[论文解读] mTORC1 regulates cytokinesis through activation of Rho-ROCK signaling

Timothy R. Peterson, Mathieu Laplante+4 more|arXiv (Cornell University)|Jun 14, 2015

Microtubule and mitosis dynamics被引用 5

一句话总结

本研究鉴定出一条新型的mTORC1-Rho-ROCK信号通路，该通路调控哺乳动物细胞的胞质分裂。mTORC1的过度激活导致Rho-GTP过度装载、肌动蛋白-肌球蛋白收缩性增强以及质膜泡状隆起，从而引起胞质分裂失败和多倍体化，而该表型可通过抑制mTORC1或ROCK得到逆转，表明mTORC1设定了细胞分裂的大小阈值。

ABSTRACT

Understanding the mechanisms by which cells coordinate their size with their ability to divide has long attracted the interest of biologists. The Target of Rapamycin (TOR) pathway is becoming increasingly recognized as a master regulator of cell size, however less is known how TOR activity might be coupled with the cell cycle. Here, we establish that mTOR complex 1 (mTORC1) promotes cytokinesis through activation of a Rho GTPase-Rho Kinase (ROCK) signaling cascade. Hyperactivation of mTORC1 signaling by depletion of any of its negative regulators: TSC1, TSC2, PTEN, or DEPTOR, induces polyploidy in a rapamycin-sensitive manner. mTORC1 hyperactivation-mediated polyploidization occurs by a prolonged, but ultimately failed attempt at abcission followed by re-fusion. Similar to the effects of ROCK2 overexpression, these mTORC1-driven aberrant cytokinesis events are accompanied by increased Rho-GTP loading, extensive plasma membrane blebbing, and increased actin-myosin contractility, all of which can be rescued by either mTORC1 or ROCK inhibition. These results provide evidence for the existence of a novel mTORC1-Rho-ROCK pathway during cytokinesis and suggest that mTORC1 might play a critical role in setting the size at which a mammalian cell divides.

研究动机与目标

• 研究mTORC1信号如何影响胞质分裂及细胞大小的协调。
• 确定mTORC1是否调控细胞分裂所需的细胞骨架装置。
• 探讨mTORC1活性与Rho GTPase/ROCK通路激活在胞质分裂期间的分子联系。
• 评估mTORC1过度激活对细胞周期完成和基因组稳定性的影响。

提出的方法

• 通过RNAi介导的敲低mTORC1负向调节因子（TSC1、TSC2、PTEN、DEPTOR）以实现mTORC1信号的过度激活。
• 进行活细胞成像，实时监测胞质分裂动态及膜泡状隆起现象。
• 通过下拉实验测定Rho-GTP水平，评估Rho GTP酶的激活状态。
• 应用药理学抑制剂（雷帕霉素、ROCK抑制剂）以检测通路依赖性。
• 利用免疫荧光染色和共聚焦显微镜观察肌动蛋白-肌球蛋白收缩性及收缩环的形成。
• 通过流式细胞术和DNA含量分析检测多倍体化率。

实验结果

研究问题

• RQ1 mTORC1信号是否调控哺乳动物细胞中胞质分裂的完成？
• RQ2 Rho-ROCK通路是否在mTORC1下游被激活于胞质分裂过程中？
• RQ3 mTORC1过度激活在细胞分裂期间导致的细胞表型是什么？
• RQ4 抑制mTORC1或ROCK是否能挽救mTORC1过度活化引起的胞质分裂失败？
• RQ5 mTORC1活性是否影响细胞分裂的大小阈值？

主要发现

• 敲低TSC1、TSC2、PTEN或DEPTOR可诱导mTORC1过度激活，并导致雷帕霉素敏感性的多倍体化。
• mTORC1过度激活引起胞质分裂尝试时间延长、膜泡状隆起以及最终细胞重新融合，形成多核细胞。
• 在mTORC1过度活化的细胞中观察到Rho-GTP装载增加及肌动蛋白-肌球蛋白收缩性增强，其表型与ROCK2过表达相似。
• 这些胞质分裂缺陷可通过mTORC1和ROCK抑制同时得到挽救，证实了通路依赖性。
• 鉴定出mTORC1-Rho-ROCK轴为调控胞质分裂和细胞大小控制的新信号级联。
• 研究结果表明mTORC1在哺乳动物细胞中起核心作用，决定细胞分裂的大小阈值。