Selective fluorescent labeling of proteins in living cells

描述（由申请人提供）：对细胞内分子事件的研究为生理学和病理学中的许多问题提供了新颖而重要的见解：细胞马达如何工作？质子泵如何产生ATP？货物是如何从细胞中分泌出来的？病毒如何进入细胞，一旦进入，它们如何组装。该应用程序有三个目标，将显着提高能力，在活细胞中跟踪单分子事件。第一个具体目标是开发将探针加载到细胞中的技术。该技术是有效的，并直接将探针递送到胞质溶胶中，而不是进入内吞途径。我们特别感兴趣的是传递内含肽的能力。第二个具体目标是产生内含肽，这些内含肽将在生理温度下以更快的速度和更高的效率在细胞内发挥作用。第三个具体目标使用两个生物学问题来测试前三个具体目标结果的优势和局限性。这两个问题与核内和核外的运输有关：我们能否跟踪单个核孔中蛋白质的构象变化，以及我们能否跟踪单个分子与核孔组分相互作用并穿过核孔时的运输。这些问题对于理解细胞生物学中的基本过程具有重要意义。它们还将提供关于我们研究活细胞中单分子事件的技术所需的进一步修改的见解。 公共卫生相关性：在过去的几年里，光学成像为我们对生物学的理解做出了非常重要的贡献。这是成像的许多优点的结果：成像使我们能够研究仍然活着的生物系统;成像使我们能够研究单个分子，单个细胞而不是平均行为;成像使我们能够研究从单个分子到整个个体的许多不同大小的尺度。通常，发生在单个分子水平上的缺陷在整个生物体水平上表现为病理学。成像技术使我们能够追踪疾病，从单个分子到分子机器，到整个细胞，到整个器官，再到个体患者。光学成像的一个主要限制是使探针进入细胞以特异性和选择性地标记单个分子的能力。这一应用将有助于我们研究细胞中单个分子事件的能力，为我们提供了将探针放入细胞的新方法，而不会干扰细胞和标记细胞成分的新方法，对细胞的影响最小，通常没有。

DESCRIPTION (provided by applicant): Studies of molecular events inside of cells have provided novel and important insights into many questions in physiology and pathology: How do cellular motors work? How does a proton pump generate ATP? How is cargo secreted from cells? How do viruses enter a cell and, once in, how do they assemble. This application has three aims that will significantly advance ability to follow single molecular events in living cells. The first specific aim develops the technology for loading probes into cells. The technique is efficient and directly delivers probe into the cytosol rather than into the endocytic pathway. Of particular interest for us is the ability to deliver inteins. The second specific aim will generate inteins that will function inside of cells at physiological temperatures at faster rates and higher efficiencies. The third specific aim uses two biological questions to test the strengths and limitations of the results from the first three specific aims. The two questions relate to transport in and out of the nucleus: Can we follow the conformational changes of proteins in single nuclear pores and can we follow the transport of single molecules as they interact with the nuclear pore components and move through the pore. These questions are significant for understanding a fundamental process in cell biology. They will also provide insight as to further modifications that are needed in our technology for studying single molecular events in living cells. PUBLIC HEALTH RELEVANCE: Optical imaging has contributed very significant advances to our understanding of biology in the last few years. This is the consequence of a number of advantages of imaging: Imaging allows us to study biological systems that are still alive; Imaging allows us to study individual molecules, individual cells rather than the average behavior; Imaging allows us to study many different size scales from single molecules to whole individuals. Often a defect that occurs at the level of a single molecule represents itself as pathology at the level of the whole organism. Imaging allows us to follow the disease from the single molecule, to the molecular machine, to the whole cell, to the whole organ, to the individual patient. A major limitation of optical imaging has been the capability to get probes into cells to specifically and selectively label individual molecules. This application will contribute to our ability to study individual molecular events in the cell by giving us new methods of putting our probes into cells, without disturbing the cells and new methods of labeling cellular components with minimal, often no, effect on the cell.