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Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Cheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G - J. Cell Biol. (2001)

Bottom Line: We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM.To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1.These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

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Analysis of stu2 dam1 and dam1 bik1 double mutants reveals shared roles in spindle structure. dam1-11, stu2-10, and bik1-1 single mutants and dam1-11 stu2-10, dam1-1 stu2-10, dam1-11 bik1-1, and dam1-1 bik1-1 double mutants were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). Bar, 5 μm.
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fig5: Analysis of stu2 dam1 and dam1 bik1 double mutants reveals shared roles in spindle structure. dam1-11, stu2-10, and bik1-1 single mutants and dam1-11 stu2-10, dam1-1 stu2-10, dam1-11 bik1-1, and dam1-1 bik1-1 double mutants were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). Bar, 5 μm.

Mentions: Our previous work indicated that Dam1p is a microtubule-associated protein involved in attaching the kinetochore to spindle microtubules (Cheeseman et al., 2001). Recent work has suggested a similar role for the microtubule-associated proteins Stu2p and Bik1p (He et al., 2001). Therefore, we also conducted crosses between dam1-1 and stu2-10 (Kosco et al., 2001; Severin et al., 2001) or bik1-1 (Pellman et al., 1995). dam1-1 was synthetically sick in combination with both stu2-10 and bik1-1; however, bik1-1 dam1-1 double mutants were much sicker, growing poorly even at 25°C. To determine whether these genetic interactions indicated a shared function in kinetochore–microtubule attachments or in spindle structure, we performed tubulin immunofluorescence on bik1-1 dam1-1, bik1-1 dam1-11, stu2-10 dam1-1, and stu2-10 dam1-11 double mutants grown at 37°C (Fig. 5). In contrast to dam1-1 and dam1-11 mutants that arrest in metaphase and show premature spindle elongation (Cheeseman et al., 2001; Fig. 5), dam1-1 stu2-10 and dam1-11 stu2-10 double mutants showed shorter broken down spindles. This lack of elongated spindles is consistent with the role that has been ascribed to Stu2p in mediating microtubule dynamics and spindle elongation (Kosco et al., 2001; Severin et al., 2001). dam1-11 bik1-1 and dam1-1 bik1-1 double mutants arrested primarily with short highly abnormal spindles, although some DNA segregation was observed. This severe spindle phenotype is not observed in either dam1 or bik1 single mutants, suggesting an overlapping role for Bik1p and Dam1p in spindle structure. Therefore, although Stu2p and Bik1p may have roles in kinetochore function the results described here indicate that both proteins play important roles in spindle structure.


Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Cheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G - J. Cell Biol. (2001)

Analysis of stu2 dam1 and dam1 bik1 double mutants reveals shared roles in spindle structure. dam1-11, stu2-10, and bik1-1 single mutants and dam1-11 stu2-10, dam1-1 stu2-10, dam1-11 bik1-1, and dam1-1 bik1-1 double mutants were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199314&req=5

fig5: Analysis of stu2 dam1 and dam1 bik1 double mutants reveals shared roles in spindle structure. dam1-11, stu2-10, and bik1-1 single mutants and dam1-11 stu2-10, dam1-1 stu2-10, dam1-11 bik1-1, and dam1-1 bik1-1 double mutants were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). Bar, 5 μm.
Mentions: Our previous work indicated that Dam1p is a microtubule-associated protein involved in attaching the kinetochore to spindle microtubules (Cheeseman et al., 2001). Recent work has suggested a similar role for the microtubule-associated proteins Stu2p and Bik1p (He et al., 2001). Therefore, we also conducted crosses between dam1-1 and stu2-10 (Kosco et al., 2001; Severin et al., 2001) or bik1-1 (Pellman et al., 1995). dam1-1 was synthetically sick in combination with both stu2-10 and bik1-1; however, bik1-1 dam1-1 double mutants were much sicker, growing poorly even at 25°C. To determine whether these genetic interactions indicated a shared function in kinetochore–microtubule attachments or in spindle structure, we performed tubulin immunofluorescence on bik1-1 dam1-1, bik1-1 dam1-11, stu2-10 dam1-1, and stu2-10 dam1-11 double mutants grown at 37°C (Fig. 5). In contrast to dam1-1 and dam1-11 mutants that arrest in metaphase and show premature spindle elongation (Cheeseman et al., 2001; Fig. 5), dam1-1 stu2-10 and dam1-11 stu2-10 double mutants showed shorter broken down spindles. This lack of elongated spindles is consistent with the role that has been ascribed to Stu2p in mediating microtubule dynamics and spindle elongation (Kosco et al., 2001; Severin et al., 2001). dam1-11 bik1-1 and dam1-1 bik1-1 double mutants arrested primarily with short highly abnormal spindles, although some DNA segregation was observed. This severe spindle phenotype is not observed in either dam1 or bik1 single mutants, suggesting an overlapping role for Bik1p and Dam1p in spindle structure. Therefore, although Stu2p and Bik1p may have roles in kinetochore function the results described here indicate that both proteins play important roles in spindle structure.

Bottom Line: We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM.To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1.These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

Show MeSH