CSF : Vol. 27 (2002) , No. 6 pp.457-467

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

Kenji Sugimoto¹⁾, Takeshi Urano²⁾, Hitomi Zushi¹⁾, Kimiko Inoue¹⁾, Hiroaki Tasaka¹⁾, Makoto Tachibana³⁾ and Masaya Dotsu⁴⁾

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

ABSTRACT. 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.

Key words: Aurora-A/ centrosome/ GFP/ histone H3/ importinα/ timelapse