Multiple mechanisms regulate NuMA dynamics at spindle poles
Multiple mechanisms regulate NuMA dynamics at spindle poles
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DOI：
10.1242/jcs.01568
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发表时间：
2004-12-15
影响因子：
4
通讯作者：
Compton, DA
中科院分区：
文献类型：
--
作者：
Kisurina-Evgenieva, O;Mack, G;Compton, DA
大螺旋蛋白NuMA在脊椎动物细胞纺锤极微管负端组织中起重要作用。在这里，我们使用体内和体外方法来检查有丝分裂纺锤体极点的NuMA动力学。利用光漂白后的荧光恢复，我们发现外源表达的绿色荧光蛋白/NuMA融合在活细胞中可溶性和纺丝相关池之间持续交换。这些动态需要细胞能量，荧光恢复的平均半衰期约为3分钟。为了探索纺锤体极点的NuMA动力学是如何被调节的，我们利用了NuMA与哺乳动物有丝分裂提取物中形成的微管紫砂的关联。使用人类NuMA特异性单克隆抗体，我们用仓鼠有丝分裂提取物稀释与微管aster相关的人类NuMA的命运。与体内数据一致，该实验表明NuMA可以从预组装紫苑的核心转移到可溶性池中。在这些条件下，NuMA从紫砂位移的半衰期类似于5分钟。通过该实验，我们发现蛋白激酶活性和NuMA结合蛋白LGN调节NuMA在微管紫菀上的动态交换。因此，NuMA的动态特性受到多种机制的调控，包括蛋白质磷酸化和与LGN蛋白的结合，可溶性和微管相关池之间的交换率表明，NuMA与纺锤体极点的不溶性基质相关。
The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of similar to3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, we followed the fate of human NuMA associated with microtubule asters upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is similar to5 minutes. Using this assay, we show that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles.