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.

活细胞成像技术已取得进展。荧光显微镜的最新进展和新型荧光探针的开发使成像成为研究单个活细胞内具有高空间和时间分辨率的信号转导的强大技术。使用新一代激光扫描共焦显微镜 (LSM 510 META)，我们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化学梯度以及细胞内 Ca2+ 和 cAMP 水平变化的方法。结合成像、光谱学和定量分析，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，有必要确定亚细胞水平上 G 蛋白偶联受体 (GPCR) 的空间活性。为此，我们应用 FRET 活细胞成像来监测 GPCR G-α 和 G-β-gamma 亚基的结合和解离。由于 FRET 对的发射光谱重叠，使用基于滤光片和带通采集系统的传统系统很难在活细胞中获得可靠的 FRET 图像。我们使用激光扫描显微镜 510 META，它可以同时获取多个荧光信号的光谱图像读数。结合线性分离功能，LSM 510 META 使我们能够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-α2 和 G-β-γ 亚基分别用 ECFP 和 EYFP 标记。 GPCR 的激活引发的 G-α 和 G-β-γ 之间的解离已在单个活细胞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 CXCR1 在活细胞膜上的分布。脂筏被认为可以通过促进蛋白质之间的相互作用和防止通路之间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 CXCR1 与脂筏响应 IL-8 刺激之间的关联。我们建立了稳定的HEK 293细胞系，表达含有CXCR1和青色荧光蛋白（CFP）的重组蛋白，命名为CR1F4。在 CR1F4 细胞中，CXCR1-CFP 位于细胞膜中。在用钙指示剂 Fluo-4 标记的 CR1F4 细胞中观察到由 IL-8 刺激触发的 Ca2+ 升高，说明 CFP 标记的 CXCR1 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 引发的 Ca2+ 升高。胆固醇的补充恢复了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 分别特异性标记质膜的筏状微域和非筏微域。 FRET 用于检测 CXCR1-CFP（供体）与 DiIC16 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 DiIC16 或 Fast DiI 光漂白后 CFP 强度的增加来获得的。当 CR1F4 细胞在含有 IL-8 的培养基中培养时，CFP 标记的 CXCR1 和 DiIC16 标记的​​筏微域之间发生 FRET。 CXCR1-CFP 和 Fast DiI 之间的 FRET 信号很少可见。将细胞在缺乏IL-8的无血清培养基中饥饿后，CXCR1-CFP和DiIC16之间的FRET下降，而CXCR1-CFP和Fast DiI之间的FRET增加。我们的结果表明，CXCR1 受体在被 IL-8 激活后从非筏微域易位到筏微域。