Dynamic localisation of Ran GTPase during the cell cycle.
Dynamic localisation of Ran GTPase during the cell cycle.
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DOI：
10.1186/1471-2121-10-66
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发表时间：
2009-09-18
期刊：
影响因子：
--
通讯作者：
Clarke PR
中科院分区：
文献类型：
--
作者：
Hutchins JR;Moore WJ;Clarke PR
Ran GT3在细胞分裂周期中具有多种功能，包括核质运输、有丝分裂纺锤体组装和核膜形成。Ran的活性由其鸟嘌呤核苷酸结合状态和亚细胞定位决定。在这里，我们的特点是本地化和流动性的RAN耦合到绿色荧光蛋白（GFP）在活的人类细胞的细胞周期。Ran-GFP在间期是核的，并且在有丝分裂期间分散在整个细胞中。GFP-RanQ 69 L是一种锁定在GTP结合状态的突变体，在细胞核中的浓度较低，并与核膜内的核孔复合物相关。在有丝分裂期间，GFP-RanQ 69 L被排除在染色体之外并定位于纺锤体。相比之下，GFP-RanT 24 N，一种对核苷酸具有低亲和力的突变体，在整个细胞周期中与染色质相对稳定地相互作用，并且高度集中在有丝分裂染色体上。这些结果表明，在细胞周期中，Ran与染色质、核孔复合物和有丝分裂纺锤体动态地相互作用。这些相互作用依赖于蛋白质的核苷酸结合状态。我们的数据表明，Ran-GTP产生的染色质是高度移动的，并与远端结构，参与核运输和有丝分裂纺锤体组装动态相互作用。
Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization. Here, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanQ69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes. These results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly.
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