2-PHOTON FRET OF GFP-FUSION PROTEINS IN LIVING CELLS AND TISSUES

活细胞和组织中 GFP 融合蛋白的 2 光子干涉

• 批准号: 7365629
• 负责人: VICKIE J LAMORTE
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365629
• 关键词: PHOTON; FRET; GFP; FUSION; PROTEINS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of GFP mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Inherent to the optical sectioning capabilities of TPIS-FRET, protein-protein interactions can be identified within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们开发了一种光谱方法来监测活细胞中的荧光共振能量转移(FRET)。这种方法使用双光子激发荧光成像和光谱学(TPIS-FRET)在亚细胞成像体积内提供了出色的GFP突变信号的光谱分离。与目前基于FRET的方法不同，TPIS-FRET不依赖于光学滤光片的选择、比率图像分析或出血校正算法。由于TPIS-FRET的光学切片能力，蛋白质-蛋白质相互作用可以在离散的亚细胞结构域中识别。为了说明这项技术在检测同源二聚体形成方面的适用性，我们展示了早幼粒细胞(PML)同源二聚体在其相应核体内的体内关联。