Department of Physics, Lawrence University, Appleton, WI 54911, USA.
Biotechniques. 2011 Jul;51(1):43-8. doi: 10.2144/000113703.
Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.
细胞骨架结构的荧光成像允许研究细胞内的组织和细胞骨架本身的动态重组。微管是细胞骨架的一部分，传统的荧光标记物已被用于研究微管的定位、结构和动态，无论是在体内还是在体外。然而，现有标记物的缺点使得使用高精确度的光显微镜对微管进行成像变得困难。在本文中，我们报告了一种新的微管荧光标记技术，该技术涉及用亮有机荧光团（TAMRA、Cy3 或 Cy5）修饰的 GTP 类似物。这种荧光 GTP 以 1.0±0.4 µM 的离解常数与溶液中的微管蛋白上的一个特定位点（可交换位点）结合。此外，一旦微管蛋白聚合，该标记就会永久掺入微管晶格中。我们表明，该标记可用作具有纳米精度的单分子荧光探针，并且我们预计它对于现代亚衍射光学显微镜对微管和细胞骨架的研究将是有用的。
