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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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CENP-E is mislocalized in Cdc34-injected cells. (A) The association of CENP-E (red) with kinetochores of an uninjected cell. The kinetochores (green, closed arrows) appear yellow as a result of colocalization of anti-CENP-E and antikinetochore antibodies. (B) Injection of Cdc34CA did not affect CENP-E localization to sister kinetochores (B, closed arrows). (C and D) Cells were injected in prophase with Cdc34 protein. Both were incubated for ∼45 min after nuclear envelope breakdown was detected. Immunolabeling reveals that CENP-E is localized to distinct cytoplasmic aggregates (open arrows) but not on kinetochores (closed arrows). In some instances, yellow kinetochores in the merged image result from the aggregates of CENP-E being in close proximity to kinetochores rather than a true colocalization of anti–CENP-E and antikinetochore antibodies. (E and F) Insets show enlargements of the boxed regions of each cell and illustrate that CENP-E (red) is found on kinetochores (green) of an uninjected prometaphase LLC-Pk cell (E). CENP-E does not concentrate at kinetochores in the Cdc34-injected LLC-Pk cell (F). Each image of a cell was taken at the same focal plane. Bars, 10 μm.
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fig4: CENP-E is mislocalized in Cdc34-injected cells. (A) The association of CENP-E (red) with kinetochores of an uninjected cell. The kinetochores (green, closed arrows) appear yellow as a result of colocalization of anti-CENP-E and antikinetochore antibodies. (B) Injection of Cdc34CA did not affect CENP-E localization to sister kinetochores (B, closed arrows). (C and D) Cells were injected in prophase with Cdc34 protein. Both were incubated for ∼45 min after nuclear envelope breakdown was detected. Immunolabeling reveals that CENP-E is localized to distinct cytoplasmic aggregates (open arrows) but not on kinetochores (closed arrows). In some instances, yellow kinetochores in the merged image result from the aggregates of CENP-E being in close proximity to kinetochores rather than a true colocalization of anti–CENP-E and antikinetochore antibodies. (E and F) Insets show enlargements of the boxed regions of each cell and illustrate that CENP-E (red) is found on kinetochores (green) of an uninjected prometaphase LLC-Pk cell (E). CENP-E does not concentrate at kinetochores in the Cdc34-injected LLC-Pk cell (F). Each image of a cell was taken at the same focal plane. Bars, 10 μm.

Mentions: Inhibition of CENP-E function in mammalian cells by antibody microinjection or introduction of antisense constructs prevents congression of chromosomes to the metaphase plate (Schaar et al., 1997; Yao et al., 2000). Similar results are obtained by immunodepletion of CENP-E from Xenopus egg extracts (Wood et al., 1997). Because the chromosomes in cells injected with wild-type Cdc34 protein also fail to achieve metaphase alignment, we assessed the distribution of CENP-E in injected cells. In Fig. 4, C and D , injection of Cdc34 into Ptk1 cells inhibited CENP-E localization at kinetochores and caused CENP-E protein (red) to accumulate in irregular aggregates in the cytoplasm that did not colocalize with the kinetochores (green). Injection of the Cdc34CA enzymatically inactive mutant did not affect localization of CENP-E to kinetochores (Fig. 4 B). Mislocalization of CENP-E was not a property unique to Ptk1 cells, since injected LLC-Pk cells show similar displacement of CENP-E from the kinetochores into cytoplasmic aggregates (Fig. 4 F). Displacement of CENP-E by Cdc34 was specific for CENP-E. Mitotic centromere-associated kinesin (MCAK), another member of the kinesin superfamily (Wordeman and Mitchison, 1995; Walczak et al., 1996; Maney et al., 1998), and dynein, a minus end–directed motor protein that also localizes to kinetochores (Pfarr et al., 1990; Steuer et al., 1990) were not altered by microinjection of Cdc34 (Fig. 5, B and D) .


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)

CENP-E is mislocalized in Cdc34-injected cells. (A) The association of CENP-E (red) with kinetochores of an uninjected cell. The kinetochores (green, closed arrows) appear yellow as a result of colocalization of anti-CENP-E and antikinetochore antibodies. (B) Injection of Cdc34CA did not affect CENP-E localization to sister kinetochores (B, closed arrows). (C and D) Cells were injected in prophase with Cdc34 protein. Both were incubated for ∼45 min after nuclear envelope breakdown was detected. Immunolabeling reveals that CENP-E is localized to distinct cytoplasmic aggregates (open arrows) but not on kinetochores (closed arrows). In some instances, yellow kinetochores in the merged image result from the aggregates of CENP-E being in close proximity to kinetochores rather than a true colocalization of anti–CENP-E and antikinetochore antibodies. (E and F) Insets show enlargements of the boxed regions of each cell and illustrate that CENP-E (red) is found on kinetochores (green) of an uninjected prometaphase LLC-Pk cell (E). CENP-E does not concentrate at kinetochores in the Cdc34-injected LLC-Pk cell (F). Each image of a cell was taken at the same focal plane. Bars, 10 μm.
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Related In: Results  -  Collection

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fig4: CENP-E is mislocalized in Cdc34-injected cells. (A) The association of CENP-E (red) with kinetochores of an uninjected cell. The kinetochores (green, closed arrows) appear yellow as a result of colocalization of anti-CENP-E and antikinetochore antibodies. (B) Injection of Cdc34CA did not affect CENP-E localization to sister kinetochores (B, closed arrows). (C and D) Cells were injected in prophase with Cdc34 protein. Both were incubated for ∼45 min after nuclear envelope breakdown was detected. Immunolabeling reveals that CENP-E is localized to distinct cytoplasmic aggregates (open arrows) but not on kinetochores (closed arrows). In some instances, yellow kinetochores in the merged image result from the aggregates of CENP-E being in close proximity to kinetochores rather than a true colocalization of anti–CENP-E and antikinetochore antibodies. (E and F) Insets show enlargements of the boxed regions of each cell and illustrate that CENP-E (red) is found on kinetochores (green) of an uninjected prometaphase LLC-Pk cell (E). CENP-E does not concentrate at kinetochores in the Cdc34-injected LLC-Pk cell (F). Each image of a cell was taken at the same focal plane. Bars, 10 μm.
Mentions: Inhibition of CENP-E function in mammalian cells by antibody microinjection or introduction of antisense constructs prevents congression of chromosomes to the metaphase plate (Schaar et al., 1997; Yao et al., 2000). Similar results are obtained by immunodepletion of CENP-E from Xenopus egg extracts (Wood et al., 1997). Because the chromosomes in cells injected with wild-type Cdc34 protein also fail to achieve metaphase alignment, we assessed the distribution of CENP-E in injected cells. In Fig. 4, C and D , injection of Cdc34 into Ptk1 cells inhibited CENP-E localization at kinetochores and caused CENP-E protein (red) to accumulate in irregular aggregates in the cytoplasm that did not colocalize with the kinetochores (green). Injection of the Cdc34CA enzymatically inactive mutant did not affect localization of CENP-E to kinetochores (Fig. 4 B). Mislocalization of CENP-E was not a property unique to Ptk1 cells, since injected LLC-Pk cells show similar displacement of CENP-E from the kinetochores into cytoplasmic aggregates (Fig. 4 F). Displacement of CENP-E by Cdc34 was specific for CENP-E. Mitotic centromere-associated kinesin (MCAK), another member of the kinesin superfamily (Wordeman and Mitchison, 1995; Walczak et al., 1996; Maney et al., 1998), and dynein, a minus end–directed motor protein that also localizes to kinetochores (Pfarr et al., 1990; Steuer et al., 1990) were not altered by microinjection of Cdc34 (Fig. 5, B and D) .

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