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First published online 19 August 2003
doi: 10.1242/jcs.00707

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Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells

Henrik Appelgren, Barbara Kniola and Karl Ekwall{dagger},*

Karolinska Institute, Department of Biosciences/Department of Natural Sciences, University College Sodertorn, Sweden



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  		Fig. 1. Mitotic fission yeast centromeres are organized in cytologically separable domains. (A) Schematic diagrams of S. pombe centromere structures. Centromere 1 is shown as an example but centromeres 2 and 3 have similar symmetric organization. Top: centromere 1 DNA consists two otr regions containing a region of centromeric outer repeats (dg and dh) flanking a single central core region containing the cnt sequence. Bottom: a putative structure of centromere 1 in sister centromeres at metaphase (based on data in Fig. 1B,C,E and supplemental data at http://jcs.biologists.org/supplemental/). Kinetochores (Kin) are separated and facing opposite spindle poles whereas the otr regions are held together by sister-chromatid cohesion. The central core regions are the bases for Kin structures. (B) Deconvolved IF images of fixed metaphase cells showing two pairs of sister kinetochores. The otr DNA sequences were detected by FISH (green) and are flanked by the kinetochore protein (Ndc80) signal (red). (C) Frames from live imaging movies (see Movies 1 and 2 at http://jcs.biologists.org/supplemental) showing the spindle pole body protein Cut12-CFP (red) along with Ndc80-GFP (green) in one cell (left) and Cut12-GFP (red) along with CFP-Cnp1 (green) in another cell (right). Bottom panel: the graphs show the pixel intensities along the spindle axis in the cells of Cut12 (red) and Cnp1 or Ndc80 as indicated (green). The length of the spindle axis is indicated by large black arrowed lines below each graph and the distance between two adjacent Ndc80 (0.41 µm) or Cnp1 (0.25 µm) signals on the spindle axis are indicated by small black arrowed lines. (D) Top: frames from the movie showing Ndc80-GFP (green) along with CFP-Cnp1 and Cut12-CFP (red) in one cell. The dotted squares represent the pixel position of the Ndc80 signals (see Movies 1 and 2 at http://jcs.biologists.org/supplemental). Bottom: pixel shift control of the Hu1048 strain expressing the same protein in dual colors: Cut12-CFP (green) and Cut12-GFP (red). The perfect colocalization of the two colors of Cut12 (bottom) shows that the pixel differences between Cnp1 and Ndc80 (top) are significant. (A-C) Scale bar: 0.50 µm.

 


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  		Fig. 2. Interphase centromere DNA clustering is impaired in mis6 cells and declustered centromeres contain the heterochromatin marker Swi6 but lack the kinetochore component Ndc80. (A) Images showing cen (otr)-FISH and Ndc80 localization in wild-type and mis6 cells at the permissive and restrictive temperatures. Scale bar: 0.40 µm. (B) Diagram showing the percentage of declustered centromeres. Symbols below the diagram show that the four categories of bars represent cells with normal centromere clusters, cells with one declustered centromere, cells with two declustered centromere FISH signals and finally cells with three declustered centromere FISH signals. (C) Images showing the location of the chromodomain protein GFP-Swi6 in wild-type cells and mis6 cells, respectively. (D) The area of cen(otr)-FISH signals represented as a bar diagram. The area in rik1-302 at 25°C (strain Hu607) was 0.8±0.4 µm, mis6 (Hu633) at 36°C was 0.4±0.2 µm, mis6 at 25°C was 0.2±0.1 µm and wild type (Fy2214) at 25°C was 0.2±0.1 µm (n=50).

 


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  		Fig. 3. EM analysis of centromeres in mis6 and rik1 cells. Cells were subjected to HPF EM and immuno-gold staining for GFP-Swi6. (A,D) Different magnifications of a wild-type cell labeled with GFP-Swi6 immuno-gold. The strain used was Fy2214. (B,E) Two different magnifications of a mis6 cell grown at 36°C prior to fixation and labeled with GFP-Swi6 immuno-gold. Declustering of centromeric heterochromatin is seen. The strain used was Hu633. (C,F) Two different EM magnifications of a rik1 cell where electron dense heterochromatin is not readily detectable. Immuno-gold staining of GFP-Swi6 was not performed on this sample. The strain used was Hu607. HET, heterochromatin; N, nucleolus; SPB, spindle pole body. Swi6-Au, 10 nm gold labeling shows the presence of GFP-Swi6. Scale bar: 490 nm.

 


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  		Fig. 4. Declustering of centromeres in mis6 and nuf2-1 cells and localization of declustered centromeres to the nuclear periphery. (A) Images and statistical analysis of live cells showing the location of cen1::lacO-LacI-GFP (green) signals and the SPB marker Cut12-CFP (red) in wild-type, mis6 and nuf2-1 cells as indicated. Scale bar: 1.0 µm. cen1 at SPB indicates that cen1::lacO-LacI-GFP is within 0.2 µm from Cut12-CFP and cen1 away from SPB indicates that cen1::lacO-LacI-GFP is >0.2 µm from Cut12-CFP. (B) Images showing the location of centromeres (green) with respect to the nuclear periphery marker Pom152-GFP (white). Scale bar: 1.0 µm. See also deconvolved stacks of images (see supplemental Fig. S1 at http://jcs.biologists.org/supplemental). (C) Diagram showing the distance in one focal plane between the declustered centromere cen(otr)-FISH signal and the nuclear periphery as marked by Pom152-GFP (strains were Hu334 wild type (n=26), Hu903 rik1 (n=36) and Hu900 mis6 (n=16).

 


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  		Fig. 5. Analysis of cen1::lacO-LacI-GFP and SPBs during mitosis in live wild type, nuf2 and mis6 cells. Images from time lapse analysis of live cells showing the location of cen1::lacO-LacI-GFP (green) and the SPB marker Cut12-CFP (red) in (A) wild-type (B) mis6 and (C) nuf2-1 cells. Scale bar: 0.2 µm. The white and red arrows indicate the positions of the SPB Cut12-CFP signals (red arrows indicate inferred SPB positions from other images in the time course series) and the yellow arrows indicate the position of cen1::lacO-LacI-GFP in cases where labeling was weak. The cytoplasmic red signal is nonspecific and due to autofluorescence detected with the CFP filter. Statistical analysis of anaphase cen1::lacO-LacI-GFP movements in live wild-type, mis6-302 and nuf2-1 cells is shown in Table 2.

 

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© The Company of Biologists Ltd 2003