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.
Sugimoto
Kenji
K
Laboratory of Applied Molecular Biology, Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan. sugimoto@biochem.osakafu-u.ac.jp
Urano
Takeshi
T
Zushi
Hitomi
H
Inoue
Kimiko
K
Tasaka
Hiroaki
H
Tachibana
Makoto
M
Dotsu
Masaya
M
D064127:Aurora Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D018385:Centrosome / Q000201:enzymology; D002843:Chromatin / Q000378:metabolism; D005057:Eukaryotic Cells / Q000166:cytology / Q000201:enzymology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins; D018715:Microscopy, Video; D008938:Mitosis / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D008941:Spindle Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism; D029867:Xenopus Proteins; D028901:alpha Karyopherins / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D029867:Xenopus Proteins; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases