Optimization of Bimolecular Fluorescence Complementation Probes for New Imaging F

描述（由申请人提供）：细胞高分辨率成像的主要目的是可视化细胞组分的分子组织，并确定特定组分是否参与相同的大分子复合物。解决这些问题的努力从两个相反的角度解决了这个问题：1。显微镜的改进使得在理想条件下能够在分子尺度上分辨光谱上不同的信号;以及2.能量转移方法允许检测两种蛋白质非常紧密接触的复合物。该提案引入了第三种方法，该方法填补了前两种方法之间分辨率的差距，并避免了其中固有的许多妥协。这种方法是基于荧光复合物的形成，当两个非荧光蛋白片段之间的关联是通过拴在一个大分子复合物的片段促进。这种方法的早期适应被称为双分子荧光互补（BiFC）和泛素介导的荧光互补（UbFC）。这些早期的方法具有显著的局限性，这是由于缺乏用于测定目的的荧光蛋白片段（BiFC探针）的优化。拟议的研究旨在通过系统的体外进化和蛋白质设计策略来消除这些限制。该研究小组将开发新型BiFC探针，使以前由于现有方法固有的局限性而无法成像的细胞过程成像成为可能。为了验证BiFC探针的通用性，它们将被用于对涉及细菌、植物和动物细胞的分子事件进行成像。公共卫生相关性：许多细胞功能需要多个组件聚集在一起形成一个复合体。我们建议开发新的方法，使我们能够看到由组分的特定组合形成的复合物。这些方法类似于将灯泡连接到一个组件，将电池连接到另一个组件。这项工作将提高我们对细胞组分之间相互作用的理解，这些组分对细胞和生物体的健康至关重要。

DESCRIPTION (provided by applicant): A major objective of high resolution imaging of cells is to visualize the molecular organization of cellular components and to determine if specific components participate in the same macromolecular complex. Efforts to address these issues have tackled the problem from two opposite perspectives: 1. Improvements in microscopy have enabled resolution of spectrally distinct signals at a molecular scale under ideal conditions; and 2. Energy transfer approaches have allowed detection of complexes where two proteins are in very close contact. This proposal introduces a third approach that fills the gap in resolution between the two former methods and avoids many of the compromises inherent in them. This approach is based on formation of a fluorescent complex when the association between two non-fluorescent protein fragments is facilitated by tethering the fragments in a macromolecular complex. Earlier adaptations of this approach are known as bimolecular fluorescence complementation (BiFC) and ubiquitin-mediated fluorescence complementation (UbFC). These earlier approaches have significant limitations that are due to the lack of optimization of the fluorescent protein fragments (BiFC probes) for purposes of the assay. The proposed research seeks to eliminate these limitations through systematic in vitro evolution and protein design strategies. The research team will develop novel BiFC probes that will enable imaging of cellular processes that have not been previously imaged due to limitations inherent in existing methods. To validate the universal utility of the BiFC probes, they will be used to image molecular events involving bacteria, plants and animal cells. Public Health Relevance: Many cellular functions require that multiple components come together to form a complex. We propose to develop new methods that will enable us to see the complexes formed by specific combinations of components. These methods are analogous to linking a light bulb to one component and a battery to the other. This work will improve our understanding of interactions among cellular components that are important for the health of the cell and the organism.