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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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Bipolar spindles and normal kinetochores form in cells injected with Cdc34. (A) Cells were injected with Cdc34 protein, observed to undergo nuclear envelope breakdown, and were further observed for ∼45 min before fixation. Both fixed cells were labeled with antitubulin (red) and anticentromere (green) antibodies. (B) Ultrastructure of kinetochores in injected cells. The chromosome distribution of an uninjected prometaphase cell is shown (a) with an enlargement of a kinetochore from that cell (b). The inner and outer plates of the trilaminar structure are denoted (b, arrows). The chromosome distribution of a cell injected with Cdc34 (c) appears similar to the pattern in the uninjected cell. However, as often seen in Cdc34-injected cells a chromosome is situated very close to the spindle pole (arrowhead). Kinetochores of the injected cell (d) show a clear trilaminar structure with microtubules embedded in the outer plate. Bars: (A and B, a) 10 μm; (B, b) 2 μm.
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fig3: Bipolar spindles and normal kinetochores form in cells injected with Cdc34. (A) Cells were injected with Cdc34 protein, observed to undergo nuclear envelope breakdown, and were further observed for ∼45 min before fixation. Both fixed cells were labeled with antitubulin (red) and anticentromere (green) antibodies. (B) Ultrastructure of kinetochores in injected cells. The chromosome distribution of an uninjected prometaphase cell is shown (a) with an enlargement of a kinetochore from that cell (b). The inner and outer plates of the trilaminar structure are denoted (b, arrows). The chromosome distribution of a cell injected with Cdc34 (c) appears similar to the pattern in the uninjected cell. However, as often seen in Cdc34-injected cells a chromosome is situated very close to the spindle pole (arrowhead). Kinetochores of the injected cell (d) show a clear trilaminar structure with microtubules embedded in the outer plate. Bars: (A and B, a) 10 μm; (B, b) 2 μm.

Mentions: Spindles that formed in the Cdc34-injected cells were bipolar and contained astral, kinetochore, and nonkinetochore spindle microtubules (Fig. 3 A). This morphology resembled that of uninjected cells fixed in prometaphase. Ultrastructurally, kinetochores in cells injected with Cdc34 appeared identical to kinetochores of uninjected cells (Fig. 3 B). In injected cells, some chromosomes were found situated unusually close to the centriole at the spindle pole (Fig. 3 B, c, arrowhead). However, sister kinetochores (Fig. 3 B, c, white arrows) of chromosomes situated between the spindle poles (Fig. 3 B, c, black arrows) were widely separated, indicating the kinetochore area was under bipolar tension. We quantified the distances between sister kinetochores of chromosomes at the midplane and near the spindle poles in Cdc34-injected and uninjected cells (Table II). For both bioriented or monooriented chromosomes, we found no significant differences in the interkinetochore distances, comparing injected and uninjected cells. Nocodazole treatment reduced the interkinetochore distances of both injected and noninjected cells similarly (Table II.). These results suggest that the excess Cdc34 protein did not interfere with kinetochore attachment to microtubules or spindle assembly or cause structural alterations in the centromeric chromatin between sister kinetochores. Cells injected with Cdc34 protein were stably arrested in M phase as determined by several markers of M phase arrest including expression of phosphorylated histone H3 (Hendzel et al., 1997) and dispersed distribution of nuclear pore antigens (Snow et al., 1987) (Fig. S1 available at http://www.jcb.org/content/vol154/issue4/707).


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)

Bipolar spindles and normal kinetochores form in cells injected with Cdc34. (A) Cells were injected with Cdc34 protein, observed to undergo nuclear envelope breakdown, and were further observed for ∼45 min before fixation. Both fixed cells were labeled with antitubulin (red) and anticentromere (green) antibodies. (B) Ultrastructure of kinetochores in injected cells. The chromosome distribution of an uninjected prometaphase cell is shown (a) with an enlargement of a kinetochore from that cell (b). The inner and outer plates of the trilaminar structure are denoted (b, arrows). The chromosome distribution of a cell injected with Cdc34 (c) appears similar to the pattern in the uninjected cell. However, as often seen in Cdc34-injected cells a chromosome is situated very close to the spindle pole (arrowhead). Kinetochores of the injected cell (d) show a clear trilaminar structure with microtubules embedded in the outer plate. Bars: (A and B, a) 10 μm; (B, b) 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196447&req=5

fig3: Bipolar spindles and normal kinetochores form in cells injected with Cdc34. (A) Cells were injected with Cdc34 protein, observed to undergo nuclear envelope breakdown, and were further observed for ∼45 min before fixation. Both fixed cells were labeled with antitubulin (red) and anticentromere (green) antibodies. (B) Ultrastructure of kinetochores in injected cells. The chromosome distribution of an uninjected prometaphase cell is shown (a) with an enlargement of a kinetochore from that cell (b). The inner and outer plates of the trilaminar structure are denoted (b, arrows). The chromosome distribution of a cell injected with Cdc34 (c) appears similar to the pattern in the uninjected cell. However, as often seen in Cdc34-injected cells a chromosome is situated very close to the spindle pole (arrowhead). Kinetochores of the injected cell (d) show a clear trilaminar structure with microtubules embedded in the outer plate. Bars: (A and B, a) 10 μm; (B, b) 2 μm.
Mentions: Spindles that formed in the Cdc34-injected cells were bipolar and contained astral, kinetochore, and nonkinetochore spindle microtubules (Fig. 3 A). This morphology resembled that of uninjected cells fixed in prometaphase. Ultrastructurally, kinetochores in cells injected with Cdc34 appeared identical to kinetochores of uninjected cells (Fig. 3 B). In injected cells, some chromosomes were found situated unusually close to the centriole at the spindle pole (Fig. 3 B, c, arrowhead). However, sister kinetochores (Fig. 3 B, c, white arrows) of chromosomes situated between the spindle poles (Fig. 3 B, c, black arrows) were widely separated, indicating the kinetochore area was under bipolar tension. We quantified the distances between sister kinetochores of chromosomes at the midplane and near the spindle poles in Cdc34-injected and uninjected cells (Table II). For both bioriented or monooriented chromosomes, we found no significant differences in the interkinetochore distances, comparing injected and uninjected cells. Nocodazole treatment reduced the interkinetochore distances of both injected and noninjected cells similarly (Table II.). These results suggest that the excess Cdc34 protein did not interfere with kinetochore attachment to microtubules or spindle assembly or cause structural alterations in the centromeric chromatin between sister kinetochores. Cells injected with Cdc34 protein were stably arrested in M phase as determined by several markers of M phase arrest including expression of phosphorylated histone H3 (Hendzel et al., 1997) and dispersed distribution of nuclear pore antigens (Snow et al., 1987) (Fig. S1 available at http://www.jcb.org/content/vol154/issue4/707).

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