Yamagishi Yuka, Oya Kazumasa, Matsuura Akira, Abe Hiroshi
Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, Chiba 263-8522, Japan.
Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
Biochem Biophys Res Commun. 2018 Feb 12;496(3):834-839. doi: 10.1016/j.bbrc.2018.01.143. Epub 2018 Feb 1.
Two types of Arp2/3 complex inhibitors, CK-666/636 and CK-548/869, are commonly used to study Arp2/3 complex-dependent actin assembly both in vitro and in vivo. However, we found that CK-548 and CK-869 directly suppress microtubule (MT) assembly independent of the actin cytoskeleton. Treatment of cultured mammalian cells with 50 μM CK-869 dramatically decreased MT networks and, instead, accumulated tubulin at the cell periphery, as did nocodazole that inhibits MT assembly. An in vitro MT-sedimentation assay revealed that CK-548 and CK-869 significantly suppressed MT polymerization. In budding yeast, although CK-548 and CK-869 are reported to lack binding abilities in the yeast Arp3, CK-548 treatment decreased cytoplasmic MT at several tens of micromolar concentrations. In addition, we found that the effects of CK-548 and CK-869 on MT assembly varied according to species. We propose that CK-548 and CK-869 are not suitable for studying the cytoskeleton in living cells.
两种Arp2/3复合体抑制剂CK-666/636和CK-548/869常用于在体外和体内研究Arp2/3复合体依赖性肌动蛋白组装。然而，我们发现CK-548和CK-869直接抑制微管（MT）组装，且与肌动蛋白细胞骨架无关。用50μM CK-869处理培养的哺乳动物细胞会显著减少MT网络，相反，会使微管蛋白在细胞周边积累，抑制MT组装的诺考达唑也会产生同样的效果。一项体外MT沉降试验表明，CK-548和CK-869显著抑制MT聚合。在芽殖酵母中，尽管据报道CK-548和CK-869在酵母Arp3中缺乏结合能力，但CK-548在几十微摩尔浓度下会减少细胞质MT。此外，我们发现CK-548和CK-869对MT组装的影响因物种而异。我们认为CK-548和CK-869不适用于研究活细胞中的细胞骨架。
