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DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement.

Jang CY, Wong J, Coppinger JA, Seki A, Yates JR, Fang G - J. Cell Biol. (2008)

Bottom Line: DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase.Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles.Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.

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DDA3 directly interacts with Kif2a. (A) Myc-DDA3 was cotransfected with GFP-Kif2a or GFP into 293T cells. Lysates of transfected 293T cells were immunoprecipitated (IP) with anti-GFP antibodies followed by Western blotting. (B) HeLa/GFP–DDA3 cells were synchronized by a double-thymidine treatment, released, and harvested at 9 h after release (TT9). Alternatively, 1 μM Taxol was added to cells at 8 h after release from the double-thymidine arrest and then harvested at 12 h after release (Taxol sample). Cell lysates were immunoprecipitated with nonspecific IgG (lanes 4, 6, and 8) or with anti-GFP antibodies (lanes 5, 7, and 9) followed by Western blotting. AS, asynchronous cells. (C and D) HeLa/GFP–DDA3 (C, lanes 1–5) or HeLa (C, lane 6) cells were synchronized by a double-thymidine arrest, released, and harvested at 9 h after release. The levels of Kif2a and DDA3 in total cell lysates were determined by Western blotting (D). GFP–DDA3 was immunoprecipitated with anti-GFP antibody beads, incubated with or without the Tev protease, separated into supernatant (sup) and beads, and then assayed by Western blotting (C). (E) HeLa/GFP–DDA3 cells were synchronized by a thymidine arrest and release into fresh media. 1 μM taxol or 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at 12 h after release. GFP–DDA3 was immunoprecipitated with anti-GFP antibodies and associated Kif2a was analyzed by Western blotting. (F and G) Cells stably expressing DDA3-S-GFP were synchronized by a single thymidine arrest and harvested at 8 h after release to enrich G2 and M cells (G2/M). Alternatively, cells were harvested after incubation with 250 ng/ml nocodazole for 18 h (Noc). The DDA3 complex was tandem affinity purified from the DDA3-S-GFP cells (F, lanes 1 and 3) or from the control parental cells (lanes 2 and 4), separated by SDS–PAGE, and visualized by silver staining. Arrowhead points to the precision protease used during purification to cleave its recognition site engineered between S and GFP tags. Peptides of Kif2a identified in the DDA3 complex are listed in G with XCorr and DeltaCN scores indicated. (H) Recombinant GST-DDA3 and GST were incubated with 35S-Kif2a that had been synthesized in reticulocyte lysates in vitro. GST and GST-DDA3 were then purified by glutathione beads and associated Kif2a analyzed by SDS-PAGE. CB, Coomassie blue staining.
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fig7: DDA3 directly interacts with Kif2a. (A) Myc-DDA3 was cotransfected with GFP-Kif2a or GFP into 293T cells. Lysates of transfected 293T cells were immunoprecipitated (IP) with anti-GFP antibodies followed by Western blotting. (B) HeLa/GFP–DDA3 cells were synchronized by a double-thymidine treatment, released, and harvested at 9 h after release (TT9). Alternatively, 1 μM Taxol was added to cells at 8 h after release from the double-thymidine arrest and then harvested at 12 h after release (Taxol sample). Cell lysates were immunoprecipitated with nonspecific IgG (lanes 4, 6, and 8) or with anti-GFP antibodies (lanes 5, 7, and 9) followed by Western blotting. AS, asynchronous cells. (C and D) HeLa/GFP–DDA3 (C, lanes 1–5) or HeLa (C, lane 6) cells were synchronized by a double-thymidine arrest, released, and harvested at 9 h after release. The levels of Kif2a and DDA3 in total cell lysates were determined by Western blotting (D). GFP–DDA3 was immunoprecipitated with anti-GFP antibody beads, incubated with or without the Tev protease, separated into supernatant (sup) and beads, and then assayed by Western blotting (C). (E) HeLa/GFP–DDA3 cells were synchronized by a thymidine arrest and release into fresh media. 1 μM taxol or 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at 12 h after release. GFP–DDA3 was immunoprecipitated with anti-GFP antibodies and associated Kif2a was analyzed by Western blotting. (F and G) Cells stably expressing DDA3-S-GFP were synchronized by a single thymidine arrest and harvested at 8 h after release to enrich G2 and M cells (G2/M). Alternatively, cells were harvested after incubation with 250 ng/ml nocodazole for 18 h (Noc). The DDA3 complex was tandem affinity purified from the DDA3-S-GFP cells (F, lanes 1 and 3) or from the control parental cells (lanes 2 and 4), separated by SDS–PAGE, and visualized by silver staining. Arrowhead points to the precision protease used during purification to cleave its recognition site engineered between S and GFP tags. Peptides of Kif2a identified in the DDA3 complex are listed in G with XCorr and DeltaCN scores indicated. (H) Recombinant GST-DDA3 and GST were incubated with 35S-Kif2a that had been synthesized in reticulocyte lysates in vitro. GST and GST-DDA3 were then purified by glutathione beads and associated Kif2a analyzed by SDS-PAGE. CB, Coomassie blue staining.

Mentions: A functional connection between DDA3 and Kif2a was confirmed in GFP–DDA3 cells. The levels of DDA3 in GFP–DDA3 cells (endogenous DDA3 plus GFP–DDA3) were approximately doubled compared with the parental cells, whereas the levels of Kif2a were not altered (Fig. 1 C and see Fig. 7 D). Interestingly, the amounts of spindle and spindle pole–associated Kif2a in GFP–DDA3 cells were higher than those in GFP cells (Fig. 5, E and F). Thus, DDA3 recruits Kif2a to the mitotic spindle and spindle poles and controls spindle dynamics.


DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement.

Jang CY, Wong J, Coppinger JA, Seki A, Yates JR, Fang G - J. Cell Biol. (2008)

DDA3 directly interacts with Kif2a. (A) Myc-DDA3 was cotransfected with GFP-Kif2a or GFP into 293T cells. Lysates of transfected 293T cells were immunoprecipitated (IP) with anti-GFP antibodies followed by Western blotting. (B) HeLa/GFP–DDA3 cells were synchronized by a double-thymidine treatment, released, and harvested at 9 h after release (TT9). Alternatively, 1 μM Taxol was added to cells at 8 h after release from the double-thymidine arrest and then harvested at 12 h after release (Taxol sample). Cell lysates were immunoprecipitated with nonspecific IgG (lanes 4, 6, and 8) or with anti-GFP antibodies (lanes 5, 7, and 9) followed by Western blotting. AS, asynchronous cells. (C and D) HeLa/GFP–DDA3 (C, lanes 1–5) or HeLa (C, lane 6) cells were synchronized by a double-thymidine arrest, released, and harvested at 9 h after release. The levels of Kif2a and DDA3 in total cell lysates were determined by Western blotting (D). GFP–DDA3 was immunoprecipitated with anti-GFP antibody beads, incubated with or without the Tev protease, separated into supernatant (sup) and beads, and then assayed by Western blotting (C). (E) HeLa/GFP–DDA3 cells were synchronized by a thymidine arrest and release into fresh media. 1 μM taxol or 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at 12 h after release. GFP–DDA3 was immunoprecipitated with anti-GFP antibodies and associated Kif2a was analyzed by Western blotting. (F and G) Cells stably expressing DDA3-S-GFP were synchronized by a single thymidine arrest and harvested at 8 h after release to enrich G2 and M cells (G2/M). Alternatively, cells were harvested after incubation with 250 ng/ml nocodazole for 18 h (Noc). The DDA3 complex was tandem affinity purified from the DDA3-S-GFP cells (F, lanes 1 and 3) or from the control parental cells (lanes 2 and 4), separated by SDS–PAGE, and visualized by silver staining. Arrowhead points to the precision protease used during purification to cleave its recognition site engineered between S and GFP tags. Peptides of Kif2a identified in the DDA3 complex are listed in G with XCorr and DeltaCN scores indicated. (H) Recombinant GST-DDA3 and GST were incubated with 35S-Kif2a that had been synthesized in reticulocyte lysates in vitro. GST and GST-DDA3 were then purified by glutathione beads and associated Kif2a analyzed by SDS-PAGE. CB, Coomassie blue staining.
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fig7: DDA3 directly interacts with Kif2a. (A) Myc-DDA3 was cotransfected with GFP-Kif2a or GFP into 293T cells. Lysates of transfected 293T cells were immunoprecipitated (IP) with anti-GFP antibodies followed by Western blotting. (B) HeLa/GFP–DDA3 cells were synchronized by a double-thymidine treatment, released, and harvested at 9 h after release (TT9). Alternatively, 1 μM Taxol was added to cells at 8 h after release from the double-thymidine arrest and then harvested at 12 h after release (Taxol sample). Cell lysates were immunoprecipitated with nonspecific IgG (lanes 4, 6, and 8) or with anti-GFP antibodies (lanes 5, 7, and 9) followed by Western blotting. AS, asynchronous cells. (C and D) HeLa/GFP–DDA3 (C, lanes 1–5) or HeLa (C, lane 6) cells were synchronized by a double-thymidine arrest, released, and harvested at 9 h after release. The levels of Kif2a and DDA3 in total cell lysates were determined by Western blotting (D). GFP–DDA3 was immunoprecipitated with anti-GFP antibody beads, incubated with or without the Tev protease, separated into supernatant (sup) and beads, and then assayed by Western blotting (C). (E) HeLa/GFP–DDA3 cells were synchronized by a thymidine arrest and release into fresh media. 1 μM taxol or 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at 12 h after release. GFP–DDA3 was immunoprecipitated with anti-GFP antibodies and associated Kif2a was analyzed by Western blotting. (F and G) Cells stably expressing DDA3-S-GFP were synchronized by a single thymidine arrest and harvested at 8 h after release to enrich G2 and M cells (G2/M). Alternatively, cells were harvested after incubation with 250 ng/ml nocodazole for 18 h (Noc). The DDA3 complex was tandem affinity purified from the DDA3-S-GFP cells (F, lanes 1 and 3) or from the control parental cells (lanes 2 and 4), separated by SDS–PAGE, and visualized by silver staining. Arrowhead points to the precision protease used during purification to cleave its recognition site engineered between S and GFP tags. Peptides of Kif2a identified in the DDA3 complex are listed in G with XCorr and DeltaCN scores indicated. (H) Recombinant GST-DDA3 and GST were incubated with 35S-Kif2a that had been synthesized in reticulocyte lysates in vitro. GST and GST-DDA3 were then purified by glutathione beads and associated Kif2a analyzed by SDS-PAGE. CB, Coomassie blue staining.
Mentions: A functional connection between DDA3 and Kif2a was confirmed in GFP–DDA3 cells. The levels of DDA3 in GFP–DDA3 cells (endogenous DDA3 plus GFP–DDA3) were approximately doubled compared with the parental cells, whereas the levels of Kif2a were not altered (Fig. 1 C and see Fig. 7 D). Interestingly, the amounts of spindle and spindle pole–associated Kif2a in GFP–DDA3 cells were higher than those in GFP cells (Fig. 5, E and F). Thus, DDA3 recruits Kif2a to the mitotic spindle and spindle poles and controls spindle dynamics.

Bottom Line: DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase.Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles.Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.

Show MeSH
Related in: MedlinePlus