Molecular Dynamics of Aurora-A Kinase in Living Mitotic Cells Simultaneously Visualized with Histone H3 and Nuclear Membrane Protein Importinα

Cell Structure and Function

Takeshi Urano³  Makoto Tachibana²  Kenji Sugimoto⁴  Hiroaki Tasaka⁴  Hitomi Zushi⁴  Kimiko Inoue⁴  Masaya Dotsu¹ 

[1] Visual System Department, System Development Division, Mitani Corporation;Laboratory of Signal Transduction, Institute for Virus Research, Kyoto University;Department of Biochemistry II, Nagoya University School of Medicine;Laboratory of Applied Molecular Biology, Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University

关键词: Aurora-A;    centrosome;    GFP;    histone H3;    importinα;    timelapse;   

DOI  :  10.1247/csf.27.457

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(31)Cited-By(19)Supplementary materials(2)Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinα were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importin α beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinα membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importin α membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-α or γ-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.

【 授权许可】

Unknown