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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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Immunofluorescence analysis of microtubules in  temperature-sensitive duo1  mutants. Wild-type cells,  chromosomally integrated  duo1-2 mutant cells, and  duo-1 mad2Δ double mutant  cells were grown to log phase  at 25°C, shifted to 37°C for 90  min, and fixed for immunofluorescence microscopy. a–c  show microtubule fluorescence for wild-type and  duo1-2 and duo1-2 mad2Δ  double mutant cells, respectively. d–f show DNA (DAPI)  staining for the same cells  shown in a–c. Bar, 10 μm.
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Figure 3: Immunofluorescence analysis of microtubules in temperature-sensitive duo1 mutants. Wild-type cells, chromosomally integrated duo1-2 mutant cells, and duo-1 mad2Δ double mutant cells were grown to log phase at 25°C, shifted to 37°C for 90 min, and fixed for immunofluorescence microscopy. a–c show microtubule fluorescence for wild-type and duo1-2 and duo1-2 mad2Δ double mutant cells, respectively. d–f show DNA (DAPI) staining for the same cells shown in a–c. Bar, 10 μm.

Mentions: When cells carrying integrated duo1-2 were grown at the restrictive temperature for 90 min, essentially all of the large-budded cells contained short spindles (100%, n = 23) (Fig. 3 b). In wild-type cells, most large-budded cells contain elongated spindles (Fig. 3 a). Thus, the earliest defect to develop in the duo1 mutants seems to be the inability to elongate the spindle. When duo1-2 mutant cells were grown for 3 h or 4 h at the restrictive temperature, a large-budded arrest was evident in 81 and 90% of the cells, respectively. After 3 h at the restrictive temperature, 92% (n = 169) of large-budded duo1-2 cells contained a short spindle.


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)

Immunofluorescence analysis of microtubules in  temperature-sensitive duo1  mutants. Wild-type cells,  chromosomally integrated  duo1-2 mutant cells, and  duo-1 mad2Δ double mutant  cells were grown to log phase  at 25°C, shifted to 37°C for 90  min, and fixed for immunofluorescence microscopy. a–c  show microtubule fluorescence for wild-type and  duo1-2 and duo1-2 mad2Δ  double mutant cells, respectively. d–f show DNA (DAPI)  staining for the same cells  shown in a–c. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132964&req=5

Figure 3: Immunofluorescence analysis of microtubules in temperature-sensitive duo1 mutants. Wild-type cells, chromosomally integrated duo1-2 mutant cells, and duo-1 mad2Δ double mutant cells were grown to log phase at 25°C, shifted to 37°C for 90 min, and fixed for immunofluorescence microscopy. a–c show microtubule fluorescence for wild-type and duo1-2 and duo1-2 mad2Δ double mutant cells, respectively. d–f show DNA (DAPI) staining for the same cells shown in a–c. Bar, 10 μm.
Mentions: When cells carrying integrated duo1-2 were grown at the restrictive temperature for 90 min, essentially all of the large-budded cells contained short spindles (100%, n = 23) (Fig. 3 b). In wild-type cells, most large-budded cells contain elongated spindles (Fig. 3 a). Thus, the earliest defect to develop in the duo1 mutants seems to be the inability to elongate the spindle. When duo1-2 mutant cells were grown for 3 h or 4 h at the restrictive temperature, a large-budded arrest was evident in 81 and 90% of the cells, respectively. After 3 h at the restrictive temperature, 92% (n = 169) of large-budded duo1-2 cells contained a short spindle.

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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