Department of Chemistry, Yale University, New Haven, CT, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2022;2430:73-91. doi: 10.1007/978-1-0716-1983-4_5.
The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules-growth, shrinkage, nucleation, and severing-are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide-field imaging technique that allows label-free visualization of microtubules with high contrast and speed. IRM circumvents several problems associated with fluorescence microscopy including the high concentrations of tubulin required for fluorescent labeling, the potential perturbation of function caused by the fluorophores, and the risks of photodamage. IRM can be implemented on a standard epifluorescence microscope at low cost and can be combined with fluorescence techniques like total-internal-reflection-fluorescence (TIRF) microscopy. Here we describe the experimental procedure to image microtubule dynamics and severing using IRM , providing practical tips and guidelines to resolve possible experimental hurdles.
微管细胞骨架的动态结构对于细胞分裂、运动和形态发生至关重要。微管的动态特性——生长、收缩、成核和切割——受到微管相关蛋白 (MAP) 库的调节。许多这些 MAP 的活性已经在体外使用显微镜测定法重新构建。作为荧光显微镜的替代方法，干涉反射显微镜 (IRM) 已被引入作为一种易于使用的宽场成像技术，可实现微管的无标记可视化，具有高对比度和速度。IRM 避免了与荧光显微镜相关的几个问题，包括荧光标记所需的高浓度微管蛋白、荧光团可能引起的功能干扰以及光损伤的风险。IRM 可以以低成本在标准落射荧光显微镜上实现，并可以与荧光技术（如全内反射荧光 (TIRF) 显微镜）结合使用。在这里，我们描述了使用 IRM 成像微管动力学和切割的实验程序，提供了实用的技巧和指南，以解决可能出现的实验障碍。
