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SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans.

Yamamoto TG, Watanabe S, Essex A, Kitagawa R - J. Cell Biol. (2008)

Bottom Line: In two-cell-stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole.In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo.These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

ABSTRACT
The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD-ZW10-ZWILCH complex. In two-cell-stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

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Molecular dependency of kinetochore localization of SPDL-1 and other kinetochore proteins. (A) Embryos dissected from adult hermaphrodites untreated (WT) or injected with dsRNA of indicated genes were fixed and analyzed by immunofluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. KNL-1 and CZW-1 are required but NDC-80 or BUB-1 are not for SPDL-1 targeting to kinetochores. Bar, 20 μm. (B) Lysate from wild-type worms untreated (WT) or injected with dsRNA of indicated genes were analyzed by Western blotting with antibody to indicated proteins. To each lane, whole-worm lysates from 20 injected worms was loaded. (C) Embryos expressing indicated protein fused with GFP were dissected from adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA and analyzed by time-lapse fluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. For BUB-1, wild-type embryos were dissected from wild-type adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA, fixed, and stained with anti–BUB-1 antibody. Kinetochore localization of proteins was not affected in SPDL-1–depleted embryos. Bars, 10 μm.
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fig2: Molecular dependency of kinetochore localization of SPDL-1 and other kinetochore proteins. (A) Embryos dissected from adult hermaphrodites untreated (WT) or injected with dsRNA of indicated genes were fixed and analyzed by immunofluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. KNL-1 and CZW-1 are required but NDC-80 or BUB-1 are not for SPDL-1 targeting to kinetochores. Bar, 20 μm. (B) Lysate from wild-type worms untreated (WT) or injected with dsRNA of indicated genes were analyzed by Western blotting with antibody to indicated proteins. To each lane, whole-worm lysates from 20 injected worms was loaded. (C) Embryos expressing indicated protein fused with GFP were dissected from adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA and analyzed by time-lapse fluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. For BUB-1, wild-type embryos were dissected from wild-type adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA, fixed, and stained with anti–BUB-1 antibody. Kinetochore localization of proteins was not affected in SPDL-1–depleted embryos. Bars, 10 μm.

Mentions: We analyzed the molecular dependency of kinetochore localization of SPDL-1 on other kinetochore proteins. RNAi-mediated depletion of KNL-1 (Desai et al., 2003), a core outer kinetochore component, or CZW-1/ZW10 (Starr et al., 1997), a component of the RZZ complex, significantly reduced the targeting of SPDL-1 on chromosomes (Fig. 2 A) without affecting localization of SPDL-1 on microtubules (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200805185/DC1) and total amount of cellular SPDL-1 in whole-worm lysate (Fig. 2 B), suggesting that both are specifically required for SPDL-1 targeting to kinetochores. However, in embryos depleted substantially of endogenous BUB-1 (Oegema et al., 2001) or NDC-80 (Fig. 2 B and Fig. S3 B), SPDL-1 remained associated with chromosomes (Fig. 2 A and Fig. S3 A).


SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans.

Yamamoto TG, Watanabe S, Essex A, Kitagawa R - J. Cell Biol. (2008)

Molecular dependency of kinetochore localization of SPDL-1 and other kinetochore proteins. (A) Embryos dissected from adult hermaphrodites untreated (WT) or injected with dsRNA of indicated genes were fixed and analyzed by immunofluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. KNL-1 and CZW-1 are required but NDC-80 or BUB-1 are not for SPDL-1 targeting to kinetochores. Bar, 20 μm. (B) Lysate from wild-type worms untreated (WT) or injected with dsRNA of indicated genes were analyzed by Western blotting with antibody to indicated proteins. To each lane, whole-worm lysates from 20 injected worms was loaded. (C) Embryos expressing indicated protein fused with GFP were dissected from adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA and analyzed by time-lapse fluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. For BUB-1, wild-type embryos were dissected from wild-type adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA, fixed, and stained with anti–BUB-1 antibody. Kinetochore localization of proteins was not affected in SPDL-1–depleted embryos. Bars, 10 μm.
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fig2: Molecular dependency of kinetochore localization of SPDL-1 and other kinetochore proteins. (A) Embryos dissected from adult hermaphrodites untreated (WT) or injected with dsRNA of indicated genes were fixed and analyzed by immunofluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. KNL-1 and CZW-1 are required but NDC-80 or BUB-1 are not for SPDL-1 targeting to kinetochores. Bar, 20 μm. (B) Lysate from wild-type worms untreated (WT) or injected with dsRNA of indicated genes were analyzed by Western blotting with antibody to indicated proteins. To each lane, whole-worm lysates from 20 injected worms was loaded. (C) Embryos expressing indicated protein fused with GFP were dissected from adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA and analyzed by time-lapse fluorescence microscopy. Images of embryos at metaphase of the first mitosis are shown. For BUB-1, wild-type embryos were dissected from wild-type adult hermaphrodites untreated (WT) or injected with spdl-1 dsRNA, fixed, and stained with anti–BUB-1 antibody. Kinetochore localization of proteins was not affected in SPDL-1–depleted embryos. Bars, 10 μm.
Mentions: We analyzed the molecular dependency of kinetochore localization of SPDL-1 on other kinetochore proteins. RNAi-mediated depletion of KNL-1 (Desai et al., 2003), a core outer kinetochore component, or CZW-1/ZW10 (Starr et al., 1997), a component of the RZZ complex, significantly reduced the targeting of SPDL-1 on chromosomes (Fig. 2 A) without affecting localization of SPDL-1 on microtubules (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200805185/DC1) and total amount of cellular SPDL-1 in whole-worm lysate (Fig. 2 B), suggesting that both are specifically required for SPDL-1 targeting to kinetochores. However, in embryos depleted substantially of endogenous BUB-1 (Oegema et al., 2001) or NDC-80 (Fig. 2 B and Fig. S3 B), SPDL-1 remained associated with chromosomes (Fig. 2 A and Fig. S3 A).

Bottom Line: In two-cell-stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole.In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo.These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

ABSTRACT
The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD-ZW10-ZWILCH complex. In two-cell-stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

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