微图案引导的重叠动态微管对的组装。
Micropattern-guided assembly of overlapping pairs of dynamic microtubules.
作者信息
Fourniol Franck J, Li Tai-De, Bieling Peter, Mullins R Dyche, Fletcher Daniel A, Surrey Thomas
机构信息
London Research Institute, Cancer Research UK, London, United Kingdom.
Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA.
出版信息
Methods Enzymol. 2014;540:339-60. doi: 10.1016/B978-0-12-397924-7.00019-4.
Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein.
反平行微管之间的相互作用对于分裂细胞中纺锤体的组织至关重要。体外形成固定的反平行微管对的能力，与通过全内反射荧光显微镜（TIRF显微镜）对其成像的能力相结合，使得能够对微管交联蛋白及其对微管动力学的影响进行详细的生化表征。在这里，我们描述了在玻璃表面上对微管种子进行化学微图案化的方法，其构型能够在体外特异性促进反平行微管重叠的形成。我们证明，该检测方法特别适合于重组由反平行微管交联蛋白PRC1及其结合伙伴稳定的最小中间区重叠。微图案化方法适用于多种蛋白质，并且该检测方法通常适用于任何微管交联蛋白。
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