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BACKGROUND: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance.
METHODS: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work.
RESULTS: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells.
CONCLUSIONS: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.
Fig. 1.
Disappearance of Aurora A from the centrosome region in XL2 cells. (A) Analysis of relative Aurora A quantity in different phases of the cell cycle. Western blot analysis of cell fractions enriched by G0, G1, S, G2-phases, and mitosis (M). (B) Comparative quantitative analysis of Aurora A in different cell cycle phases from Western blot (ratio Aurora A to beta-tubulin in G1 was considered as 1). (C) Phase contract (a,d,g,j) and Aurora A (red) immunofluorescent labeling (b,c,e,f,h,i,k,l), or gamma-tubulin (green) immunofluorescent labeling (m,n). Images c, f, i, l, and n show the enlarged centrosome region from images b, e, h, k and m, respectively. Bars: 10 µm (a,b,d,e,g,h,j,k,m) and 1 µm (c,f,i,l,n). (D) Diagram of the ratio Aurora A-positive and Aurora A-negative centrosomes from immunofluorescent data of XL2 cells in A—anaphase, T—telophase of mitosis, and post-mitotic cells. (E) Analysis of Aurora A degradation after mitosis. Cells were synchronized in mitosis, and the level of Aurora A was estimated during 1–4 h of the cell cycle. (F) Cell cycle phases of XL2 cells and the presence of Aurora A.
Fig. 2.
Dependence of the presence of Aurora A kinase in the centrosome region and centrosome separation from the size of nuclei in the XL2 cell line. The area of the nucleus in cells with Aurora A-negative centrosomes (n = 226), Aurora A-positive non-separated centrosomes (n = 67), and Aurora A-positive separated centrosomes (n = 19). * marks statistically significant differences between the groups (p
<
0.001).
Fig. 3.
Recovery of Aurora A in the centrosome region in S-phase. Phase contrast (a,e) and immunofluorescent labeling with antibodies against Bromodeoxyuridine (BrdU) (b,f) or antibodies against Aurora A (c,g, and inserts). Inserts (in g) present enlarged centrosomal regions of Aurora A-positive cells. On schemes (d,h), nuclei of Aurora A-negative cells are shown in green, and nuclei of Aurora A-positive cells are shown in orange. Bar: 10 µm (a–h) and 1 µm (inserts in g).
Fig. 4.
Cells were monitored after mitosis to define exact cell cycle timing. Cells in the middle of the G1-phase (8 h 10 min after mitosis—(a–d)) and G2-phase (25 h after mitosis—(e–h)) are presented. (a,e) combined phase-contrast images/4′,6-Diamidino-2-Phenylindole (DAPI) labeling of DNA for nuclear localization/double immunofluorescent labeling using polyclonal antibodies against gamma-tubulin and monoclonal antibodies against Aurora A are presented; (b,f) enlarged centrosome regions of these two cells after anti-gamma-tubulin labeling; (c,g) after anti-Aurora A labeling and (d,h) overlapping gamma-tubulin and Aurora A are shown. Centrosome position in whole-cell images marked by arrows (a,e). Bar 10 µm (a,e) and 1 µm (b–d, f–h).
