微管在许多细胞功能中起着至关重要的作用,部分是通过驱动蛋白和动力蛋白作为细胞内运输的轨道。微管履行这一角色的能力从根本上取决于它们是极性的事实,马达沿着它们向它们的正端或负端移动。鉴于微管细胞骨架在不同细胞类型中采用各种专门的结构,精确绘制微管在这些细胞中的取向和组织方式是很重要的。为此,电机漆已经开发出来,但是在目前的实施中,它依赖于全内反射荧光(TIRF)显微镜,因此仅限于在紧邻盖玻片的细胞薄切片中成像微管。这里,我们报告了一种运动PAINT的变体,它使用晶格光片显微镜来克服这一点,允许在三维样品中绘制微管组织和方向。我们描述了纯化的必要步骤,标签,使用,和用于motor-PAINT的图像驱动电机,并概述用于可视化结果数据的分析管道。此处描述的方法可用于将来研究(厚)极化细胞如肠上皮细胞中的微管细胞骨架。
Microtubules play an essential role in many cellular functions, in part by serving as tracks for intracellular transport by kinesin and dynein. The ability of microtubules to fulfill this role fundamentally depends on the fact that they are polar, with motors moving along them toward either their plus or minus end. Given that the microtubule cytoskeleton adopts a variety of specialized architectures in different cell types, it is important to map precisely how microtubules are oriented and organized in these cells. To this end, motor-PAINT has been developed, but in its current implementation, it relies on total internal reflection fluorescence (TIRF) microscopy and is thus restricted to imaging microtubules in a thin section of the cell immediately adjacent to the coverslip. Here, we report a variant of motor-PAINT that uses lattice light-sheet microscopy to overcome this, allowing for the mapping of microtubule organization and orientation in three-dimensional samples. We describe the necessary steps to purify, label, use, and image kinesin motors for motor-PAINT and outline the analysis pipeline used to visualize the resulting data. The method described here can be used in the future to study the microtubule cytoskeleton in (thick) polarized cells such as intestinal epithelial cells.