| 题名 | 活细胞荧光共振能量转移探测方法与单分子光学显微术的探索和应用 |
| 作者 | 付国 |
| 学位类别 | 博士 |
| 答辩日期 | 2008 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 徐至展 |
| 关键词 | FRET 单分子光学显微术 Mac-1 Dok5 |
| 其他题名 | Studies and applications of fluorescence resonance energy transfer and single-molecule optical microscopy in living cells |
| 中文摘要 | 蛋白质-蛋白质相互作用在整个细胞生命过程中占有重要地位，能够在活细胞中观察蛋白质-蛋白质相互作用一直都是生物学家的追求，也是生物学发展的必然要求，利用荧光共振能量转移（FRET）能够实时、直观、无损地探测活细胞中蛋白质-蛋白质相互作用，因此FRET成为活细胞中探测蛋白质-蛋白质相互作用的主要手段。与此同时，我们对于细胞生物学和分子生物学中有关分子事件和相互作用的认识，大部分是集团平均的研究结果，集团研究往往会掩盖各个分子的个性，因此集团平均的研究结果很需要从实际的单个分子活动加以验证。由于光学探测方法对于被研究分子的扰动最轻微，因此光学探测的结果更加可靠和直观，就活细胞单分子实时视见研究而言，则光学探测方法在目前几乎是唯一的方法。 本论文工作依据国家自然科学基金重大项目“单分子Mac-1的构象与亲和力变化的实时定量动态研究”和国家973课题“活细胞单分子实时视见研究”的要求，目标是建立活细胞FRET和活细胞单分子实时视见的光学探测方法，并尝试去解决一些基本的生物学问题。我们首先建立了集团水平的活细胞FRET探测方法，利用已经建立的活细胞FRET探测方法探测了整合素Mac-1亚基之间的相互作用。接下来，研究工作从集团平均研究转移到单分子水平上的研究，我们建立了活细胞单分子光学显微术，利用已经建立的活细胞单分子光学显微术研究了聚丙烯酰胺中的单个EGFP分子的光物理特性，探测了整合素Mac-1α和β亚基之间的缔合，研究了单分子 Dok5的细胞膜靶向和细胞膜微结构域定位的特性。概括起来，本论文工作取得以下几个方面的创新性研究进展： 1、 首次利用FRET在活细胞中探测到整合素Mac-1α和β亚基之间的缔合。结合了三通道FRET光学显微术和测量FRET光谱的方法，分别在CHO细胞和HEK293T细胞中探测了整合素Mac-1α和β亚基之间的缔合，验证了以往离体的研究结果。利用数值模拟的方法，首次对整合素Mac-1α和β亚基之间的距离进行了估算。 2、 首次利用FRET在活细胞中探测到整合素Mac-1α亚基之间以及β亚基之间的缔合。结合了三通道FRET光学显微术和测量FRET光谱的方法在CHO细胞中，对全长的整合素Mac-1α和β亚基来研究，克服了以往离体条件下仅选择跨膜区和胞浆区进行研究的不足。本研究结果对于揭示整合素聚集的可能机制具有重要的启示意义。 3、 提出较为简单的在活细胞中测量三通道FRET显微镜G参数以及CFP与YFP浓度比的方法。在活细胞中测量了三通道FRET显微镜G参数，整合素Mac-1亚基之间的表观FRET效率及亚基的浓度比。本方法不需要对活细胞进行固定和对荧光团进行光漂白，因此对活细胞的损害最小，并且实现起来也较容易。 4、 首次利用单分子光学显微术探测到表达在CHO细胞表面的单个Mac-1亚基，首次在单分子水平上对整合素Mac-1α和β亚基之间的缔合进行了验证。 5、 首次利用单分子光学显微术探测到表达在CHO细胞膜内表面的单个Dok5分子，首次在单分子水平上对Dok5组成性的细胞膜靶向和细胞膜微结构域定位进行了研究。 |
| 英文摘要 | The protein-protein interactions play important role on the process of cell life activities. The ability to observe the protein-protein interactions in living cell is the dream of biologist and the requirement of the development of biology. As fluorescence resonance energy transfer (FRET) method can realize the straightforward, real-time, and noninvasive detection of the protein-protein interactions in living cells, it has become a major method for the detection of protein-protein interactions in living cells. At the same time, our knowledge of molecular events and interactions in cell biology and molecular biology are mostly based on ensemble averaging studies. However, ensemble averaging studies always mask the property of individual molecule, so the ensemble averaging studies need to be proved at the single-molecule level. Because optical method has the least perturbation to biomolecule, results abtained by optical method are more credible and straightforward. Now, for the visualization of individual molecule in living cells, optical method almost is the only approach. The research in this thesis was based on the requirements of the project of “The studies of conformational and affinity changes of single-molecule Mac-1 in real-time” supported by National Natural Science Foundation of China and the project of “visualizing the single molecule in live cells” supported by National Basic Research Program of China. My research focuses on establishing FRET and single-molecule optical microscopy in living cells as well as using these methods to address the basic issues in biology. First of all, we established the optical method for FRET detection in living cells at the ensemble level, and detected the associations between integrin Mac-1 subunits. Second, we established the single-molecule optical microscopy, detected the photophysical properties of single-molecule EGFP immobilized in polyacrylamide gel, detected the associations between integrin Mac-1α and β subunits, and detected the plasma membrane microdomain localization of Dok5 in living cells. In summary, the research in this thesis has achieved the following innovative progresses: 1. Detected the associations between integrin Mac-1 α and β subunits in living cells by FRET. Three-channel FRET microscopy in combination with FRET spectra were used to detect the associations between integrin Mac-1α and β subunits in CHO and HEK293T cells, respectively. In addition, we estimated the distance of integrin Mac-1α and β subunits by numerical simulation. 2. Detected the associations between integrin Mac-1α and α subunits as well as β and β subunits in living cells by FRET. Three-channel FRET microscopy in combination with FRET spectra were used to detect the associations between integrin Mac-1α and α subunits as well as β and β subunits in CHO cells. In our study, the full length of integrin Mac-1α and β were used, which can overcome the inherent disadvantages of the use of transmembrane domain or cytoplasmic domain. 3. Proposed a simple method to measure the G factor for three-channel FRET microscope and the ratio of the concentration of CFP to YFP in living cells. Measured the G factor three-channel FRET microscope, FRET efficiency between integrin Mac-1 subunits and the ratio of the concentration of integrin Mac-1 subunits in living cells. The method for calculating the G factor developed by us does not require photobleaching, thus it does the least harm to cells and is easy to be realized. 4. Detected individual integrin Mac-1 subunits expressed at the ventral membrane of CHO cells by single-molecule optical microscopy, and detected the associations between Integrin Mac-1α and β subunits at the single-molecule level. 5. Detected individual Dok5 expressed at the cell inner membrane of CHO cells by single-molecule optical microscopy, and investigated the constitutive plasma membrane targeting and microdomain localization of Dok5 at the single-molecule level. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/15214]  |
| 专题 | 上海光学精密机械研究所_学位论文 |
| 推荐引用方式 GB/T 7714 | 付国. 活细胞荧光共振能量转移探测方法与单分子光学显微术的探索和应用[D]. 中国科学院上海光学精密机械研究所. 2008. |
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