Buđa R, Vukušić K, Tolić I M
Ruđer Bošković Institute, Zagreb, Croatia.
Methods Cell Biol. 2017;139:81-101. doi: 10.1016/bs.mcb.2016.11.007. Epub 2017 Jan 6.
The mitotic spindle is a highly organized and dynamic structure required for segregation of the genetic material into two daughter cells. Although most of the individual players involved in building the spindle have been characterized in vitro, a general understanding of how all of the spindle players act together in vivo is still missing. Hence, in recent years, experiments have focused on introducing mechanical perturbations of the spindle on a micron scale, thereby providing insight into its function and organization, as well as into forces acting in the spindle. Among different types of mechanical perturbations, optical ones are more flexible, less invasive, and more precise than other approaches. In this chapter, we describe a detailed protocol for cutting the microtubule bundles in human cells using a near-infrared femtosecond laser. This type of laser microsurgery provides the ability to precisely sever a single microtubule bundle while preserving spindle integrity and dynamics. Furthermore, we describe quantitative measurements obtained from the response of a severed microtubule bundle to laser ablation, which reveal the structure and function of individual parts of the spindle, such as the bridging fiber connecting sister k-fibers. Finally, the method described here can be easily combined with other quantitative techniques to address the complexity of the spindle.
有丝分裂纺锤体是一种高度有序且动态的结构，对于将遗传物质分离到两个子细胞中至关重要。尽管参与构建纺锤体的大多数个体成分已在体外得到表征，但对于所有纺锤体成分在体内如何协同作用仍缺乏全面的了解。因此，近年来，实验集中于在微米尺度上对纺锤体进行机械扰动，从而深入了解其功能、组织以及纺锤体内的作用力。在不同类型的机械扰动中，光学扰动比其他方法更灵活、侵入性更小且更精确。在本章中，我们描述了一种使用近红外飞秒激光切割人类细胞中微管束的详细方案。这种激光显微手术能够精确切断单个微管束，同时保持纺锤体的完整性和动态性。此外，我们描述了从切断的微管束对激光消融的反应中获得的定量测量结果，这些结果揭示了纺锤体各个部分的结构和功能，例如连接姐妹动粒微管的桥连纤维。最后，这里描述的方法可以很容易地与其他定量技术相结合，以解决纺锤体的复杂性问题。
