Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Biophys J. 2013 Apr 16;104(8):1642-51. doi: 10.1016/j.bpj.2013.03.024.
The formation of an activity gradient of the small G-protein Ran around chromatin depends on the differential partitioning of the opposing enzyme activities of the Ran guanine nucleotide exchange factor RCC1 that resides on chromatin, and the cytoplasmic Ran GTPase activating protein RanGAP. We studied the time-dependent interaction kinetics between RCC1 and chromatin and the mobility of the Ran-RCC1 complex in living cells by fluorescence correlation spectroscopy to investigate whether binding of RCC1 to chromatin regulates the exchange activity of RCC1, and whether the stability of the RCC1-chromatin interaction is regulated during the cell cycle. We found that RCC1 mobility is dominated by two states: a highly mobile state that is trapped within chromatin, and a transiently immobilized state that is stabilized during mitosis. We show that only the immobilized state of RCC1 interacts with Ran and conclude that its guanine nucleotide exchange activity is restricted to specific sites on chromatin.
Ran 是一种小分子 G 蛋白，其在染色质周围形成活性梯度，这取决于 Ran 鸟嘌呤核苷酸交换因子 RCC1 的两种拮抗酶活性在染色质上的不同分配，以及位于细胞质中的 RanGAP。我们通过荧光相关光谱法研究了活细胞中 RCC1 与染色质之间的时间依赖性相互作用动力学和 Ran-RCC1 复合物的迁移率，以探究 RCC1 与染色质的结合是否调节 RCC1 的交换活性，以及 RCC1-染色质相互作用的稳定性是否在细胞周期中受到调控。我们发现，RCC1 的迁移率主要由两种状态决定：一种是高度迁移的状态，被困在染色质内；另一种是短暂固定的状态，在有丝分裂期间稳定。我们表明，只有 RCC1 的固定状态与 Ran 相互作用，并且可以得出结论，其鸟嘌呤核苷酸交换活性仅限于染色质上的特定位置。
