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Control of localization of a spindle checkpoint protein, Mad2, in fission yeast

Amy E. Ikui1,*, Kanji Furuya2,*,{ddagger}, Mitsuhiro Yanagida2 and Tomohiro Matsumoto1,3,§

1 Departments of Radiation Oncology and Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Graduate School of Biostudies, Department of Gene Mechanisms, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
3 Radiation Biology Center, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan
* These authors contributed equally to this work
{ddagger} Present address: MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, BN1 9RR, UK



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  		Fig. 1. Mad2 localization in mitosis. Samples were collected 20, 30, 40 or 60 minutes after the release from the cdc25-block. The cells were stained with anti-tubulin antibody (red) and DAPI. Mad2-GFP (green) was visualized in the same cells.

 


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  		Fig. 2. Mad2 on unattached kinetochores. Mad2 was localized in the nda3-KM311 mutant cells that were expressing Mis6-HA (top panels). Mis6-HA was visualized by indirect immunofluorescent staining (red), DNA by DAPI (blue) and Mad2 by GFP (green). As a negative control, indirect immunofluorescent staining for HA epitope was performed with cells not expressing Mis6-HA (lower panels).

 


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  		Fig. 3. (A) Mad2-GFP as a speckle in nda3-KM311 mutant. The cells were collected every hour during the synchronization at the restrictive temperature. After incubation for 6 hours (indicated by the arrowhead), the cells were released into the permissive temperature. Note that the samples were collected every 3 minutes after the point indicated by the arrowhead. Mad2-GFP was visualized under the microscope, and the percentage of the cells showing Mad2 as speckles versus total cell number was calculated. Over 100 cells were examined at each time point. (B) Mad2 is released from kinetochores after metaphase. A block and release experiment using the nda3-KM311 mutant was performed to stain tubulin by indirect immunofluorescence (red) and DNA by DAPI (blue). Mad2-GFP (green) was visualized in the same cells. (C) Slp1 and Mad2 protein expression after metaphase arrest. Slp1 and Mad2 protein expression in the nda3-KM311 mutant cells was analyzed 0, 3, 6, 9, 12 and 15 minutes after the release. The proteins were blotted using anti-Slp1 or anti-Mad2 antibody.

 


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  		Fig. 4. Mad2-GFP in mitotic mutants. Mad2-GFP was visualized in cut7, dis1, cut4 and nuc2 mutants at their restrictive temperature.

DAPI (blue) was used to stain DNA, and the GFP-fluorescence was converted to red.

 


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  		Fig. 5. (A) The Slp1-Mad2 complex formed in nda3-KM311 mutant cells. The protein binding was observed by a coimmunoprecipitation experiment using the anti-Slp1 antibody in cdc25-22, nda3-KM311, nuc2, cut9 or mts3 mutants (left). Mad2-GFP and Slp1 were blotted using an anti-GFP (lower panel) or anti-Slp1 antibody (upper panel), respectively. The total amount of protein in the cells was analyzed by straight western blotting (right). (B) Visualization of Mad2 and Slp1 in nda3-KM311, cdc25-22, nuc2, cut9 or mts3 mutants. The cells were stained using an anti-Slp1 antibody (red) or DAPI (blue).

Mad2-GFP was observed in the same cells as those used for Slp1 staining and DAPI (green).

 


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  		Fig. 6. (A) The Mad1-Mad2 complex in the wild-type strain. A coimmunoprecipitation experiment using an anti-Mad2 antibody was performed in order to visualize the Mad1-Mad2 protein interaction (right). The total amount of proteins was analyzed by straight western blotting (left). Mad1 and Mad2 precipitates were analyzed using an anti-Mad2 antibody (lower panels), whereas Mad1-HA was blotted using an anti-HA antibody (top panels). Mad2 knockout strain ({Delta}mad2) was used as a negative control. (B) Localization of Mad2-GFP in the {Delta}mad1 strain. Mad2-GFP (middle) was observed in the wild-type strain (upper panel) or {Delta}mad1 strain (lower panel). DNA was stained with DAPI in the same cells (left). (C) Mad2-GFP expresses at the same level in both the wild-type and {Delta}mad1 strain. Mad2-GFP protein level was analyzed by western blot using an anti-GFP antibody (upper panel). GTPase was blotted using anti-Spi1 antibody as a loading control (lower panel). A non-tagged Mad2 strain was used as a negative control for Mad2.

 

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© The Company of Biologists Ltd 2002