Department of Biology, McGill University, Montreal, Quebec, Canada.
PLoS One. 2011;6(9):e24698. doi: 10.1371/journal.pone.0024698. Epub 2011 Sep 9.
Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.
To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.
Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.
生长停滞特异性基因 2（Growth-arrest-specific 2 gene）最初在生长停滞条件下的小鼠成纤维细胞中被鉴定出来。此外，血清刺激静止、非分裂细胞会导致 gas2 的下调，并导致细胞周期重新进入。细胞骨架重排对于细胞周期进程和细胞分裂至关重要，Gas2 蛋白已被证明在有丝分裂哺乳动物细胞中与肌动蛋白和微管共定位。尽管有这些发现，但没有报道直接证据支持 Gas2 在细胞分裂机制中的作用。
为了确定 Gas2 蛋白是否在细胞分裂中发挥作用，我们在非洲爪蟾胚胎中过表达全长 Gas2 蛋白和包含肌动蛋白结合 CH 结构域或微管结合 Gas2 结构域的 Gas2 截断体。我们发现全长 Gas2 蛋白和 Gas2 结构域都能抑制细胞分裂并导致多核细胞，而不是 CH 结构域。Gas2 结构域本身就能阻止细胞分裂的观察结果表明，Gas2 功能是通过微管结合介导的。使用冷冻共聚焦显微镜，Gas2 在 Gas2 注射的非洲爪蟾胚胎的细胞皮层与微管共定位，并且在细胞骨架共沉淀测定中与微管共沉淀。为了研究 Gas2 诱导的细胞分裂阻滞的机制，我们使用伤口诱导的收缩阵列测定表明，Gas2 稳定了微管。最后，电子显微镜研究表明 Gas2 将微管束成更高阶的结构。
我们的实验表明 Gas2 在非洲爪蟾胚胎中抑制细胞分裂。我们提出 Gas2 功能是通过结合和束微管来介导的，导致细胞分裂阻滞。
