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The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore

Frédéric Scaërou1,*, Daniel A. Starr2,*, Fabio Piano2, Ophelia Papoulas3, Roger E. Karess1,{ddagger} and Michael L. Goldberg2,{ddagger}

1 CNRS, Centre de Génétique Moléculaire, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
2 Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
3 Department of Biology, Sinsheimer Labs, University of California at Santa Cruz, Santa Cruz, CA 95064, USA
* The first two authors contributed equally to this work



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  		Fig. 1. Drosophila ROD and ZW10 protein localization. Drosophila ROD and ZW10 proteins colocalize throughout mitosis. (a-d) Wild-type larval brains were fixed and stained to detect ROD (red), ZW10 (green) and DNA (blue). (a) Prometaphase cell; (b) metaphase; (c) early anaphase; (d) late anaphase. The superimposition of ROD and ZW10 signals is shown in the merged images of each set. ROD and ZW10 are found together on prometaphase kinetochores, on the spindle fibers in metaphase, and on kinetochores of the segregating chromatids in anaphase. ROD fails to localize in a zw10 mutant background (e,f). Larval brains from zw10 mutants were fixed and stained to detect ROD (red) and DNA (blue). (e) Metaphase zw10 cell with chromosomes at the equator. (f) Anaphase zw10 cell. No discrete ROD staining can be seen in either cell. By western blot, ROD is still present in zw10 mutant brains at normal levels (data not shown). ROD localization is dependent on tension (g). In metaphase I spermatocytes, ROD distribution differs on bivalent (bi-oriented) and on univalent (mono-oriented) chromosomes. (left) DNA; (center) merged image with DNA in blue and ROD in red; (right) same image with tubulin in green. Spindle fibers stain with ROD only on kMTs attached to bi-oriented bivalents. The attached-4 univalent chromosome (arrow) shows no staining of kMTs, but has a prominent ROD signal on the presumptive kinetochore. Bars, 5 µm.

 


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  		Fig. 2. Subcellular localization of ROD and ZW10 throughout HeLa cell mitosis. (a) Prophase cell. (b) Prometaphase cell. (c,g) Metaphase cells. (d) Early anaphase cell. (e) Late anaphase cell. (f) Telophase cell. The panels show from left to right: DNA, CREST staining, HsROD staining, HsZW10 staining and the superimposition of HsROD and HsZW10 with DNA (b-f) or without DNA (a). For the merged pictures at the right, HsROD was colored in red, HsZW10 in green and DNA in blue. The insets in A and B show higher magnification superimpositions of HsROD (red)/CREST (blue), HsZW10 (green)/CREST (blue) and HsROD (red)/HsZW10 (green). During early prophase (a), HsROD and HsZW10 begin to accumulate near the centromeres of some condensing chromosomes. The HsROD and HsZW10 staining is superimposable and external to the CREST staining. Certain centromeres stain with HsZW10 but not HsROD (arrowhead, inset). During prometaphase (b), both proteins are found in a double dot pattern at every centromere. In metaphase (c,g), when chromosomes are at the spindle equator, both HsROD and HsZW10 decorate kinetochore spindle fibers in an irregular pattern (g), particularly near the poles. By early anaphase (D), HsROD largely disappears from kinetochores, whereas HsZW10 is still detectable on the kinetochores of the segregating chromosomes. As anaphase (d,e) progresses to telophase (f), HsZW10 diminishes at the kinetochore, but gains prominence at the spindle midzone. By contrast, HsROD persists at the spindle poles. By telophase (f), the major HsROD and HsZW10 signals are at the spindle poles and midzone, respectively. All images are projections of 10-15 optical section stacks. Bars, 5 µm.

 


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  		Fig. 3. Chromosome segregation defects in embryos from worms injected with Cerod dsRNA. (A,B,C) Defective anaphase figures in embryos from worms injected with double stranded Cerod RNA. (D) Normal anaphase figure in an embryo obtained from a wild-type hermaphrodite injected with water. Chromatin is stained with DAPI in all panels. Bar, 2 µm.

 


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  		Fig. 4. ZW10 and ROD co-immunoprecipitate from Drosophila embryo and HeLa cell extracts. (A) Drosophila embryo extracts. Western blot probed with antibodies against DmZW10 shows the presence or absence of DmZW10 in immunoprecipitations from Drosophila embryo extracts. Immunoprecipitations used anti-DmZW10 crude (lane 2) or purified sera (lane 3), pre-immune serum from the DmZW10 injected rabbit (lane 1), anti-DmROD crude serum (lane 5), or pre-immune serum from the DmROD injected rabbit (lane 4). (B) HeLa cell extracts. Western blots were probed with antibodies against HsZW10. Lane 1 is crude HeLa cell extract. Other lanes on the blot show immunoprecipitations from the same HeLa cell extract: lane 3, anti-HsZW10 antibody; lane 2, pre-immune serum from the same rabbit; lane 5, anti-HsROD IgY; lane 4, pre-immune IgY from the same chicken. All samples are from the same western blot probed with the same anti-HsZW10 antibody, but the film exposure containing lanes 4 and 5 was for a longer period.

 


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  		Fig. 5. ZW10 and ROD co-fractionate from a sizing column. (Top) Fractions of a Drosophila embryo extract were eluted from a Superose 6 sizing column, transferred to a western blot, and probed with antibodies against DmROD. (Bottom) The same fractions probed with DmZW10 antibodies on a separate blot. Lanes at the far right show DmROD and DmZW10 in the material loaded onto the column. 0.5 ml fractions were collected; the void was at fraction 13, and the salt front in fraction 42. Positions of standards are marked below the blot; 669 kDa (thyroglobulin), 66 kDa (bovine serum albumin), 158 kDa (aldolase).

 

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