Limits...
Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint.

Famulski JK, Vos L, Sun X, Chan G - J. Cell Biol. (2008)

Bottom Line: In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s).However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein-hZW10 becomes a stable component of the kinetochore.Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD-ZW10-Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1-noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein-hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

Show MeSH

Related in: MedlinePlus

siRNA knockdown of hZW10 results in abrogation of the mitotic checkpoint. (a) Immunoblot of HeLa lysates from cells transfected with (+) or without (−) 50 nM anti-hZW10 siRNA for 72 h and probed with a rabbit hZW10 polyclonal antibody and a mouse monoclonal (B512) tubulin antibody using the Odyssey IR imaging system. A significant (∼90%) reduction in the hZW10 signal was observed. (b) HeLa cells transfected with (+) or without (−) anti-hZW10 siRNA duplexes for 72 h and stained with rabbit anti-hZW10 polyclonal antibodies, human ACA sera, and DAPI. hZW10 kinetochore signal was not detected in cells transfected with the anti-hZW10 siRNA after 72 h. Bar, 10 μm. (c) HeLa cells transfected with anti-hZW10 siRNA duplexes for 72 h and arrested with 25 μM vinblastine for 16 h were analyzed for the accumulation of mitotic cells. DNA was stained with DAPI. Representative images indicate an accumulation of mitotic cells after 16 h vinblastine arrest in the control cells but not in the hZW10 siRNA knockdown cells. Bar, 100 μm. A histogram of the percentage of mitotic cells is shown (n = 3 experiments; >300 cells per experiment; error bars show ± SD).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234252&req=5

fig4: siRNA knockdown of hZW10 results in abrogation of the mitotic checkpoint. (a) Immunoblot of HeLa lysates from cells transfected with (+) or without (−) 50 nM anti-hZW10 siRNA for 72 h and probed with a rabbit hZW10 polyclonal antibody and a mouse monoclonal (B512) tubulin antibody using the Odyssey IR imaging system. A significant (∼90%) reduction in the hZW10 signal was observed. (b) HeLa cells transfected with (+) or without (−) anti-hZW10 siRNA duplexes for 72 h and stained with rabbit anti-hZW10 polyclonal antibodies, human ACA sera, and DAPI. hZW10 kinetochore signal was not detected in cells transfected with the anti-hZW10 siRNA after 72 h. Bar, 10 μm. (c) HeLa cells transfected with anti-hZW10 siRNA duplexes for 72 h and arrested with 25 μM vinblastine for 16 h were analyzed for the accumulation of mitotic cells. DNA was stained with DAPI. Representative images indicate an accumulation of mitotic cells after 16 h vinblastine arrest in the control cells but not in the hZW10 siRNA knockdown cells. Bar, 100 μm. A histogram of the percentage of mitotic cells is shown (n = 3 experiments; >300 cells per experiment; error bars show ± SD).

Mentions: hZW10 has been shown to be required for mitotic checkpoint fidelity in fly, frog, and human cells (Williams et al., 1992; Basto et al., 2000; Chan et al., 2000; Scaerou et al., 2001; Kops et al., 2005). We therefore investigated whether the interaction between hZW10 and hZwint-1, which we have shown to regulate hZW10 kinetochore dynamics in prometaphase, is functionally required for mitotic checkpoint fidelity. To assess the fidelity of the mitotic checkpoint in the presence of an hZwint-1–noninteracting hZW10 mutant, endogenous hZW10 was depleted by siRNA while the cells were rescued with either a wild-type EGFP-hZW10 or EGFP-hZW10N1 siRNA-resistant construct. Fig. 4 A depicts the knockdown of endogenous hZW10 protein by immunoblot, whereas Fig. 4 B illustrates immunofluorescence staining for hZW10 siRNA knockdown. In either case, hZW10 is clearly depleted after 72 h of siRNA transfection. To assay mitotic checkpoint function in cells lacking hZW10, cells depleted of hZW10 using siRNA for 72 h were arrested with vinblastine for 16 h and analyzed using fluorescence microscopy. In control cells, the vinblastine-induced mitotic arrest resulted in a mitotic index of ∼45% (Fig. 4 C). However, in cells knocked down for hZW10 and subsequently arrested with vinblastine, the mitotic index dropped to ∼10%, thus indicating escape from mitotic checkpoint arrest (Fig. 4 C). To test whether EGFP-hZW10 or EGFP-hZW10N1 can rescue the hZW10 siRNA knockdown phenotype, we generated HeLa cell lines stably expressing EGFP-hZW10 or EGFP-hZW10N1 siRNA-resistant constructs. We subsequently depleted endogenous hZW10 in the siRNA-resistant cell lines for 72 h, arrested the cells with vinblastine for 16 h, and analyzed using fluorescence microscopy. Fig. 5 A shows the depletion of endogenous hZW10 protein and the expression of EGFP-hZW10 siRNA-resistant constructs. In control cells expressing either siRNA-resistant EGFP-hZW10 or EGFP-hZW10N1, the vinblastine-induced mitotic arrest resulted in a mitotic index of ∼42 and 43%, respectively. However, when we depleted endogenous hZW10, the EGFP-hZW10N1–expressing cells reached a mitotic index of only ∼16%, whereas the mitotic index of the EGFP-hZW10–expressing cells reached ∼45%, which was similar to the control HeLa cells (Figs. 4 and Figs.5). In conclusion, our results indicate that although wild-type EGFP-hZW10 can rescue the depletion of endogenous hZW10, EGFP-hZW10N1 is unable to support a sustained mitotic checkpoint arrest in response to vinblastine treatment. We therefore believe that the interaction between hZW10 and hZwint-1, which stabilizes hZW10 at prometaphase kinetochores, is required for mitotic checkpoint fidelity.


Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint.

Famulski JK, Vos L, Sun X, Chan G - J. Cell Biol. (2008)

siRNA knockdown of hZW10 results in abrogation of the mitotic checkpoint. (a) Immunoblot of HeLa lysates from cells transfected with (+) or without (−) 50 nM anti-hZW10 siRNA for 72 h and probed with a rabbit hZW10 polyclonal antibody and a mouse monoclonal (B512) tubulin antibody using the Odyssey IR imaging system. A significant (∼90%) reduction in the hZW10 signal was observed. (b) HeLa cells transfected with (+) or without (−) anti-hZW10 siRNA duplexes for 72 h and stained with rabbit anti-hZW10 polyclonal antibodies, human ACA sera, and DAPI. hZW10 kinetochore signal was not detected in cells transfected with the anti-hZW10 siRNA after 72 h. Bar, 10 μm. (c) HeLa cells transfected with anti-hZW10 siRNA duplexes for 72 h and arrested with 25 μM vinblastine for 16 h were analyzed for the accumulation of mitotic cells. DNA was stained with DAPI. Representative images indicate an accumulation of mitotic cells after 16 h vinblastine arrest in the control cells but not in the hZW10 siRNA knockdown cells. Bar, 100 μm. A histogram of the percentage of mitotic cells is shown (n = 3 experiments; >300 cells per experiment; error bars show ± SD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234252&req=5

fig4: siRNA knockdown of hZW10 results in abrogation of the mitotic checkpoint. (a) Immunoblot of HeLa lysates from cells transfected with (+) or without (−) 50 nM anti-hZW10 siRNA for 72 h and probed with a rabbit hZW10 polyclonal antibody and a mouse monoclonal (B512) tubulin antibody using the Odyssey IR imaging system. A significant (∼90%) reduction in the hZW10 signal was observed. (b) HeLa cells transfected with (+) or without (−) anti-hZW10 siRNA duplexes for 72 h and stained with rabbit anti-hZW10 polyclonal antibodies, human ACA sera, and DAPI. hZW10 kinetochore signal was not detected in cells transfected with the anti-hZW10 siRNA after 72 h. Bar, 10 μm. (c) HeLa cells transfected with anti-hZW10 siRNA duplexes for 72 h and arrested with 25 μM vinblastine for 16 h were analyzed for the accumulation of mitotic cells. DNA was stained with DAPI. Representative images indicate an accumulation of mitotic cells after 16 h vinblastine arrest in the control cells but not in the hZW10 siRNA knockdown cells. Bar, 100 μm. A histogram of the percentage of mitotic cells is shown (n = 3 experiments; >300 cells per experiment; error bars show ± SD).
Mentions: hZW10 has been shown to be required for mitotic checkpoint fidelity in fly, frog, and human cells (Williams et al., 1992; Basto et al., 2000; Chan et al., 2000; Scaerou et al., 2001; Kops et al., 2005). We therefore investigated whether the interaction between hZW10 and hZwint-1, which we have shown to regulate hZW10 kinetochore dynamics in prometaphase, is functionally required for mitotic checkpoint fidelity. To assess the fidelity of the mitotic checkpoint in the presence of an hZwint-1–noninteracting hZW10 mutant, endogenous hZW10 was depleted by siRNA while the cells were rescued with either a wild-type EGFP-hZW10 or EGFP-hZW10N1 siRNA-resistant construct. Fig. 4 A depicts the knockdown of endogenous hZW10 protein by immunoblot, whereas Fig. 4 B illustrates immunofluorescence staining for hZW10 siRNA knockdown. In either case, hZW10 is clearly depleted after 72 h of siRNA transfection. To assay mitotic checkpoint function in cells lacking hZW10, cells depleted of hZW10 using siRNA for 72 h were arrested with vinblastine for 16 h and analyzed using fluorescence microscopy. In control cells, the vinblastine-induced mitotic arrest resulted in a mitotic index of ∼45% (Fig. 4 C). However, in cells knocked down for hZW10 and subsequently arrested with vinblastine, the mitotic index dropped to ∼10%, thus indicating escape from mitotic checkpoint arrest (Fig. 4 C). To test whether EGFP-hZW10 or EGFP-hZW10N1 can rescue the hZW10 siRNA knockdown phenotype, we generated HeLa cell lines stably expressing EGFP-hZW10 or EGFP-hZW10N1 siRNA-resistant constructs. We subsequently depleted endogenous hZW10 in the siRNA-resistant cell lines for 72 h, arrested the cells with vinblastine for 16 h, and analyzed using fluorescence microscopy. Fig. 5 A shows the depletion of endogenous hZW10 protein and the expression of EGFP-hZW10 siRNA-resistant constructs. In control cells expressing either siRNA-resistant EGFP-hZW10 or EGFP-hZW10N1, the vinblastine-induced mitotic arrest resulted in a mitotic index of ∼42 and 43%, respectively. However, when we depleted endogenous hZW10, the EGFP-hZW10N1–expressing cells reached a mitotic index of only ∼16%, whereas the mitotic index of the EGFP-hZW10–expressing cells reached ∼45%, which was similar to the control HeLa cells (Figs. 4 and Figs.5). In conclusion, our results indicate that although wild-type EGFP-hZW10 can rescue the depletion of endogenous hZW10, EGFP-hZW10N1 is unable to support a sustained mitotic checkpoint arrest in response to vinblastine treatment. We therefore believe that the interaction between hZW10 and hZwint-1, which stabilizes hZW10 at prometaphase kinetochores, is required for mitotic checkpoint fidelity.

Bottom Line: In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s).However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein-hZW10 becomes a stable component of the kinetochore.Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD-ZW10-Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1-noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein-hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

Show MeSH
Related in: MedlinePlus