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CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation

Young Y. Ou, Gary J. Mack^*, Meifeng Zhang and Jerome B. Rattner

Department of Anatomy and Cell Biology, The University of Calgary, Calgary, Alberta, Canada, T2N 4N1
^* Present address: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA

View larger version (46K): [in a new window]   Fig. 1. Typical views of the centrosomes as seen at different time points during the cell cycle when stained by an autoimmune serum (for details, see Ou and Rattner, 2000). (A-C) Diagrammatic representation of the centrosome before duplication (A), the centrosome in the process of duplication (B), and the centrosomes after duplication and separation (C). Arrowheads denote the site from which a new tubular centrosome (daughter centrosome) is formed. The upper panel shows the 3D view of the centrosome. The lower panel illustrates both longitudinal and cross-sectional views of the centrosome tube shown at the upper panel. CV, cross-sectional view; LV, longitudinal view. Note that the longitudinal view represents the middle sections whereas the cross-sectional view represents the top end of the centrosome tube. (D-F) Overlay of whole cell DIC images and the fluorescent centrosomes (red spots) in HeLa cells at the cell cycle stages G1 (D), S (E) and G2 (F). The inserts show high magnification views of centrosomes from the corresponding cells. Note the centrosomes seen at D-F provide the basis for the diagrammatic views of the centrosomes shown in A-C. Bar, 10 µM (for DIC images only).

View larger version (21K): [in a new window]   Fig. 2. Localization of CEP110 within the centrosome tube. HeLa centrosomes were stained with the autoimmune serum M4491 (red) and an anti-CEP110 antibody (green). The diagram on the left shows the views of the centrosomes seen in the images on the right. (a,b) The centrosomes before duplication. (c,d) The centrosomes in the process of duplication. (e) The centrosomes after duplication and separation. The arrow denotes the mother centrosomes when the centrosome is still in the process of duplication (c,d) or when duplication and separation are completed (e). The arrowhead denotes the daughter centrosomal bud (c,d) or the fully-formed daughter centrosome (e). Bar, 5 µM.

View larger version (14K): [in a new window]   Fig. 3. The relationship between CEP110 and Cep250/c-Nap1. HeLa centrosomes were triple-stained with the autoimmune serum M4491 (red), the anti-CEP110 antibody (purple) and an antibody against the centriolar protein Cep250/c-Nap1 (green). (a) Centrosome in the process of duplication. (b) Centrosomes after duplication and separation. The diagrams on the left illustrate the views of the centrosomes seen in the images. The arrow denotes the co-localization of CEP110 and Cep250/c-Nap1 at the daughter centrosomal bud (a) or the well-formed daughter centrosome tube (b). The arrowhead points to the co-localization of the two proteins at the mother centrosomes when centrosome duplication is still going on (a) or after duplication and separation (b). Bar, 5 µM.

View larger version (22K): [in a new window]   Fig. 4. The distribution of ninein within the centrosome tube. The centrosomes were stained with the autoimmune serum M4491 (red) and the anti-ninein antibody (green). The diagrams on the left illustrate the views of the centrosomes seen in the images. (a,b) Centrosomes before duplication. (c,d) The centrosomes in the process of duplication. (e) The centrosomes after duplication and separation. The arrow points to the mother centrosome when the centrosome is in the process of duplication (c,d) or duplication is completed (e). The arrowhead denotes the daughter centrosomal bud (c,d) or the well-formed daughter centrosome (e). Bar, 5 µM.

View larger version (26K): [in a new window]   Fig. 5. Co-localization of CEP110 and ninein. The centrosomes were co-stained with anti-CEP110 (green) and anti-ninein (red) antibodies. The diagrams on the left illustrate the views of the centrosomes seen in the images. (a) The centrosome before duplication. (b,c) The centrosomes in the process of duplication. (d) The centrosomes after duplication and separation. The arrow points to the mother centrosome when the centrosome is in the process of duplication (b,c) or duplication is completed (d). The arrowhead denotes the daughter centrosomal bud (b,c) or the well-formed daughter centrosome (d). Bar, 5 µM.

View larger version (10K): [in a new window]   Fig. 6. Microinjection of anti-CEP110 antibodies into mitotic HeLa cells disrupts the centrosome reassembly following mitosis. The cells were fixed at 24 hours after the injection and stained with the autoimmune serum M4491. (a-c) Centrosome morphology in cells injected with the anti-CEP110 antibody. (d) Centrosome morphology in cells injected with the nonspecific IgG. Bar, 5 µM.

View larger version (80K): [in a new window]   Fig. 9. Loss of centrosome MTOC function in post-mitotic PtK2 cells injected with anti-ninein antibodies. Control, cell injected with normal serum (a,b); ninein, cell injected with anti-ninein antibodies (c-f). (a,c,e) The injected cells revealed by staining with secondary antibodies. (b,d) The injected cells were stained with anti-ß-tubulin antibodies to illustrate microtubules. The arrowhead points to microtubules nucleated from the centrosome in the control cell. (f) The injected cell was stained with M4491 for the centrosome. The arrow points to the centrosome. (Inset) Enlarged view of the centrosome. Bar, 10 µM.

View larger version (21K): [in a new window]   Fig. 7. Comparison of control and CEP110 injected cells stained with M4491 for the centrosome and an antibody to ninein. The CEP110-injected cells shows a disrupted centrosome tube organization as well as a dispersed ninein distribution. Bar, 3 µM.

View larger version (90K): [in a new window]   Fig. 8. Loss of centrosome MTOC function in PtK2 cells injected with anti-CEP110 antibodies at mitosis. Control, cells injected with normal serum (a,b); CEP110, cells injected with anti-CEP110 antibodies (c,d). (a,c) The injected cells revealed by staining with secondary antibodies. (b,d) The injected cells were stained with anti-ß-tubulin antibodies to illustrate microtubules. Arrows denote microtubule nucleation from the centrosomes in the control cells. (Inset) Higher magnification of the centrosome from the upper cell. The arrowhead points to microtubules. Panel b has been printed with high contrast to better illustrate the microtubules growing from the centrosome. Abundant microtubules are present in the cytoplasm of control cells and increase over time following drug reversal. Bar, 10 µM.

View larger version (6K): [in a new window]   Fig. 10. Summary of the distribution of CEP110 and ninein during the cell cycle and their relationship to centrosome duplication. Red, the centrosome tube; green, CEP110 and ninein.