Selective fluorescent labeling of proteins in living cells

对细胞内分子事件的研究提供了新的和重要的见解 生理学和病理学中的许多问题：细胞马达是如何工作的？多么 质子泵能产生三磷酸腺苷吗？细胞是如何分泌货物的？病毒是如何 输入一个单元格，一旦进入，它们是如何组合的。这项提议有三个目标，将 显著提高了在活细胞中跟踪单分子事件的能力。第一 特定的目的是开发将探针加载到细胞中的技术。这项技术是 高效并直接将探针送入胞浆而不是胞内 路径。我们特别感兴趣的是传递整数的能力。第二个具体问题 AIM将产生在生理温度下在细胞内发挥作用的内联蛋白 更快的速度和更高的效率。第三个具体目标使用了两个生物学问题 来检验前三个具体目标的结果的优点和局限性。这个 有两个问题与进出原子核的运输有关：我们能遵循 蛋白质在单个核孔中的构象变化以及我们是否可以遵循 单分子与核孔成分相互作用时的传输 穿过毛孔。这些问题对于理解 细胞生物学中的基本过程。它们还将提供关于进一步 我们研究单分子事件所需的技术改进 活细胞。

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 proposal 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.