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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 is a spindle-associated cell cycle protein. (A–C) Shown are maximum projections from deconvolved z stacks of representative cells stained for DDA3 (A and C, green)/GFP (B, green), β-tubulin (red), and DNA (blue). A, HeLa cells; B, HeLa/GFP–DDA3 cells; C, HeLa cells transfected with siControl or siDDA3-A. Bars, 5 μm. (D) HeLa cells were synchronized by a double-thymidine treatment and released into fresh media. 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at the indicated times. Cells were analyzed by FACS and Western blotting. AS, unsynchronized cells. (E) HeLa cells were transfected with siRNAs, harvested at 62 h as asynchronous cells, and analyzed to identify DDA3-specific bands on Western blot. (F) Lysates of thymidine-nocodazole–treated mitotic HeLa cells were incubated with or without λ-phosphatase (λ-PPase) before immunoblotting to identify phosphorylated DDA3. Arrows point to hyperphosphorylated mitotic DDA3 and arrowheads point to two forms of hypo- or unphosphorylated DDA3.
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fig2: DDA3 is a spindle-associated cell cycle protein. (A–C) Shown are maximum projections from deconvolved z stacks of representative cells stained for DDA3 (A and C, green)/GFP (B, green), β-tubulin (red), and DNA (blue). A, HeLa cells; B, HeLa/GFP–DDA3 cells; C, HeLa cells transfected with siControl or siDDA3-A. Bars, 5 μm. (D) HeLa cells were synchronized by a double-thymidine treatment and released into fresh media. 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at the indicated times. Cells were analyzed by FACS and Western blotting. AS, unsynchronized cells. (E) HeLa cells were transfected with siRNAs, harvested at 62 h as asynchronous cells, and analyzed to identify DDA3-specific bands on Western blot. (F) Lysates of thymidine-nocodazole–treated mitotic HeLa cells were incubated with or without λ-phosphatase (λ-PPase) before immunoblotting to identify phosphorylated DDA3. Arrows point to hyperphosphorylated mitotic DDA3 and arrowheads point to two forms of hypo- or unphosphorylated DDA3.

Mentions: To elucidate its role in mitosis, we determined the cellular localization of endogenous DDA3 across the cell cycle. DDA3 punctately colocalized with spindle MTs from prophase to anaphase A and with the central spindle and midbody MTs from anaphase B to the end of cytokinesis (Fig. 2 A). DDA3 was diffusively distributed throughout the cell without obvious localization to MTs in interphase cells, indicating that the association of DDA3 with the mitotic spindle is under active regulation. This cell cycle–dependent localization of DDA3 is specific, as stably expressed GFP–DDA3 also localized to the mitotic spindle (Fig. 2 B; Hsieh et al., 2007) and as knockdown of DDA3 completely abolished its localization on the spindle (Fig. 2 C).


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 is a spindle-associated cell cycle protein. (A–C) Shown are maximum projections from deconvolved z stacks of representative cells stained for DDA3 (A and C, green)/GFP (B, green), β-tubulin (red), and DNA (blue). A, HeLa cells; B, HeLa/GFP–DDA3 cells; C, HeLa cells transfected with siControl or siDDA3-A. Bars, 5 μm. (D) HeLa cells were synchronized by a double-thymidine treatment and released into fresh media. 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at the indicated times. Cells were analyzed by FACS and Western blotting. AS, unsynchronized cells. (E) HeLa cells were transfected with siRNAs, harvested at 62 h as asynchronous cells, and analyzed to identify DDA3-specific bands on Western blot. (F) Lysates of thymidine-nocodazole–treated mitotic HeLa cells were incubated with or without λ-phosphatase (λ-PPase) before immunoblotting to identify phosphorylated DDA3. Arrows point to hyperphosphorylated mitotic DDA3 and arrowheads point to two forms of hypo- or unphosphorylated DDA3.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315673&req=5

fig2: DDA3 is a spindle-associated cell cycle protein. (A–C) Shown are maximum projections from deconvolved z stacks of representative cells stained for DDA3 (A and C, green)/GFP (B, green), β-tubulin (red), and DNA (blue). A, HeLa cells; B, HeLa/GFP–DDA3 cells; C, HeLa cells transfected with siControl or siDDA3-A. Bars, 5 μm. (D) HeLa cells were synchronized by a double-thymidine treatment and released into fresh media. 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at the indicated times. Cells were analyzed by FACS and Western blotting. AS, unsynchronized cells. (E) HeLa cells were transfected with siRNAs, harvested at 62 h as asynchronous cells, and analyzed to identify DDA3-specific bands on Western blot. (F) Lysates of thymidine-nocodazole–treated mitotic HeLa cells were incubated with or without λ-phosphatase (λ-PPase) before immunoblotting to identify phosphorylated DDA3. Arrows point to hyperphosphorylated mitotic DDA3 and arrowheads point to two forms of hypo- or unphosphorylated DDA3.
Mentions: To elucidate its role in mitosis, we determined the cellular localization of endogenous DDA3 across the cell cycle. DDA3 punctately colocalized with spindle MTs from prophase to anaphase A and with the central spindle and midbody MTs from anaphase B to the end of cytokinesis (Fig. 2 A). DDA3 was diffusively distributed throughout the cell without obvious localization to MTs in interphase cells, indicating that the association of DDA3 with the mitotic spindle is under active regulation. This cell cycle–dependent localization of DDA3 is specific, as stably expressed GFP–DDA3 also localized to the mitotic spindle (Fig. 2 B; Hsieh et al., 2007) and as knockdown of DDA3 completely abolished its localization on the spindle (Fig. 2 C).

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