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Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Robbins JA, Cross FR - Mol. Biol. Cell (2010)

Bottom Line: Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation.Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1.Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

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Restoration of mitotic cyclin Clb2 promotes spindle pole body separation and restores isotropic growth in CDH1-m11 cells. (A) MET3pr-Clb2-kd cells, with either CDH1 or CDH1-m11, were synchronized in α-factor, released, and Clb2-kd induced 60 min after release; images were obtained 180 min after α-factor release. Bars, 5 μm. (B) Clb2 immunoblot for cells in A. Clb2 antibody detects both endogenous Clb2 and Clb2-kd. Pgk1 serves as a loading control. (C) Quantification of cells with separated SPBs from A. (D) Single-cell time-lapse microscopy of strains of the indicated genotypes (all exact gene replacements), with minutes after release from α-factor indicated.
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Figure 4: Restoration of mitotic cyclin Clb2 promotes spindle pole body separation and restores isotropic growth in CDH1-m11 cells. (A) MET3pr-Clb2-kd cells, with either CDH1 or CDH1-m11, were synchronized in α-factor, released, and Clb2-kd induced 60 min after release; images were obtained 180 min after α-factor release. Bars, 5 μm. (B) Clb2 immunoblot for cells in A. Clb2 antibody detects both endogenous Clb2 and Clb2-kd. Pgk1 serves as a loading control. (C) Quantification of cells with separated SPBs from A. (D) Single-cell time-lapse microscopy of strains of the indicated genotypes (all exact gene replacements), with minutes after release from α-factor indicated.

Mentions: The Cdh1-m11 arrest is associated with degradation of cell cycle regulators (mitotic cyclins, Cdc5) as well as spindle components (see above). Mitotic cyclins modulate numerous cell cycle processes. Some cell cycle defects in CDH1-m11 cells could be due specifically and solely to mitotic cyclin proteolysis. To test this, we placed Clb2-kd, an undegradable version of Clb2 lacking both KEN and destruction boxes and therefore immune to APC-mediated proteolysis (Wäsch and Cross, 2002) under the control of the MET3 promoter, and turned on expression by methionine deprivation in synchronized CDH1-m11 cells, after they were released from α-factor and allowed to bud. Clb2-kd had striking though variable effects on SPB and tubulin morphology. The majority of CDH1-m11 cells in which Clb2-kd was expressed had separated their SPBs as indicated by at least two SPC42-CFP foci, instead of the single focus predominantly observed in controls without Clb2-kd (Figure 4, A and C). This effect was detectable when Clb2-kd levels were similar to those attained with Clb2-kd expressed from the endogenous locus (this level was attained transiently at 30 min after induction; fully induced Clb2-kd levels from the MET3 promoter plateau at ∼ 3-fold the level of Clb2-kd under its endogenous promoter).


Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Robbins JA, Cross FR - Mol. Biol. Cell (2010)

Restoration of mitotic cyclin Clb2 promotes spindle pole body separation and restores isotropic growth in CDH1-m11 cells. (A) MET3pr-Clb2-kd cells, with either CDH1 or CDH1-m11, were synchronized in α-factor, released, and Clb2-kd induced 60 min after release; images were obtained 180 min after α-factor release. Bars, 5 μm. (B) Clb2 immunoblot for cells in A. Clb2 antibody detects both endogenous Clb2 and Clb2-kd. Pgk1 serves as a loading control. (C) Quantification of cells with separated SPBs from A. (D) Single-cell time-lapse microscopy of strains of the indicated genotypes (all exact gene replacements), with minutes after release from α-factor indicated.
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Related In: Results  -  Collection

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Figure 4: Restoration of mitotic cyclin Clb2 promotes spindle pole body separation and restores isotropic growth in CDH1-m11 cells. (A) MET3pr-Clb2-kd cells, with either CDH1 or CDH1-m11, were synchronized in α-factor, released, and Clb2-kd induced 60 min after release; images were obtained 180 min after α-factor release. Bars, 5 μm. (B) Clb2 immunoblot for cells in A. Clb2 antibody detects both endogenous Clb2 and Clb2-kd. Pgk1 serves as a loading control. (C) Quantification of cells with separated SPBs from A. (D) Single-cell time-lapse microscopy of strains of the indicated genotypes (all exact gene replacements), with minutes after release from α-factor indicated.
Mentions: The Cdh1-m11 arrest is associated with degradation of cell cycle regulators (mitotic cyclins, Cdc5) as well as spindle components (see above). Mitotic cyclins modulate numerous cell cycle processes. Some cell cycle defects in CDH1-m11 cells could be due specifically and solely to mitotic cyclin proteolysis. To test this, we placed Clb2-kd, an undegradable version of Clb2 lacking both KEN and destruction boxes and therefore immune to APC-mediated proteolysis (Wäsch and Cross, 2002) under the control of the MET3 promoter, and turned on expression by methionine deprivation in synchronized CDH1-m11 cells, after they were released from α-factor and allowed to bud. Clb2-kd had striking though variable effects on SPB and tubulin morphology. The majority of CDH1-m11 cells in which Clb2-kd was expressed had separated their SPBs as indicated by at least two SPC42-CFP foci, instead of the single focus predominantly observed in controls without Clb2-kd (Figure 4, A and C). This effect was detectable when Clb2-kd levels were similar to those attained with Clb2-kd expressed from the endogenous locus (this level was attained transiently at 30 min after induction; fully induced Clb2-kd levels from the MET3 promoter plateau at ∼ 3-fold the level of Clb2-kd under its endogenous promoter).

Bottom Line: Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation.Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1.Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

Show MeSH
Related in: MedlinePlus