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-importinα 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 importinα 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/timelapseIn mitotic cells, we observe drastic morphological changes of subcellular structures, such as nuclear membrane, centrosome, mitotic spindle and chromosome. Many studies have shown that centrosomes and kinetochores play an important role in mitosis. Centrosomes locate on the surface of nuclear membrane in interphase, duplicate in late S, and migrate to the opposite poles in G2/M (for a review see Goepfert and Brinkley, 2000). Upon entry into prophase, they act as microtubule-organizing centers (MTOC) and microtubules nucleate from the duplicated centrosomes and form a bipolar spindle. Kinetochores, on the other hand, are morphologically defined as the microtubule attachment sites of mitotic chromosomes. Interestingly, prekinetochore structures are present in interphase nuclei, duplicate in G2 and maturate into the fully differentiated kinetochores that are visible in metaphase chromosomes (Brenner et al., 1981;Sugimoto et al., 2000). Although these two representative organelles are physically separated by nuclear
