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Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

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DUO1 overexpression phenotypes. (A) a–c are  phase micrographs, d–f are  fluorescence micrographs  showing microtubule staining,  and g–i are fluorescence micrographs showing DNA  (DAPI) staining. The first  column shows wild-type cells  (a, d, and g), the second column shows cells after 8 h of  DUO1 overexpression (b, e,  and h), and the third column  shows cells after 16 h of overexpression (c, f, and i). For  overexpression studies, cells  were grown in glucose medium until log phase, washed,  and grown in raffinose medium for 12 h, and then galactose was added to the raffinose-containing medium. (B)  The effect of DUO1 overexpression on spindle pole bodies. a and c show Tub4p staining, and b and d show nuclear  (DAPI) staining. a and b  show wild-type cells, and c  and d show cells overexpressing DUO1 for 16 h. (C)  FACS® analysis of nuclear  DNA. a shows FACS® data  for a wild-type control strain.  b shows FACS® data before  DUO1 overexpression. c  shows FACS® data for the  same cell line in b 8 h after  overexpression of DUO1 was  initiated. Bars, 5 μm.
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Figure 1: DUO1 overexpression phenotypes. (A) a–c are phase micrographs, d–f are fluorescence micrographs showing microtubule staining, and g–i are fluorescence micrographs showing DNA (DAPI) staining. The first column shows wild-type cells (a, d, and g), the second column shows cells after 8 h of DUO1 overexpression (b, e, and h), and the third column shows cells after 16 h of overexpression (c, f, and i). For overexpression studies, cells were grown in glucose medium until log phase, washed, and grown in raffinose medium for 12 h, and then galactose was added to the raffinose-containing medium. (B) The effect of DUO1 overexpression on spindle pole bodies. a and c show Tub4p staining, and b and d show nuclear (DAPI) staining. a and b show wild-type cells, and c and d show cells overexpressing DUO1 for 16 h. (C) FACS® analysis of nuclear DNA. a shows FACS® data for a wild-type control strain. b shows FACS® data before DUO1 overexpression. c shows FACS® data for the same cell line in b 8 h after overexpression of DUO1 was initiated. Bars, 5 μm.

Mentions: Yeast cells were grown to early log phase in minimal or rich medium. For galactose induction, cells were grown to early log phase (∼2 × 106 cells/ml) in medium containing glucose. Using a Millipore 150-ml sterilizing filter flask (Bedford, MA), cells grown on glucose were washed twice with minimal medium without a carbon source and resuspended into medium containing glycerol. After incubating the cells in medium containing glycerol in a shaking water bath for 10–12 h, the cells were washed twice again with minimal medium without a carbon source and then resuspended from the filter surface with minimal medium containing galactose. Galactose induction for the experiment shown in Fig. 1 was instead as described in the Fig. 1 legend. Fixation and immunofluorescence procedures were carried out as described by Drubin et al. (1988). The YOL134 antitubulin antibody was used at 1:200, and the anti-Duo1p antibody (preparation described below) was used at 1:2,000. Fluorescein-conjugated anti-IgG heavy chain secondary antisera were obtained from Cappel/Organon Teknika (Malvern, PA).


Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

DUO1 overexpression phenotypes. (A) a–c are  phase micrographs, d–f are  fluorescence micrographs  showing microtubule staining,  and g–i are fluorescence micrographs showing DNA  (DAPI) staining. The first  column shows wild-type cells  (a, d, and g), the second column shows cells after 8 h of  DUO1 overexpression (b, e,  and h), and the third column  shows cells after 16 h of overexpression (c, f, and i). For  overexpression studies, cells  were grown in glucose medium until log phase, washed,  and grown in raffinose medium for 12 h, and then galactose was added to the raffinose-containing medium. (B)  The effect of DUO1 overexpression on spindle pole bodies. a and c show Tub4p staining, and b and d show nuclear  (DAPI) staining. a and b  show wild-type cells, and c  and d show cells overexpressing DUO1 for 16 h. (C)  FACS® analysis of nuclear  DNA. a shows FACS® data  for a wild-type control strain.  b shows FACS® data before  DUO1 overexpression. c  shows FACS® data for the  same cell line in b 8 h after  overexpression of DUO1 was  initiated. Bars, 5 μm.
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Figure 1: DUO1 overexpression phenotypes. (A) a–c are phase micrographs, d–f are fluorescence micrographs showing microtubule staining, and g–i are fluorescence micrographs showing DNA (DAPI) staining. The first column shows wild-type cells (a, d, and g), the second column shows cells after 8 h of DUO1 overexpression (b, e, and h), and the third column shows cells after 16 h of overexpression (c, f, and i). For overexpression studies, cells were grown in glucose medium until log phase, washed, and grown in raffinose medium for 12 h, and then galactose was added to the raffinose-containing medium. (B) The effect of DUO1 overexpression on spindle pole bodies. a and c show Tub4p staining, and b and d show nuclear (DAPI) staining. a and b show wild-type cells, and c and d show cells overexpressing DUO1 for 16 h. (C) FACS® analysis of nuclear DNA. a shows FACS® data for a wild-type control strain. b shows FACS® data before DUO1 overexpression. c shows FACS® data for the same cell line in b 8 h after overexpression of DUO1 was initiated. Bars, 5 μm.
Mentions: Yeast cells were grown to early log phase in minimal or rich medium. For galactose induction, cells were grown to early log phase (∼2 × 106 cells/ml) in medium containing glucose. Using a Millipore 150-ml sterilizing filter flask (Bedford, MA), cells grown on glucose were washed twice with minimal medium without a carbon source and resuspended into medium containing glycerol. After incubating the cells in medium containing glycerol in a shaking water bath for 10–12 h, the cells were washed twice again with minimal medium without a carbon source and then resuspended from the filter surface with minimal medium containing galactose. Galactose induction for the experiment shown in Fig. 1 was instead as described in the Fig. 1 legend. Fixation and immunofluorescence procedures were carried out as described by Drubin et al. (1988). The YOL134 antitubulin antibody was used at 1:200, and the anti-Duo1p antibody (preparation described below) was used at 1:2,000. Fluorescein-conjugated anti-IgG heavy chain secondary antisera were obtained from Cappel/Organon Teknika (Malvern, PA).

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

Show MeSH
Related in: MedlinePlus