RAN1基因过表达抑制嗜热四膜虫大核无丝分裂

Overexpression of RAN1 Inhibits Macronuclear Amitosis in Tetrahymena thermophila

Ran GTPase通过RanGTP/RanGDP循环的形式,参与调控多种细胞增殖方式:包括有丝分裂和减数分裂.敲减RAN1基因可导致嗜热四膜虫大核内微管组装紊乱,从而抑制大核无丝分裂.为进一步分析Ran1在无丝分裂中的功能,本研究将野生型Ran1以及模拟GTP(Ran1Q70L)和GDP(Ran1T25N)锁定形式的Ran1突变体在嗜热四膜虫中过量表达,均导致四膜虫细胞增殖速率下降,并引起大核无丝分裂异常,且这种核异常细胞比率与Ran1过表达量呈正相关.免疫荧光定位结果显示,过表达的HA-Ran1在整个细胞中弥散分布,破坏了正常的Ran1分布形式;而过表达的HA-Ran1Q70L明显集中在大核核膜和胞质中,HA-Ran1T25N则主要定位在大核和小核内,分别与Ran1GTP/Ran1GDP循环的辅助调节因子定位模式一致.以上结果表明,过表达Ran1及其突变体可能影响嗜热四膜虫细胞中正常的Ran1GTP/Ran1GDP循环,进而导致大核无丝分裂异常.

Ran is an evolutionary conserved GTPase and involved in the regulation of cell proliferation during mitosis and meiosis through RanGTP/RanGDP cycle. Knockdown of RAN1 may cause abnormal organization of intramacronuclear microtubule and inhibit macronuclear amitosis in Tetrahymena thermophila. To analyze the roles of Ran1 in amitosis, we overexpressed Ran1 mutants and investigated the GTP-bound Ran1-mimetic （Ran1Q70L） and GDP-bound Ran1-mimetic （Ran1T25N） on macronuclear amitosis in T. thermophila. The results showed that Ran1 mutant overexpression decreased the cell proliferation rate and lead to abnormal macronuclear amitosis. The percentage of abnormal amitotic cells positively correlated with the Ran1 overexpression levels. Immuno-fluorescence analysis showed that the overexpressed HA-Ran1 was distributed throughout the cell during amitosis which disrupted the Ran gradient with high concentrations in the nucleus. The overexpressed HA-Ran1Q70L was localized in both the macronuclear envelope and the cytoplasm; whereas the overexpressed HA- Ran1T25N predominantly in the macronucleus and micronucleus. The localization of the Ran1 mutants was the same with where the regulatory proteins of Ran1GTP/Ran1GDP cycle localized. Our results suggested that the overexpression of Ran1 and its mutants inhibited the macronuclear amitotic progress by disrupting the Ran1GTP/Ran1GDP cycle in T. thermophila.