Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha.

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 importinalpha 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-importinalpha beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinalpha 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 importinalpha 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-alpha or gamma-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.