G-protein Coupled Receptor Mediated Directional Sensing

活细胞成像技术已取得进展。荧光显微镜技术的最新进展和新型荧光探针的开发使成像技术成为研究单个活细胞内部信号转导的一种强有力的技术，具有高空间和时间分辨率。利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来监测两种蛋白质在亚细胞水平上的动态相互作用。此外，我们正在开发可视化细胞内Ca2+和cAMP水平的化学梯度和变化的方法。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。

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.