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A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

Jensen S, Segal M, Clarke DJ, Reed SI - J. Cell Biol. (2001)

Bottom Line: Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding.In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells.Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

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Cell cycle–dependent regulation of Esp1. (A) Esp1 protein level during the cell cycle. Strain carrying epitope-tagged Esp1 integrated at the chromosomal locus (SY108) was arrested in G1 with α-factor. Cells were released into YEPDextrose at 25°C and aliquots removed at indicated times for analysis of cell morphology, Esp1 and Cdc28 protein levels, and FACS® analysis. (B) Cell cycle changes in Esp1 localization in live cells. Strain carrying GAL1-inducible ESP1GFP (SY101) was arrested in G1 with α-factor. 30 min before release, 2% galactose was added to induce Esp1GFP. Cells were released into YEPDextrose at 30°C, and aliquots were removed at intervals for analysis by real time microscopy, determination of budding index and cell-cycle progression by DAPI stain. (□) Cells with preanaphase nuclear morphology; (▪) cells with anaphase and telophase nuclear morphology; (▴) budding index; (♦) nuclear localization of Esp1; (○) spindle localization of Esp1.
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Figure 1: Cell cycle–dependent regulation of Esp1. (A) Esp1 protein level during the cell cycle. Strain carrying epitope-tagged Esp1 integrated at the chromosomal locus (SY108) was arrested in G1 with α-factor. Cells were released into YEPDextrose at 25°C and aliquots removed at indicated times for analysis of cell morphology, Esp1 and Cdc28 protein levels, and FACS® analysis. (B) Cell cycle changes in Esp1 localization in live cells. Strain carrying GAL1-inducible ESP1GFP (SY101) was arrested in G1 with α-factor. 30 min before release, 2% galactose was added to induce Esp1GFP. Cells were released into YEPDextrose at 30°C, and aliquots were removed at intervals for analysis by real time microscopy, determination of budding index and cell-cycle progression by DAPI stain. (□) Cells with preanaphase nuclear morphology; (▪) cells with anaphase and telophase nuclear morphology; (▴) budding index; (♦) nuclear localization of Esp1; (○) spindle localization of Esp1.

Mentions: To gain insight into the regulation and function of Esp1 in S. cerevisiae, we initiated a study of the localization of this protein in live cells. Unlike Pds1, the level of Esp1 is not highly regulated in the cell cycle. The abundance of Esp1 protein was examined in synchronized cells expressing endogenous Esp1 fused to 18 myc epitopes at its COOH-terminus after release from an α-factor–induced G1 block (Fig. 1 A). The level of this fully functional fusion protein is approximately threefold lower in G1 than in the rest of the cell cycle, where it appears to be constant. The synchrony of the cells was verified by flow cytometry (data not shown).


A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

Jensen S, Segal M, Clarke DJ, Reed SI - J. Cell Biol. (2001)

Cell cycle–dependent regulation of Esp1. (A) Esp1 protein level during the cell cycle. Strain carrying epitope-tagged Esp1 integrated at the chromosomal locus (SY108) was arrested in G1 with α-factor. Cells were released into YEPDextrose at 25°C and aliquots removed at indicated times for analysis of cell morphology, Esp1 and Cdc28 protein levels, and FACS® analysis. (B) Cell cycle changes in Esp1 localization in live cells. Strain carrying GAL1-inducible ESP1GFP (SY101) was arrested in G1 with α-factor. 30 min before release, 2% galactose was added to induce Esp1GFP. Cells were released into YEPDextrose at 30°C, and aliquots were removed at intervals for analysis by real time microscopy, determination of budding index and cell-cycle progression by DAPI stain. (□) Cells with preanaphase nuclear morphology; (▪) cells with anaphase and telophase nuclear morphology; (▴) budding index; (♦) nuclear localization of Esp1; (○) spindle localization of Esp1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193664&req=5

Figure 1: Cell cycle–dependent regulation of Esp1. (A) Esp1 protein level during the cell cycle. Strain carrying epitope-tagged Esp1 integrated at the chromosomal locus (SY108) was arrested in G1 with α-factor. Cells were released into YEPDextrose at 25°C and aliquots removed at indicated times for analysis of cell morphology, Esp1 and Cdc28 protein levels, and FACS® analysis. (B) Cell cycle changes in Esp1 localization in live cells. Strain carrying GAL1-inducible ESP1GFP (SY101) was arrested in G1 with α-factor. 30 min before release, 2% galactose was added to induce Esp1GFP. Cells were released into YEPDextrose at 30°C, and aliquots were removed at intervals for analysis by real time microscopy, determination of budding index and cell-cycle progression by DAPI stain. (□) Cells with preanaphase nuclear morphology; (▪) cells with anaphase and telophase nuclear morphology; (▴) budding index; (♦) nuclear localization of Esp1; (○) spindle localization of Esp1.
Mentions: To gain insight into the regulation and function of Esp1 in S. cerevisiae, we initiated a study of the localization of this protein in live cells. Unlike Pds1, the level of Esp1 is not highly regulated in the cell cycle. The abundance of Esp1 protein was examined in synchronized cells expressing endogenous Esp1 fused to 18 myc epitopes at its COOH-terminus after release from an α-factor–induced G1 block (Fig. 1 A). The level of this fully functional fusion protein is approximately threefold lower in G1 than in the rest of the cell cycle, where it appears to be constant. The synchrony of the cells was verified by flow cytometry (data not shown).

Bottom Line: Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding.In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells.Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Show MeSH
Related in: MedlinePlus