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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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Subcellular localization of Duo1p. To demonstrate antibody specificity, a shows an immunoblot in which a yeast whole cell extract was probed with the Duo1p antiserum. A log phase culture of wild-type strain DDY898 was fixed and stained with antibodies  against tubulin (b), Duo1p (c), and with DAPI to visualize DNA (d). The Duo1p localization by immunofluorescence was confirmed by  localization of GFP–Duo1p (not shown). (e) A longitudinal EM section through a mitotic spindle, passing through one of the spindle  pole bodies (S) and showing numerous spindle microtubules. Duo1p localization is shown by the 10-nm gold particles that appear to associate primarily with microtubules. Bars: (b–d) 5 μm; (e) 0.2 μm.
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Figure 2: Subcellular localization of Duo1p. To demonstrate antibody specificity, a shows an immunoblot in which a yeast whole cell extract was probed with the Duo1p antiserum. A log phase culture of wild-type strain DDY898 was fixed and stained with antibodies against tubulin (b), Duo1p (c), and with DAPI to visualize DNA (d). The Duo1p localization by immunofluorescence was confirmed by localization of GFP–Duo1p (not shown). (e) A longitudinal EM section through a mitotic spindle, passing through one of the spindle pole bodies (S) and showing numerous spindle microtubules. Duo1p localization is shown by the 10-nm gold particles that appear to associate primarily with microtubules. Bars: (b–d) 5 μm; (e) 0.2 μm.

Mentions: A rabbit antibody was raised against bacterially expressed Duo1p containing a six His-tag. After affinity purification, the antibody recognized a single band at the predicted size for Duo1p (32 kD) in yeast whole-cell extracts (Fig. 2 a). Indirect immunofluorescence microscopy experiments using this antibody showed that the protein is located along nuclear microtubules and appears concentrated at spindle pole bodies (Fig. 2). Duo1p was not detected along cytoplasmic microtubules. Consistent with this observation, in G1, a cell cycle stage during which spindle assembly has not yet occurred, Duo1p staining is only seen in the vicinity of the spindle pole body. Localization of a GFP–Duo1p fusion protein yielded the same results (not shown).


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)

Subcellular localization of Duo1p. To demonstrate antibody specificity, a shows an immunoblot in which a yeast whole cell extract was probed with the Duo1p antiserum. A log phase culture of wild-type strain DDY898 was fixed and stained with antibodies  against tubulin (b), Duo1p (c), and with DAPI to visualize DNA (d). The Duo1p localization by immunofluorescence was confirmed by  localization of GFP–Duo1p (not shown). (e) A longitudinal EM section through a mitotic spindle, passing through one of the spindle  pole bodies (S) and showing numerous spindle microtubules. Duo1p localization is shown by the 10-nm gold particles that appear to associate primarily with microtubules. Bars: (b–d) 5 μm; (e) 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Subcellular localization of Duo1p. To demonstrate antibody specificity, a shows an immunoblot in which a yeast whole cell extract was probed with the Duo1p antiserum. A log phase culture of wild-type strain DDY898 was fixed and stained with antibodies against tubulin (b), Duo1p (c), and with DAPI to visualize DNA (d). The Duo1p localization by immunofluorescence was confirmed by localization of GFP–Duo1p (not shown). (e) A longitudinal EM section through a mitotic spindle, passing through one of the spindle pole bodies (S) and showing numerous spindle microtubules. Duo1p localization is shown by the 10-nm gold particles that appear to associate primarily with microtubules. Bars: (b–d) 5 μm; (e) 0.2 μm.
Mentions: A rabbit antibody was raised against bacterially expressed Duo1p containing a six His-tag. After affinity purification, the antibody recognized a single band at the predicted size for Duo1p (32 kD) in yeast whole-cell extracts (Fig. 2 a). Indirect immunofluorescence microscopy experiments using this antibody showed that the protein is located along nuclear microtubules and appears concentrated at spindle pole bodies (Fig. 2). Duo1p was not detected along cytoplasmic microtubules. Consistent with this observation, in G1, a cell cycle stage during which spindle assembly has not yet occurred, Duo1p staining is only seen in the vicinity of the spindle pole body. Localization of a GFP–Duo1p fusion protein yielded the same results (not shown).

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