Hinkle Beth, Slepchenko Boris, Rolls Melissa M, Walther Tobias C, Stein Pascal A, Mehlmann Lisa M, Ellenberg Jan, Terasaki Mark
Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032, USA.
J Cell Sci. 2002 Dec 1;115(Pt 23):4685-93. doi: 10.1242/jcs.00136.
Recent studies in Xenopus egg extracts indicate that the small G protein Ran has a central role in spindle assembly and nuclear envelope reformation. We determined Ran localization and dynamics in cells during M phase. By immunofluorescence, Ran is accumulated on the chromosomes of meiosis-II-arrested Xenopus eggs. In living cells, fluorescently labeled Ran associated with the chromosomes in Xenopus and remained associated during anaphase when eggs were artificially activated. Fluorescent Ran associated with chromosomes in mouse eggs, during meiotic maturation and early embryonic divisions in starfish, and to a lesser degree during mitosis of a cultured mammalian cell line. Chromosomal Ran undergoes constant flux. From photobleach experiments in immature starfish oocytes, chromosomal Ran has a k(off) of approximately 0.06 second(-1), and binding analysis suggests that there is a single major site. The chromosomal interactions may serve to keep Ran-GTP in the vicinity of the chromosomes for spindle assembly and nuclear envelope reformation.
近期对非洲爪蟾卵提取物的研究表明,小G蛋白Ran在纺锤体组装和核膜重塑过程中发挥着核心作用。我们确定了M期细胞中Ran的定位和动态变化。通过免疫荧光法发现,Ran聚集在减数分裂II期阻滞的非洲爪蟾卵的染色体上。在活细胞中,荧光标记的Ran与非洲爪蟾的染色体相关联,并且在人工激活卵母细胞的后期仍保持这种关联。荧光Ran在小鼠卵母细胞减数分裂成熟过程中、海星早期胚胎分裂期间与染色体相关联,在培养的哺乳动物细胞系有丝分裂期间也有一定程度的关联。染色体上的Ran处于持续流动状态。从不成熟海星卵母细胞的光漂白实验可知,染色体上的Ran的解离常数k(off)约为0.06秒-1,结合分析表明存在一个主要结合位点。染色体相互作用可能有助于使Ran-GTP保持在染色体附近,以进行纺锤体组装和核膜重塑。