Limits...
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.

Show MeSH

Related in: MedlinePlus

Pds1 is required to load Esp1 onto the spindle. (A) A version of Esp1GFP fused at its COOH terminus to the SV40 NLS was integrated into a wild-type strain (SY105, top), a pds1-128 mutant (SY107, middle), and a pds1Δ mutant (SY106, bottom). The Esp1GFPNLS protein was induced for 20 min from the GAL1 promoter on solid YEPGalactose medium. (Left) GFP fluorescence; (right) DIC. Scale bar: 10 μm. (B) Expression of a nondestructable Pds1 (Pds1Δdb) leads to premature nuclear localization of Esp1. A strain carrying GAL1-inducible integrated ESP1GFP and PDS1Δdb alleles (SY121) (b) was grown in YEPRaffinose. After 1-h galactose induction at 30°C, cells were fixed 30 min in 3.75% formaldehyde and stained with DAPI to visualize nuclei. (Left) Esp1GFP fluorescence; (right) DAPI/DIC overlay. A control strain carrying only a GAL1-inducible ESP1GFP allele (SY104) was analyzed in a similar fashion (a).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193664&req=5

Figure 5: Pds1 is required to load Esp1 onto the spindle. (A) A version of Esp1GFP fused at its COOH terminus to the SV40 NLS was integrated into a wild-type strain (SY105, top), a pds1-128 mutant (SY107, middle), and a pds1Δ mutant (SY106, bottom). The Esp1GFPNLS protein was induced for 20 min from the GAL1 promoter on solid YEPGalactose medium. (Left) GFP fluorescence; (right) DIC. Scale bar: 10 μm. (B) Expression of a nondestructable Pds1 (Pds1Δdb) leads to premature nuclear localization of Esp1. A strain carrying GAL1-inducible integrated ESP1GFP and PDS1Δdb alleles (SY121) (b) was grown in YEPRaffinose. After 1-h galactose induction at 30°C, cells were fixed 30 min in 3.75% formaldehyde and stained with DAPI to visualize nuclei. (Left) Esp1GFP fluorescence; (right) DAPI/DIC overlay. A control strain carrying only a GAL1-inducible ESP1GFP allele (SY104) was analyzed in a similar fashion (a).

Mentions: To address whether the lack of Esp1 spindle association in strains compromised for Pds1 function could simply reflect the absence of nuclear accumulation of Esp1, we investigated the effect of fusing a nuclear localization sequence (NLS) to Esp1 on its localization. For this purpose, a peptide encoding the potent SV40 NLS (KKKRKV) was fused to the COOH terminus of the Esp1GFP protein. This chimeric protein retains the ability to complement an esp1ts mutation when overexpressed from the GAL1 promoter (not shown). In wild-type cells, the Esp1GFPNLS protein exhibited a localization profile similar to that of Esp1 fused to GFP alone (Fig. 5 A, top). In contrast, expression of Esp1GFPNLS in the pds1-128 mutant now resulted in accumulation of the protein in the nucleus and subsequent labeling of the SPBs and weak spindle association. However, increasing the nuclear import of Esp1 in pds1-128 cells was not sufficient to achieve a strong spindle fluorescence, as seen in wild-type cells (Fig. 5 A, middle), suggesting that Pds1 association may also be required to obtain efficient Esp1 spindle interaction. This is consistent with the observation that there is no detectable spindle fluorescence in pds1Δ cells expressing Esp1GFPNLS (data not shown). Esp1 did label the SPBs in this mutant, demonstrating that Esp1 has the ability to bind the SPBs in the absence of Pds1 (Fig. 5 A, bottom). This is perhaps not surprising as mutants deleted for PDS1 are viable at low temperatures and unless the essential function of Esp1 is independent of its spindle localization, Esp1 must have intrinsic spindle binding activity sufficient for viability. This association, however, is clearly more efficient when Pds1 is present. Expressing the Esp1GFPNLS version in a pds1Δ mutant leads to partial suppression of the temperature-sensitive phenotype at 30°C, whereas the pds1Δ mutant expressing Esp1GFP is dead at this temperature (not shown). This further supports the notion that an important function of Pds1 is to target Esp1 to the nucleus and subsequently to the spindle.


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)

Pds1 is required to load Esp1 onto the spindle. (A) A version of Esp1GFP fused at its COOH terminus to the SV40 NLS was integrated into a wild-type strain (SY105, top), a pds1-128 mutant (SY107, middle), and a pds1Δ mutant (SY106, bottom). The Esp1GFPNLS protein was induced for 20 min from the GAL1 promoter on solid YEPGalactose medium. (Left) GFP fluorescence; (right) DIC. Scale bar: 10 μm. (B) Expression of a nondestructable Pds1 (Pds1Δdb) leads to premature nuclear localization of Esp1. A strain carrying GAL1-inducible integrated ESP1GFP and PDS1Δdb alleles (SY121) (b) was grown in YEPRaffinose. After 1-h galactose induction at 30°C, cells were fixed 30 min in 3.75% formaldehyde and stained with DAPI to visualize nuclei. (Left) Esp1GFP fluorescence; (right) DAPI/DIC overlay. A control strain carrying only a GAL1-inducible ESP1GFP allele (SY104) was analyzed in a similar fashion (a).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Pds1 is required to load Esp1 onto the spindle. (A) A version of Esp1GFP fused at its COOH terminus to the SV40 NLS was integrated into a wild-type strain (SY105, top), a pds1-128 mutant (SY107, middle), and a pds1Δ mutant (SY106, bottom). The Esp1GFPNLS protein was induced for 20 min from the GAL1 promoter on solid YEPGalactose medium. (Left) GFP fluorescence; (right) DIC. Scale bar: 10 μm. (B) Expression of a nondestructable Pds1 (Pds1Δdb) leads to premature nuclear localization of Esp1. A strain carrying GAL1-inducible integrated ESP1GFP and PDS1Δdb alleles (SY121) (b) was grown in YEPRaffinose. After 1-h galactose induction at 30°C, cells were fixed 30 min in 3.75% formaldehyde and stained with DAPI to visualize nuclei. (Left) Esp1GFP fluorescence; (right) DAPI/DIC overlay. A control strain carrying only a GAL1-inducible ESP1GFP allele (SY104) was analyzed in a similar fashion (a).
Mentions: To address whether the lack of Esp1 spindle association in strains compromised for Pds1 function could simply reflect the absence of nuclear accumulation of Esp1, we investigated the effect of fusing a nuclear localization sequence (NLS) to Esp1 on its localization. For this purpose, a peptide encoding the potent SV40 NLS (KKKRKV) was fused to the COOH terminus of the Esp1GFP protein. This chimeric protein retains the ability to complement an esp1ts mutation when overexpressed from the GAL1 promoter (not shown). In wild-type cells, the Esp1GFPNLS protein exhibited a localization profile similar to that of Esp1 fused to GFP alone (Fig. 5 A, top). In contrast, expression of Esp1GFPNLS in the pds1-128 mutant now resulted in accumulation of the protein in the nucleus and subsequent labeling of the SPBs and weak spindle association. However, increasing the nuclear import of Esp1 in pds1-128 cells was not sufficient to achieve a strong spindle fluorescence, as seen in wild-type cells (Fig. 5 A, middle), suggesting that Pds1 association may also be required to obtain efficient Esp1 spindle interaction. This is consistent with the observation that there is no detectable spindle fluorescence in pds1Δ cells expressing Esp1GFPNLS (data not shown). Esp1 did label the SPBs in this mutant, demonstrating that Esp1 has the ability to bind the SPBs in the absence of Pds1 (Fig. 5 A, bottom). This is perhaps not surprising as mutants deleted for PDS1 are viable at low temperatures and unless the essential function of Esp1 is independent of its spindle localization, Esp1 must have intrinsic spindle binding activity sufficient for viability. This association, however, is clearly more efficient when Pds1 is present. Expressing the Esp1GFPNLS version in a pds1Δ mutant leads to partial suppression of the temperature-sensitive phenotype at 30°C, whereas the pds1Δ mutant expressing Esp1GFP is dead at this temperature (not shown). This further supports the notion that an important function of Pds1 is to target Esp1 to the nucleus and subsequently to the spindle.

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