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Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.

Topper LM, Bastians H, Ruderman JV, Gorbsky GJ - J. Cell Biol. (2001)

Bottom Line: Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure.Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores.These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

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Injection of Cdc34 protein into prophase cells causes prometaphase arrest, and injection into prometaphase cells causes a delay at metaphase. Phase–contrast images of injected Ptk1 cells. (A) Buffer was injected into the cytoplasm of this cell during prophase. The nuclear envelope broke down within 2 min of injection, and chromosomes aligned ∼20 min later. The cell spent ∼8 min at metaphase and is shown ∼2 min after anaphase onset. (B) Recombinant Cdc34CA (a catalytically inactive form of Cdc34 protein) was injected into the cytoplasm of this cell during prophase. Progression through mitosis occurred with timing similar to the cell in A. (C and D) Injection of wild-type Cdc34 into cells at prophase. Both cells were injected in the cytoplasm just before capture of the first images. Nuclear envelope breakdown occurred normally in each cell. Many of the chromosomes of cells shown in B and C remained monooriented and never aligned at the metaphase plate during the 1-h observation period. (E) The prometaphase cell was injected with Cdc34CA protein immediately before the 0-min image was acquired. Chromosomes aligned, and the cell spent ∼10 min at metaphase then initiated anaphase. (F) The prometaphase cell was injected immediately before the first image was taken. After ∼40 min at metaphase, anaphase began. The cell is shown ∼3 min after anaphase onset. Bar, 5 μm.
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fig1: Injection of Cdc34 protein into prophase cells causes prometaphase arrest, and injection into prometaphase cells causes a delay at metaphase. Phase–contrast images of injected Ptk1 cells. (A) Buffer was injected into the cytoplasm of this cell during prophase. The nuclear envelope broke down within 2 min of injection, and chromosomes aligned ∼20 min later. The cell spent ∼8 min at metaphase and is shown ∼2 min after anaphase onset. (B) Recombinant Cdc34CA (a catalytically inactive form of Cdc34 protein) was injected into the cytoplasm of this cell during prophase. Progression through mitosis occurred with timing similar to the cell in A. (C and D) Injection of wild-type Cdc34 into cells at prophase. Both cells were injected in the cytoplasm just before capture of the first images. Nuclear envelope breakdown occurred normally in each cell. Many of the chromosomes of cells shown in B and C remained monooriented and never aligned at the metaphase plate during the 1-h observation period. (E) The prometaphase cell was injected with Cdc34CA protein immediately before the 0-min image was acquired. Chromosomes aligned, and the cell spent ∼10 min at metaphase then initiated anaphase. (F) The prometaphase cell was injected immediately before the first image was taken. After ∼40 min at metaphase, anaphase began. The cell is shown ∼3 min after anaphase onset. Bar, 5 μm.

Mentions: Mitotic progression of injected cells is shown in Fig. 1 and Table I. Cells injected during prophase with buffer required on average ∼30 min to reach metaphase after chromosomes achieved bipolar attachment. Initiation of anaphase began ∼14 min after metaphase, and cytokinesis was completed ∼20 min later. Cells injected in the cytoplasm at prophase with the inactive Cdc34CA protein progressed through mitosis at rates similar to buffer-injected cells (Fig. 1 B). In contrast, cells injected in the cytoplasm during prophase with wild-type Cdc34 arrested at prometaphase (Fig. 1, C and D). The level of Cdc34 protein expression does not appear to vary with cell cycle (Goebl et al., 1988; Reymond et al., 2000). By quantifying immunoblots and comparing a known amount of recombinant Cdc34 with extracts prepared from a known number of cells, we estimate that an individual Ptk1 cell contains ∼0.2 picograms of Cdc34 protein. Thus, the minimum amount of injected Cdc34 necessary to observe effects on chromosome movement was approximately five times the endogenous level. In cells injected with Cdc34, the chromosomes attached to microtubules of the mitotic spindle and oriented toward the poles after nuclear envelope breakdown. In most cells observed after injection of Cdc34, some chromosomes showed typical prometaphase chromosome oscillations (Fig. 2) . However, other chromosomes showed little movement, and many never aligned at the metaphase plate during the observation period. A few cells that were injected with wild-type Cdc34 in the early stages of mitosis did successfully align their chromosomes at the metaphase plate and arrested at metaphase (Table I). 12 of the cells injected at prophase with Cdc34 (> 3 mg/mL) included in Table I were followed for 2–6 h beyond nuclear envelope breakdown. 11 remained arrested in a prometaphase-like state, and 1 arrested at metaphase. Injection during late prometaphase usually delayed the onset of anaphase (Table I and Fig. 1 F). Injection of Cdc34 protein after anaphase onset did not impair completion of mitosis or cytokinesis (Table I). The prometaphase arrest was also observed when LLC-Pk cells in prophase were injected with Cdc34 (unpublished data).


Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.

Topper LM, Bastians H, Ruderman JV, Gorbsky GJ - J. Cell Biol. (2001)

Injection of Cdc34 protein into prophase cells causes prometaphase arrest, and injection into prometaphase cells causes a delay at metaphase. Phase–contrast images of injected Ptk1 cells. (A) Buffer was injected into the cytoplasm of this cell during prophase. The nuclear envelope broke down within 2 min of injection, and chromosomes aligned ∼20 min later. The cell spent ∼8 min at metaphase and is shown ∼2 min after anaphase onset. (B) Recombinant Cdc34CA (a catalytically inactive form of Cdc34 protein) was injected into the cytoplasm of this cell during prophase. Progression through mitosis occurred with timing similar to the cell in A. (C and D) Injection of wild-type Cdc34 into cells at prophase. Both cells were injected in the cytoplasm just before capture of the first images. Nuclear envelope breakdown occurred normally in each cell. Many of the chromosomes of cells shown in B and C remained monooriented and never aligned at the metaphase plate during the 1-h observation period. (E) The prometaphase cell was injected with Cdc34CA protein immediately before the 0-min image was acquired. Chromosomes aligned, and the cell spent ∼10 min at metaphase then initiated anaphase. (F) The prometaphase cell was injected immediately before the first image was taken. After ∼40 min at metaphase, anaphase began. The cell is shown ∼3 min after anaphase onset. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196447&req=5

fig1: Injection of Cdc34 protein into prophase cells causes prometaphase arrest, and injection into prometaphase cells causes a delay at metaphase. Phase–contrast images of injected Ptk1 cells. (A) Buffer was injected into the cytoplasm of this cell during prophase. The nuclear envelope broke down within 2 min of injection, and chromosomes aligned ∼20 min later. The cell spent ∼8 min at metaphase and is shown ∼2 min after anaphase onset. (B) Recombinant Cdc34CA (a catalytically inactive form of Cdc34 protein) was injected into the cytoplasm of this cell during prophase. Progression through mitosis occurred with timing similar to the cell in A. (C and D) Injection of wild-type Cdc34 into cells at prophase. Both cells were injected in the cytoplasm just before capture of the first images. Nuclear envelope breakdown occurred normally in each cell. Many of the chromosomes of cells shown in B and C remained monooriented and never aligned at the metaphase plate during the 1-h observation period. (E) The prometaphase cell was injected with Cdc34CA protein immediately before the 0-min image was acquired. Chromosomes aligned, and the cell spent ∼10 min at metaphase then initiated anaphase. (F) The prometaphase cell was injected immediately before the first image was taken. After ∼40 min at metaphase, anaphase began. The cell is shown ∼3 min after anaphase onset. Bar, 5 μm.
Mentions: Mitotic progression of injected cells is shown in Fig. 1 and Table I. Cells injected during prophase with buffer required on average ∼30 min to reach metaphase after chromosomes achieved bipolar attachment. Initiation of anaphase began ∼14 min after metaphase, and cytokinesis was completed ∼20 min later. Cells injected in the cytoplasm at prophase with the inactive Cdc34CA protein progressed through mitosis at rates similar to buffer-injected cells (Fig. 1 B). In contrast, cells injected in the cytoplasm during prophase with wild-type Cdc34 arrested at prometaphase (Fig. 1, C and D). The level of Cdc34 protein expression does not appear to vary with cell cycle (Goebl et al., 1988; Reymond et al., 2000). By quantifying immunoblots and comparing a known amount of recombinant Cdc34 with extracts prepared from a known number of cells, we estimate that an individual Ptk1 cell contains ∼0.2 picograms of Cdc34 protein. Thus, the minimum amount of injected Cdc34 necessary to observe effects on chromosome movement was approximately five times the endogenous level. In cells injected with Cdc34, the chromosomes attached to microtubules of the mitotic spindle and oriented toward the poles after nuclear envelope breakdown. In most cells observed after injection of Cdc34, some chromosomes showed typical prometaphase chromosome oscillations (Fig. 2) . However, other chromosomes showed little movement, and many never aligned at the metaphase plate during the observation period. A few cells that were injected with wild-type Cdc34 in the early stages of mitosis did successfully align their chromosomes at the metaphase plate and arrested at metaphase (Table I). 12 of the cells injected at prophase with Cdc34 (> 3 mg/mL) included in Table I were followed for 2–6 h beyond nuclear envelope breakdown. 11 remained arrested in a prometaphase-like state, and 1 arrested at metaphase. Injection during late prometaphase usually delayed the onset of anaphase (Table I and Fig. 1 F). Injection of Cdc34 protein after anaphase onset did not impair completion of mitosis or cytokinesis (Table I). The prometaphase arrest was also observed when LLC-Pk cells in prophase were injected with Cdc34 (unpublished data).

Bottom Line: Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure.Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores.These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

Show MeSH
Related in: MedlinePlus