Davies T, Sundaramoorthy S, Jordan S N, Shirasu-Hiza M, Dumont J, Canman J C
Columbia University, New York, NY, United States.
Institut Jacques Monod, Paris, France.
Methods Cell Biol. 2017;137:283-306. doi: 10.1016/bs.mcb.2016.05.004. Epub 2016 Jun 14.
Fast-acting temperature-sensitive (ts) mutations are powerful conditional tools for studying transient cellular processes such as cytokinesis. Fast-acting ts cytokinesis-defective mutants are functional at the permissive temperature; yet show a fully penetrant loss-of-function cytokinesis failure phenotype when upshifted to the restrictive temperature. Fast-acting ts mutations thus allow functional tunability and rapid and reversible protein inactivation by simply shifting the temperature at precise times throughout cell division. In this chapter, we describe several techniques and discuss various approaches for harnessing the power of fast-acting ts mutants to study cytokinesis in Caenorhabditis elegans using both simple passive heat transfer and more advanced fluidic-based thermal control systems. We also provide detailed protocols for standard dissection, mounting, and imaging of early worm embryos.
快速作用的温度敏感（ts）突变是研究诸如胞质分裂等瞬时细胞过程的强大条件工具。快速作用的ts胞质分裂缺陷型突变体在允许温度下具有功能；但当温度上调至限制温度时，会表现出完全显性的功能丧失型胞质分裂失败表型。因此，快速作用的ts突变通过在细胞分裂的精确时间简单地改变温度，实现了功能可调性以及蛋白质的快速和可逆失活。在本章中，我们描述了几种技术，并讨论了利用快速作用的ts突变体的力量，通过简单的被动热传递和更先进的基于流体的热控制系统来研究秀丽隐杆线虫胞质分裂的各种方法。我们还提供了早期线虫胚胎的标准解剖、固定和成像的详细方案。
