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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.008938

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Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2

¹ Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA
² Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. Localization of GFP-centrin to centrosomes. Exponentially growing, non-synchronized CHO cells possess centrosomes revealed as two (A-C) or sometimes four (D-F) fluorescent dots with varying distance between them. Arrowheads indicate a small centrosome next to the mother centrosomes (G-I,I'). In addition to the centriole, GFP-tagged centrin labels the PCM, which appears morphologically varied, from a fuzzy indistinct mass (J) to discrete dots or lines (arrows, K-N). Areas outlined in I' and J' are seen in I and J at high magnification. Bars, 2 µm (A-N); 10 µm (I'-J').

View larger version (80K): [in this window] [in a new window]   Fig. 2. Immunofluorescence staining of GFP-centrin-expressing CHO cells (A-C,D1-D5,E1-E3,F1-F3,G1-G3) with antibodies specific to pericentrin (A'), Cep135 (B'), PCM1 (C'), hCenexin (D1'-D5'), Nek2 (E1'-E3'), ninein (F1'-F3') and -tubulin (G1'-G3'). Merged images are shown in A"-C",D1"-D5",E1"-E3",F1"-F3",G1"-G3". Centrin is distributed differently than these molecules in the centrosome. Bars, 5 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Centrosome replication in HU-arrested CHO cells. Cells were fixed at different time points during the experiment summarized in P. One to four centrosomes are shown in A-F. Particularly prominent is the centrosome surrounded by the prominent PCM (G). New daughter centrosomes can be seen emerging next to a larger mother centrosome (arrows in H,J). This frequently occurs in an asynchronous manner, resulting in the formation of highly heterogeneous numbers of centrosomes in cells treated with HU for extended periods of time (H-M). Cells shown in N and O include numerous cytoplasmic foci dispersed to varying degrees throughout the cell. Note the presence of GFP-centrin inside the nucleus at later stages of HU treatment (M-O). (P) Quantification of the frequency of CHO cells with one to two (blue), three to four (purple) and more than five (yellow) centrosomal dots as detected by centrin-tagged GFP fluorescence. Cells were synchronized in M phase and plated into a dish containing 2 mM HU (0 hours). Centrosomal dots were counted at different times during HU incubation, after extraction of cells with detergent-containing medium. More than 200 cells were counted for each sample. Bars, 5 µm (A-C,E-L); 10 µm (D,M-O); 1 µm (inset of D).

View larger version (73K): [in this window] [in a new window]   Fig. 4. (A) Time-lapse fluorescence microscopy of centrosomes labeled with GFP-centrin in HU-arrested CHO cells. Fluorescence images were taken at indicated time points after incubation with 2 mM HU. (B) The centrosomes were frequently surrounded by the prominent PCM (arrows). Bar, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Time-lapse fluorescence microscopy of centrosomes tagged with GFP-centrin in a HU-arrested CHO cell in which two, three, four and five centrosomal dots (arrows) are detected at 19:46, 25:52, 28:59 and 31:38 hours after incubation with HU. Arrowheads indicate cytoplasmic foci of smaller sizes. Bars, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Time-lapse fluorescence microscopy of centrosomes labeled with GFP-centrin in a HU-arrested CHO cell. Images were taken at different time points after incubation with HU. Centrosomal dots continuously change their position relative to one another. Bars, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. Movement of the centrin-containing cytoplasmic foci toward the centrosome. Images were taken from supplementary material Movie 2. One fluorescent focus appears to the focal plane (yellow arrow in frame 85"), and moves back and forth before reaching the centrosome. Another focus indicated by red arrows also emerges to the plane and approaches the centrosome. Bar, 10 µm.

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