Spatial Distribution and Mobility of the Ran GTPase in Live Interphase Cells
Spatial Distribution and Mobility of the Ran GTPase in Live Interphase Cells
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DOI：
10.1016/j.bpj.2009.07.055
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发表时间：
2009-10-21
影响因子：
3.4
通讯作者：
Fradin, Cecile
中科院分区：
文献类型：
--
作者：
Abu-Arish, Asmahan;Kalab, Petr;Fradin, Cecile
GTP酶RAN是核孔复合体(NPC)通道中分子运输的关键调节因子。为了分析RAN在其核转运功能中的作用，我们使用了几种定量荧光技术来跟踪增强型黄色荧光蛋白(EYFP)-RAN在HeLa细胞中的分布和动态。大多数EYFP-RAN分子在细胞内的扩散系数对应于一个非结合状态，揭示了显著的动态RAN调节。虽然我们在细胞中没有观察到显著的固定RAN群体，但类似于10%的细胞质EYFP-RAN和30%的核EYFP-RAN表现出低迁移率，表明分子相互作用。慢核EYFP-RAN的高比例反映了预期的核RanGTP与核运输受体的大量相互作用。然而，这还不足以支持保留机制作为观察到的RAN核积累的主要原因。在核膜上检测到最高浓度的EYFP-RAN，相当于每个NPC的200个内源性RAN分子，超过了目前估计的NPC通道转运能力。再加上EYFP-RAN在核膜上相对较长的停留时间(33+/-14ms)，这些观察表明只有一小部分RAN集中在NPC上通过NPC通道。
The GTPase Ran is a key regulator of molecular transport through nuclear pore complex (NPC) channels. To analyze the role of Ran in its nuclear transport function, we used several quantitative fluorescence techniques to follow the distribution and dynamics of an enhanced yellow fluorescent protein (EYFP)-Ran in HeLa cells. The diffusion coefficient of the majority of EYFP-Ran molecules throughout the cells corresponded to an unbound state, revealing the remarkably dynamic Ran regulation. Although we observed no significant immobile Ran populations in cells, similar to 10% of the cytoplasmic EYFP-Ran and 30% of the nuclear EYFP-Ran exhibited low mobility indicative of molecular interactions. The high fraction of slow nuclear EYFP-Ran reflects the expected numerous interactions of nuclear RanGTP with nuclear transport receptors. However, it is not high enough to support retention mechanisms as the main cause for the observed nuclear accumulation of Ran. The highest cellular concentration of EYFP-Ran was detected at the nuclear envelope, corresponding to similar to 200 endogenous Ran molecules for each NPC, and exceeding the currently estimated NPC channel transport capacity. Together with the relatively long residence time of EYFP-Ran at the nuclear envelope (33 +/- 14 ms), these observations suggest that only a fraction of the Ran concentrated at NPCs transits through NPC channels.