In Vivo Flourescence Fluctuation Spectroscopy

荧光涨落光谱（FFS）是一种有吸引力的细胞应用技术。它决定 蛋白质的动力学和分子特性，具有亚微米分辨率和单分子灵敏度， 活细胞亮度是一个独特的FFS参数，用于指定蛋白质的平均荧光强度 复杂.因为亮度与复合物中标记蛋白质的数量成正比， 鉴定标记蛋白的寡聚状态。这一概念已成功应用于 测量活细胞中蛋白质的化学计量和结合曲线。我们力求在这一成功的基础上再接再厉， 提出有可能对定量细胞研究产生重大影响的项目，继续推动 FFS方法的包络。三元或更高级蛋白质复合物的形成是普遍存在的 蛋白质相互作用的特点，并在细胞过程的调节中发挥重要作用。然而，在这方面， 目前的方法大多限于检测涉及细胞内两种蛋白质的相互作用。我们 寻求克服这一技术瓶颈，并开发一种具有检测和 描述活细胞中三元蛋白质系统的相互作用。另一个令人感兴趣的话题是核问题。 包膜（NE），由分隔细胞核和细胞质的双膜层组成 隔间现在认识到NE整合了许多重要的细胞功能， 包括信号转导途径的调节和机械力之间的转换， 细胞质和细胞核。NE有超过60种不同的膜蛋白，其物理和功能 相互作用在很大程度上尚未探索。这些相互作用的表征对于理解 NE的细胞生物学，并开发与NE相关的人类疾病的治疗方法 proteins.为了促进这种研究，我们建议开发一种定量FFS技术， NE蛋白在活细胞内的相互作用和寡聚化。东北的环境构成了一个独特的 亮度测量的挑战，这将通过一种新的分析方法来解决。我们接下来 建议用光活化定位显微镜（PALM）补充FFS，以利用 这两种技术的力量。而PALM决定膜蛋白的化学计量和表面密度 在通常更接近生理条件的低浓度下，FFS补充了这些结果， 高表面密度，便于检测弱相互作用。我们将实施联合FFS 和PALM研究，并在应用之前对简单模型系统进行测试。这些项目 有望为探索细胞中蛋白质-蛋白质相互作用开辟新的途径， 应用范围从细胞生物学的基础研究到药物筛选。FFS的进展 可以通过提供有关蛋白质相互作用的详细信息来帮助对抗疾病， 确定药物开发的目标。

Fluorescence fluctuation spectroscopy (FFS) is an attractive technique for cellular applications. It determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity in the living cell. Brightness is a unique FFS parameter that specifies the average fluorescence intensity of a protein complex. Because brightness is directly proportional to the number of labeled proteins in the complex, it identifies the oligomeric state of the labeled protein. This conceptual idea has been successfully applied to measure the stoichiometry and binding curve of proteins in the living cell. We seek to build on this success by proposing projects with the potential for significant impact on quantitative cell studies that continue to push the envelope of the FFS method. The formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions and play an important role in the regulation of cellular processes. However, current methods are mostly limited to the detection of interactions involving two proteins inside the cell. We seek to overcome this technical bottleneck and develop an FFS approach with the capacity to detect and characterize the interactions of ternary protein systems in the living cell. Another topic of interest is the nuclear envelope (NE), which consists of a double membrane layer that separates the nuclear and cytoplasmic compartments. It is now being recognized that the NE integrates a number of important cell functions, which include the regulation of signal transduction pathways and mechanical force transduction between the cytoplasm and the nucleus. The NE has over 60 distinct membrane proteins, whose physical and functional interactions remain largely unexplored. Characterization of these interactions will be crucial to understand the cell biology of the NE and to develop treatments for the growing range of human disorders linked to NE proteins. To facilitate such research, we propose to develop a quantitative FFS technique that explores the interaction and oligomerization of NE proteins inside the living cell. The environment of the NE poses a unique challenge for brightness measurements, which will be addressed by a novel analysis approach. We next propose to complement FFS with photoactivated localization microscopy (PALM) in order to harness the strength of both techniques. While PALM determines stoichiometry and surface density of membrane proteins at low concentrations, which are often closer to physiological conditions, FFS complements these results at high surface densities, which facilitates the detection of weak interactions. We will implement combined FFS and PALM studies and perform tests on simple model systems before moving to applications. These projects are expected to open up new avenues for exploring protein-protein interactions in cells with potential applications ranging from basic research in cell biology to pharmaceutical drug screening. Advances in FFS could help fight diseases by providing detailed information about protein interactions and may lead to the identification of targets for drug development.