Section of Cell and Developmental Biology, Division of Biological Sciences 0347, University of California-San Diego , La Jolla, CA, USA.
Nucleus. 2020 Dec;11(1):178-193. doi: 10.1080/19491034.2020.1798093.
egg extracts are a powerful tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or on these cell cycle assembly events. Here we show that can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.
卵提取物是研究复杂生物过程的有力工具，包括核重建、核膜和核孔组装以及纺锤体组装。提取物还被进一步用于证明核输入受体或在这些细胞周期组装事件中的兼职调节作用。在这里，我们表明 也可以在这些事件中发挥作用。添加 Crm1、Exportin-t 或 Exportin-5 会减少体外核孔组装。RanQ69L-GTP，一种组成型活性形式的 RanGTP，可改善抑制作用。Crm1 和 Exportin-t 均抑制核膜融合，再次被 RanQ69L-GTP 抵消。在有丝分裂提取物中，Crm1 和 Exportin-t 对纺锤体组装有负面影响。使用 Crm1 或 Exportin-t 珠粒从提取物中进行的下拉实验揭示了核孔和纺锤体组装所必需的核孔蛋白，RanQ69L-GTP 减少了这些靶标相互作用的一部分。本研究提出了一个模型，其中输出蛋白（如输入蛋白）可以调节主要的有丝分裂组装事件。
