title: Studying Kinetochores In Vivo Using FLIM-FRET.
abstract: 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.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA.
authors.forename: Tae Yeon
authors.initials: TY
authors.lastname: Yoo
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA. dan.needleman@gmail.com.|Departments of Applied Physics, and Molecular and Cellular Biology, Harvard University, 365.1 Northwest Building, 52 Oxford St., Cambridge, MA, 02138, USA. dan.needleman@gmail.com.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Needleman
mesh_terms: D000465:Algorithms; D001499:Bayes Theorem; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis; D008941:Spindle Apparatus / Q000378:metabolism
keywords: Bayesian analysis; FLIM-FRET; Kinetochore; Live cell imaging