Yoo Tae Yeon, Needleman Daniel J
School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA.
Departments of Applied Physics, and Molecular and Cellular Biology, Harvard University, 365.1 Northwest Building, 52 Oxford St., Cambridge, MA, 02138, USA.
Methods Mol Biol. 2016;1413:169-86. doi: 10.1007/978-1-4939-3542-0_11.
Kinetochores play essential roles in coordinating mitosis, as a mechanical connector between chromosome and microtubule and as a source of numerous biochemical signals. These mechanical and biochemical behaviors of kinetochores change dynamically in cells during mitosis. Therefore, understanding kinetochore function requires an imaging tool that quantifies the protein-protein interactions or biochemical changes with high spatiotemporal resolution. FRET has previously been used in combination with biosensors to probe protein-protein interactions and biochemical activity. In this chapter, we introduce FLIM-FRET, a lifetime-based method that quantifies FRET, and describe the use of FLIM-FRET as a method for studying dynamic kinetochore behavior in cells with high spatiotemporal resolution.
动粒在协调有丝分裂过程中发挥着至关重要的作用，它作为染色体与微管之间的机械连接体以及众多生化信号的来源。在有丝分裂期间，动粒的这些机械和生化行为在细胞中动态变化。因此，要理解动粒的功能，就需要一种成像工具，能够以高时空分辨率量化蛋白质 - 蛋白质相互作用或生化变化。此前，荧光共振能量转移（FRET）已与生物传感器结合使用，以探测蛋白质 - 蛋白质相互作用和生化活性。在本章中，我们介绍基于寿命的定量FRET方法——荧光寿命成像FRET（FLIM - FRET），并描述如何将FLIM - FRET用作一种以高时空分辨率研究细胞中动粒动态行为的方法。
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