A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1 - PubMed (original) (raw)

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

S Jensen et al. J Cell Biol. 2001.

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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Figures

Figure 3

Figure 3

Esp1 is essential for spindle elongation. Cells of wild-type (A), _scc1_Δ (SY118) (B), esp1-B3 (SY119) (C), and scc1_Δ_esp1-B3 (SY120) (D) strains containing CFP-tubulin and GFP-labeled centromeres were synchronized in G1 in YEPDextrose by addition of α-factor, and subsequently released into YEPDextrose at 31.5°C. Aliquots were removed at 15-min intervals for scoring budding index, spindle morphology, and loss of cohesion for chromosome IV. Short spindles (<2.5 μm) and elongated spindles (>4 μm) were scored by measurement of digital images. More than 200 cells were counted for each time point.

Figure 1

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.

Figure 2

Figure 2

Localization of Esp1GFP at different stages of the cell cycle. (A) Diploid cells carrying an integrated ESP1GFP allele (SY203) grown in YEPRaffinose were collected on a nitrocellulose filter and induced 15 min on a YEPGalactose plate. Cells were examined by microscopy and images acquired of GFP signal and DIC. (a and b) Nuclear Esp1GFP signal in late G2 cells; (c) metaphase spindle and SPB stain of Esp1; (d–h) anaphase spindles stained by Esp1; (i) anaphase spindle stain in cells expressing Esp1GFP from the native promoter (SY201). Scale bar: 10 μm. (B) Images of cells expressing Esp1GFP and either Spc29CFP (SY205, top) or CFPTub1 (SY206, bottom) captured and merged as described in Materials and Methods. (C) The length distribution of spindles labeled by Esp1GFP. Spindles measured on cells treated as described in A.

Figure 4

Figure 4

Proper Esp1 localization depends on Pds1. (A, a and b) Esp1 signal at the spindle midzone is absent in _ase1_Δ mutant. A diploid _ase1_Δ strain carrying _GAL1_-inducible _ESP1_GFP (SY204) was examined by microscopy after induction, as described in Fig. 2. (B, a and b) Localization of Esp1 in _pds1_Δ mutant. Haploid _pds1_Δ cells expressing Esp1GFP from the GAL1 promoter (SY102) were treated as in A. (C, a and b) Movement of Esp1 to nucleus and spindle is reduced in a pds1-128 mutant. Haploid pds1-128 cells with _ESP1_GFP integrated (SY103) were analyzed as above. (D) Comparison of Esp1/Pds1 complex formation in exponentially growing wild-type cells and pds1-128 cells. Strains carrying endogenous myc18-tagged Esp1 and HA3-tagged Pds1 (wt) (SY109) or Pds1-128 (ts) (SY110) were grown at 25°C. Extracts were immunoprecipitated with anti–myc antibody. Samples were analyzed by SDS-PAGE followed by immunostaining with anti–myc antibody and anti–HA antibody. An isogenic control strain expressing endogenous Pds1HA3 protein was included to show specificity of the anti–myc antibody (SY111). Note that the additional amino acids at the COOH terminus of the Pds1-128 protein causes it to migrate at a slightly lower mobility.

Figure 6

Figure 6

Pds1 associates with the mitotic spindle apparatus. (A) A strain carrying PDS1 fused at the COOH-terminal end to GFP integrated at the chromosomal locus (SY116) was arrested in G1 with α-factor. After release into YEPDextrose at 30°C, aliquots were taken for scoring budding index, cell-cycle progression by DAPI, and real-time localization of Pds1GFP. (□) Cells with pre-anaphase nuclear morphology; (▪) cells with anaphase and telophase nuclear morphology; (▴) budding index; (♦) nuclear localization of Pds1; (○) spindle localization of Pds1. (B, a and b) Live cell localization of Pds1GFP. Images of a diploid strain with one endogenous copy of Pds1 fused to GFP (SY202). Scale bar: 10 μm. (C) A strain expressing Pds1GFP and Spc29CFP (SY207) was examined by microscopy using GFP and CFP filter sets. Merged images show colocalization of Pds1 with Spc29 in anaphase (top). Simultaneous staining of Pds1GFP and CFPTub1 is shown at bottom (SY208). (D) The length distribution of spindles visualized by Pds1GFP. Spindles measured on cells treated as described in B.

Figure 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 _pds1_Δ 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 _ESP1_GFP and _PDS1_Δdb 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 _ESP1_GFP allele (SY104) was analyzed in a similar fashion (a).

Figure 7

Figure 7

Complex formation between Esp1 and Pds1. (A) A strain expressing endogenous myc18-tagged Esp1 and HA3-tagged Pds1 (SY109) was arrested in G1 by α-factor. After release into YEPDextrose at 22°C, samples were removed at the indicated time points. Extract was analyzed by SDS-PAGE and immunostaining with anti–myc antibody (top), anti–HA antibody (middle), and anti–PSTAIRE antibody, the latter to visualize Cdc28 protein, which serves as a loading control. (B) Extracts prepared from samples in A were subjected to immunoprecipitation using anti–HA antibody to pull down Pds1 protein. SDS-PAGE and immunostaining was carried out with anti–myc antibody to reveal the presence of Esp1 in the precipitate (top) and anti–HA antibody to show amount of precipitated Pds1 (bottom). (C) Esp1/Pds1 complex is not formed in vitro. Extract from a strain expressing Esp1myc18 protein was mixed with extract containing Pds1HA3 protein. After immunoprecipitation with anti–HA antibody and SDS-PAGE, samples were analyzed by immunostaining using anti–myc antibody (top) and anti–HA antibody (bottom).

Figure 8

Figure 8

The role of the putative calcium-binding site in the conserved COOH-terminal domain of Esp1. (A) A schematic representation of the domain structure of Esp1. The NH2-terminal domain constituting 1/3 of the protein is sufficient to interact with Pds1. The conserved COOH-terminal region contains a putative calcium binding site, which is conserved in the S. pombe homologue Cut1. (B) Complementation ability of various Esp1 mutant proteins in an esp1ts strain. Proteins were either expressed from an integrated allele driven by the GAL1 promoter (GAL1) or expressed from their own promoter on a low copy plasmid (ARS/CEN). The Esp1 mutants were tested for complementation in esp1ts strain (SY122) with or without the GFP tag with similar results. (C) Mutation in the COOH-terminal region of Esp1 does not affect the ability to interact with Pds1. A strain expressing HA3-tagged Pds1 (SY111) was transformed with integrating plasmids encoding the different Esp1GFP mutant proteins inducible from the GAL1 promoter. The resulting strains were grown in YEPRaffinose, induced for 80 min with 2% galactose, and extract prepared for immunoprecipitation with anti–HA antibody to bring down Pds1. After SDS-PAGE immunostaining was performed with anti–GFP antibody to visualize Esp1GFP in the precipitate (top) and anti–HA antibody to confirm Pds1 precipitation. (Right) The presence of Esp1GFP, Pds1HA3, and Cdc28 protein in the extract from the different strains is shown. An isogenic strain expressing only Esp1GFP protein (SY104) was included as a control. (D) Localization of mutant Esp1GFP protein in live cells. Strains expressing either _GAL1_-inducible Esp1(1-1568)GFP (SY113), Esp1(D1568A)GFP (SY114), or Esp1(D1568A/D1570A)GFP (SY115) were examined by microscopy after a 20-min induction on solid YEPGalactose media. (a) Esp1(1-1568)GFP, (b) Esp1(D1568A)GFP, and (c) Esp1(D1568A/D1570A)-GFP. (Top) GFP signals; (bottom) DIC. Scale bar: 10 μm. (E) Calcium added to the growth medium can suppress the temperature-sensitive phenotype of esp1ts mutant. The esp1-N5 mutant (SY117) and an isogenic wild-type strain grown at room temperature was replica plated onto the following plates: (a) YEPDextrose + 50 mM CaCl2, (b) YEPDextrose, (c) YEPDextrose + 50 mM EGTA/CaCl2, (d) YEPDextrose + 50 mM MgCl2. All plates were incubated for 1 d at 2°C above the restrictive temperature of the esp1-N5 mutation (37°C). The ability of calcium to suppress the lethality of an _esp1ts_mutation was confirmed in several different esp1ts alleles.

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