Limits...
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.

Show MeSH

Related in: MedlinePlus

Chromosome oscillation during Cdc34 induced prometaphase arrest. (A) Ptk1 cell injected with Cdc34 protein at prophase. The spindle poles were widely separated at the time of nuclear envelope breakdown, and all of the chromosomes assumed an initial monopolar orientation. A curved arrow indicates the lower pole. The straight white line is drawn an equal distance from the pole in all panels to serve as a position reference. The chromosome indicated by the white arrow begins at the white line and then moves repeatedly away from and toward the lower pole during the observation period. In contrast, the chromosomes indicated by the white arrowhead in the first and last images undergo very little motion relative to the spindle pole. Time is indicated in minutes and seconds. A Quicktime video of the time-lapse phase image referring to Fig. 2 (Video 1) and additional examples of chromosome behavior in cells injected with Cdc34 protein (videos 2 and 3) are available at http://www.jcb.org/content/vol154/issue4/707. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196447&req=5

fig2: Chromosome oscillation during Cdc34 induced prometaphase arrest. (A) Ptk1 cell injected with Cdc34 protein at prophase. The spindle poles were widely separated at the time of nuclear envelope breakdown, and all of the chromosomes assumed an initial monopolar orientation. A curved arrow indicates the lower pole. The straight white line is drawn an equal distance from the pole in all panels to serve as a position reference. The chromosome indicated by the white arrow begins at the white line and then moves repeatedly away from and toward the lower pole during the observation period. In contrast, the chromosomes indicated by the white arrowhead in the first and last images undergo very little motion relative to the spindle pole. Time is indicated in minutes and seconds. A Quicktime video of the time-lapse phase image referring to Fig. 2 (Video 1) and additional examples of chromosome behavior in cells injected with Cdc34 protein (videos 2 and 3) are available at http://www.jcb.org/content/vol154/issue4/707. Bar, 10 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)

Chromosome oscillation during Cdc34 induced prometaphase arrest. (A) Ptk1 cell injected with Cdc34 protein at prophase. The spindle poles were widely separated at the time of nuclear envelope breakdown, and all of the chromosomes assumed an initial monopolar orientation. A curved arrow indicates the lower pole. The straight white line is drawn an equal distance from the pole in all panels to serve as a position reference. The chromosome indicated by the white arrow begins at the white line and then moves repeatedly away from and toward the lower pole during the observation period. In contrast, the chromosomes indicated by the white arrowhead in the first and last images undergo very little motion relative to the spindle pole. Time is indicated in minutes and seconds. A Quicktime video of the time-lapse phase image referring to Fig. 2 (Video 1) and additional examples of chromosome behavior in cells injected with Cdc34 protein (videos 2 and 3) are available at http://www.jcb.org/content/vol154/issue4/707. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196447&req=5

fig2: Chromosome oscillation during Cdc34 induced prometaphase arrest. (A) Ptk1 cell injected with Cdc34 protein at prophase. The spindle poles were widely separated at the time of nuclear envelope breakdown, and all of the chromosomes assumed an initial monopolar orientation. A curved arrow indicates the lower pole. The straight white line is drawn an equal distance from the pole in all panels to serve as a position reference. The chromosome indicated by the white arrow begins at the white line and then moves repeatedly away from and toward the lower pole during the observation period. In contrast, the chromosomes indicated by the white arrowhead in the first and last images undergo very little motion relative to the spindle pole. Time is indicated in minutes and seconds. A Quicktime video of the time-lapse phase image referring to Fig. 2 (Video 1) and additional examples of chromosome behavior in cells injected with Cdc34 protein (videos 2 and 3) are available at http://www.jcb.org/content/vol154/issue4/707. Bar, 10 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