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

Abstract

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

Visualization of Aberrant Perinuclear Microtubule Aster Organization by Microtubule-Destabilizing Agents

“…5,6) However, attention was paid only to the behavior of one or two mitotic components such as the nuclear envelope and microtubules in those studies. We have constructed a multicolor cell line in which the centrosomes and chromosomes could be simultaneously visualized, 7) and using this cell line, we recently characterized the in vivo effect of the microtubule-stabilizing agents taxanes and epothilones on centrosomes at low concentrations that do not cause mitotic arrest. 8,9) Since centrosomes are the dominant microtubule organizing centers (MTOCs) for spindle formation, it is thought that microtubule-targeting agents disturb the spindle architecture by inhibiting microtubule organization.…”

“…pmCherry--tubulin, pmKO-AF8, 10) and pECFPhistone H3 7) were separately introduced into MDA-GFP-Aurora-A cells 7) with selection plasmids, pTKHyg, pPur, and pCAGGS-bsr respectively. Stable transformants were sequentially selected by hygromycin B, puromycin, and blasticidin to establish MDA AuroA/ tub/AF8/H3 (clone 7-1).…”

“…ECFP-histone H3 was used as a marker for interphase chromatin and mitotic chromosomes. 7) Live cell imaging and fluorescence microscopy were performed as described previously. 7,9) Time-lapse images of mitosis of this cell line are shown in Fig.…”

Improvement of a Venus-Based Bimolecular Fluorescence Complementation Assay to Visualize bFos-bJun Interaction in Living Cells

“…Mouse C3H10T1/2 cells were transfected with plasmids encoding Venus and its variants (L201A, L207A, V150A, and I152A) using LipofectamineÔ LTX Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Twentyfour h after transfection, the cells were fixed and observed under a fluorescence microscope, as described previously 14) (left column). BiFC images were obtained after transfection with bJun-VN155 and bFos-VC155 plasmids (right column).…”

A Rapid and Simple Method of Evaluating the Dimeric Tendency of Fluorescent Proteins in Living Cells Using a Truncated Protein of Importin α as Fusion Tag

“…Expressed as a fusion-tag, it is possible to visualize the molecular behavior of many proteins of interest. 2,3) The molecular structures of Aequorea GFP and the red fluorescent protein from the coral Discosoma sp. (DsRed) 4) have been extensively characterized.…”