Indiana University School of Medicine-Bloomington, Medical Sciences, Bloomington, IN, USA.
Methods Mol Biol. 2022;2415:221-243. doi: 10.1007/978-1-0716-1904-9_17.
Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly.
正确的纺锤体组装和染色体与纺锤体的连接对于染色体准确地分配到子细胞中至关重要。这些过程中的错误会导致非整倍体，这是非典型癌症的一个标志。了解驱动纺锤体组装的机制将为深入了解如何实现准确的染色体分离提供基本的认识。阐明纺锤体组装的复杂性的一个挑战是在空间和时间上可视化蛋白质相互作用。非洲爪蟾卵提取物系统是一种有价值的工具，可以在体外探测纺锤体组装过程中的蛋白质功能。用荧光蛋白标记蛋白质，并利用荧光方法，如Förster 共振能量转移（FRET）和荧光寿命成像显微镜（FLIM），为纺锤体组装及其调节剂的力学提供了直观的线索。然而，阐明纺锤体组装因子如何在空间上受到调节仍然具有挑战性。将卵提取物系统和可视化 FRET 方法相结合，提供了一种强大的工具来探测纺锤体组装过程中涉及的过程。在这里，我们描述了如何使用 FLIM-FRET 生物传感器来研究在非洲爪蟾卵提取物中组装的纺锤体中的蛋白质-蛋白质相互作用。这种方法应该很容易适用于各种蛋白质，从而为研究纺锤体组装的调节提供新的见解。
