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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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Overexpression of  Dam1p results in spindle abnormalities. Dam1p was overexpressed in DDY759 as a  GFP–Dam1p construct. Cells  are shown after 8 h of overproduction. Fluorescence analysis  of microtubule organization in  cells overexpressing GFP– Dam1p is shown in the middle  panels. Right panels show  DNA staining by DAPI. Left  panels show GFP–Dam1p fluorescence. Approximately  90% of cells overexpressing  Dam1p or GFP–Dam1p arrested at the large-budded  stage and displayed abnormal  spindles or apparently monopolar spindles. Bar, 10 μm.
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Figure 6: Overexpression of Dam1p results in spindle abnormalities. Dam1p was overexpressed in DDY759 as a GFP–Dam1p construct. Cells are shown after 8 h of overproduction. Fluorescence analysis of microtubule organization in cells overexpressing GFP– Dam1p is shown in the middle panels. Right panels show DNA staining by DAPI. Left panels show GFP–Dam1p fluorescence. Approximately 90% of cells overexpressing Dam1p or GFP–Dam1p arrested at the large-budded stage and displayed abnormal spindles or apparently monopolar spindles. Bar, 10 μm.

Mentions: Since overexpression of Duo1p was lethal and caused pronounced mitotic defects, the overexpression phenotype for DAM1 was determined. As with Duo1p, overexpression of Dam1p was lethal. Furthermore, overexpression of Dam1p resulted in a large-budded cell cycle arrest in ∼90% of the cells. The nuclei in the majority of the large-budded cells were undivided or only partially divided, and in either case, the microtubules were organized abnormally. Often, what appeared to be a single long microtubule and one to several short microtubules were organized around a single structure, most likely the spindle pole body (Fig. 6, bottom). In the large-budded cells with partially divided nuclei, the spindle often was bipolar but appeared bent or broken (Fig. 6, top and middle). In some cells, Dam1p localizes to a structure that appears to be a spindle, bent or broken, but which is poorly recognized or not recognized at all by antitubulin antibodies (Fig. 6, top). This may be because GFP–Dam1p physically blocks the antitubulin antibody. After 10 h of DAM1 overexpression, no microtubules were detected by immunofluorescence, a phenotype reminiscent of the effects of prolonged Duo1p overexpression (Fig. 1), and GFP–Dam1p fluorescence was also absent (not shown). Overexpression of wild-type Dam1p and GFP–Dam1p had the same effect on yeast cells, indicating some degree of functionality for the GFP fusion protein.


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)

Overexpression of  Dam1p results in spindle abnormalities. Dam1p was overexpressed in DDY759 as a  GFP–Dam1p construct. Cells  are shown after 8 h of overproduction. Fluorescence analysis  of microtubule organization in  cells overexpressing GFP– Dam1p is shown in the middle  panels. Right panels show  DNA staining by DAPI. Left  panels show GFP–Dam1p fluorescence. Approximately  90% of cells overexpressing  Dam1p or GFP–Dam1p arrested at the large-budded  stage and displayed abnormal  spindles or apparently monopolar spindles. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Overexpression of Dam1p results in spindle abnormalities. Dam1p was overexpressed in DDY759 as a GFP–Dam1p construct. Cells are shown after 8 h of overproduction. Fluorescence analysis of microtubule organization in cells overexpressing GFP– Dam1p is shown in the middle panels. Right panels show DNA staining by DAPI. Left panels show GFP–Dam1p fluorescence. Approximately 90% of cells overexpressing Dam1p or GFP–Dam1p arrested at the large-budded stage and displayed abnormal spindles or apparently monopolar spindles. Bar, 10 μm.
Mentions: Since overexpression of Duo1p was lethal and caused pronounced mitotic defects, the overexpression phenotype for DAM1 was determined. As with Duo1p, overexpression of Dam1p was lethal. Furthermore, overexpression of Dam1p resulted in a large-budded cell cycle arrest in ∼90% of the cells. The nuclei in the majority of the large-budded cells were undivided or only partially divided, and in either case, the microtubules were organized abnormally. Often, what appeared to be a single long microtubule and one to several short microtubules were organized around a single structure, most likely the spindle pole body (Fig. 6, bottom). In the large-budded cells with partially divided nuclei, the spindle often was bipolar but appeared bent or broken (Fig. 6, top and middle). In some cells, Dam1p localizes to a structure that appears to be a spindle, bent or broken, but which is poorly recognized or not recognized at all by antitubulin antibodies (Fig. 6, top). This may be because GFP–Dam1p physically blocks the antitubulin antibody. After 10 h of DAM1 overexpression, no microtubules were detected by immunofluorescence, a phenotype reminiscent of the effects of prolonged Duo1p overexpression (Fig. 1), and GFP–Dam1p fluorescence was also absent (not shown). Overexpression of wild-type Dam1p and GFP–Dam1p had the same effect on yeast cells, indicating some degree of functionality for the GFP fusion protein.

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