信号很小。细胞在缺乏IL-8的无血清培养基中饥饿后，CXCR 1-CFP和DiIC 16之间的FRET降低，而CXCR 1-CFP和Fast DiI之间的FRET增加。我们的研究结果表明，CXCR 1受体易位从非筏微域筏微域激活后，IL-8。