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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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Depletion of DDA3 prolongs metaphase and activates the spindle checkpoint. (A and B) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse starting from 50 h after transfection. Images were captured every 3 min to monitor mitotic progression. The duration from nuclear envelop breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (NEB to unaligned), from the unaligned state to metaphase without unaligned chromosomes (unaligned to metaphase), and from metaphase without unaligned chromosomes to anaphase (metaphase to anaphase) were determined for control and DDA3-depleted cells (n = 30 cells). Still frames from time-lapse movies of representative cells are shown in B. Arrowheads point to unaligned chromosomes. *, P < 0.0013; **, P < 6 × 10−5; ***, P < 0.0026 (two-tailed t test). (C) HeLa cells were transfected with siRNAs. At 50 h after transfection, cells were incubated at 4°C for 10 min, fixed, and stained for kinetochore MTs. Shown are maximum projections from deconvolved z stacks of representative cells stained for CenpA (green), β-tubulin (red), and DNA (blue). Insets show single focal planes of boxed regions. (D and E) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Mad2 (D, red)/ BubR1 (E, red), and DNA (blue). Insets show single focal planes of boxed regions. Mad2 and BubR1 signals on kinetochores were quantified in five control or DDA3-depleted cells at prometaphase (PM) or metaphase (M; n > 100 kinetochores for each quantification). For unaligned chromosomes (UC) in DDA3-depleted metaphase cells, 40 kinetochores on unaligned chromosomes from 10 metaphase cells were quantified. *, P < 6 × 10−32; **, P < 6 × 10−10 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (F and G) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Hec1 (red), and DNA (blue). Images were collected with a 100× objective lens without binning. Interkinetochore (Inter-KT) distance in prometaphase and metaphase cells was quantified from >100 kinetochore pairs in five cells for each quantification. For unaligned chromosomes in DDA3-depleted metaphase cells, 10 kinetochore pairs were quantified. Boxes 1 and 2 in D–F represent aligned and unaligned chromosomes in DDA3-depleted metaphase cells, respectively. *, P < 5 × 10−57; **, P < 1.1−4 (two-tailed t test relative to siControl metaphase cells). (H) HeLa cells stably expressing GFP-CenpA were transfected with siRNAs and imaged for GFP-CenpA by time lapse. Images were acquired every 6 s on a spinning disc confocal microscope using a 63× objective lens with 2 × 2 binning. Interkinetochore distance of individual kinetochore pairs from chromosomes aligned at the metaphase plate was tracked and plotted over 60 s. Error bars show SEM. Bars: (B) 10 μm; (C–F) 5 μm.
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fig3: Depletion of DDA3 prolongs metaphase and activates the spindle checkpoint. (A and B) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse starting from 50 h after transfection. Images were captured every 3 min to monitor mitotic progression. The duration from nuclear envelop breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (NEB to unaligned), from the unaligned state to metaphase without unaligned chromosomes (unaligned to metaphase), and from metaphase without unaligned chromosomes to anaphase (metaphase to anaphase) were determined for control and DDA3-depleted cells (n = 30 cells). Still frames from time-lapse movies of representative cells are shown in B. Arrowheads point to unaligned chromosomes. *, P < 0.0013; **, P < 6 × 10−5; ***, P < 0.0026 (two-tailed t test). (C) HeLa cells were transfected with siRNAs. At 50 h after transfection, cells were incubated at 4°C for 10 min, fixed, and stained for kinetochore MTs. Shown are maximum projections from deconvolved z stacks of representative cells stained for CenpA (green), β-tubulin (red), and DNA (blue). Insets show single focal planes of boxed regions. (D and E) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Mad2 (D, red)/ BubR1 (E, red), and DNA (blue). Insets show single focal planes of boxed regions. Mad2 and BubR1 signals on kinetochores were quantified in five control or DDA3-depleted cells at prometaphase (PM) or metaphase (M; n > 100 kinetochores for each quantification). For unaligned chromosomes (UC) in DDA3-depleted metaphase cells, 40 kinetochores on unaligned chromosomes from 10 metaphase cells were quantified. *, P < 6 × 10−32; **, P < 6 × 10−10 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (F and G) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Hec1 (red), and DNA (blue). Images were collected with a 100× objective lens without binning. Interkinetochore (Inter-KT) distance in prometaphase and metaphase cells was quantified from >100 kinetochore pairs in five cells for each quantification. For unaligned chromosomes in DDA3-depleted metaphase cells, 10 kinetochore pairs were quantified. Boxes 1 and 2 in D–F represent aligned and unaligned chromosomes in DDA3-depleted metaphase cells, respectively. *, P < 5 × 10−57; **, P < 1.1−4 (two-tailed t test relative to siControl metaphase cells). (H) HeLa cells stably expressing GFP-CenpA were transfected with siRNAs and imaged for GFP-CenpA by time lapse. Images were acquired every 6 s on a spinning disc confocal microscope using a 63× objective lens with 2 × 2 binning. Interkinetochore distance of individual kinetochore pairs from chromosomes aligned at the metaphase plate was tracked and plotted over 60 s. Error bars show SEM. Bars: (B) 10 μm; (C–F) 5 μm.

Mentions: The cellular function of DDA3 was analyzed by time-lapse imaging of HeLa cells stably expressing GFP-Histone H2B (Fig. 3, A and B). Quantitative analysis of mitotic progression indicated that depletion of DDA3 altered the duration of prometaphase (from nuclear envelope breakdown to initial formation of the metaphase plate) to some extent but more significantly prolonged the duration of metaphase (from the initial formation of the metaphase plate to anaphase onset; Fig. 3 A). This extended metaphase mainly resulted from the persistence of unaligned chromosomes in metaphase cells depleted of DDA3 (Fig. 3 A, unaligned to metaphase). However, this is only a kinetic delay at metaphase. The majority of unaligned chromosomes eventually congressed to the metaphase plate (Fig. 3, A and B), followed by anaphase initiation, even though a certain percentage (20 and 1% for siDDA3-A and siControl metaphase cells, respectively) of transfected cells with a large number of unaligned chromosomes underwent apoptosis after extended arrest at metaphase. Thus, DDA3 is required for efficient chromosome congression to the metaphase plate and for timely onset of anaphase.


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)

Depletion of DDA3 prolongs metaphase and activates the spindle checkpoint. (A and B) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse starting from 50 h after transfection. Images were captured every 3 min to monitor mitotic progression. The duration from nuclear envelop breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (NEB to unaligned), from the unaligned state to metaphase without unaligned chromosomes (unaligned to metaphase), and from metaphase without unaligned chromosomes to anaphase (metaphase to anaphase) were determined for control and DDA3-depleted cells (n = 30 cells). Still frames from time-lapse movies of representative cells are shown in B. Arrowheads point to unaligned chromosomes. *, P < 0.0013; **, P < 6 × 10−5; ***, P < 0.0026 (two-tailed t test). (C) HeLa cells were transfected with siRNAs. At 50 h after transfection, cells were incubated at 4°C for 10 min, fixed, and stained for kinetochore MTs. Shown are maximum projections from deconvolved z stacks of representative cells stained for CenpA (green), β-tubulin (red), and DNA (blue). Insets show single focal planes of boxed regions. (D and E) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Mad2 (D, red)/ BubR1 (E, red), and DNA (blue). Insets show single focal planes of boxed regions. Mad2 and BubR1 signals on kinetochores were quantified in five control or DDA3-depleted cells at prometaphase (PM) or metaphase (M; n > 100 kinetochores for each quantification). For unaligned chromosomes (UC) in DDA3-depleted metaphase cells, 40 kinetochores on unaligned chromosomes from 10 metaphase cells were quantified. *, P < 6 × 10−32; **, P < 6 × 10−10 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (F and G) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Hec1 (red), and DNA (blue). Images were collected with a 100× objective lens without binning. Interkinetochore (Inter-KT) distance in prometaphase and metaphase cells was quantified from >100 kinetochore pairs in five cells for each quantification. For unaligned chromosomes in DDA3-depleted metaphase cells, 10 kinetochore pairs were quantified. Boxes 1 and 2 in D–F represent aligned and unaligned chromosomes in DDA3-depleted metaphase cells, respectively. *, P < 5 × 10−57; **, P < 1.1−4 (two-tailed t test relative to siControl metaphase cells). (H) HeLa cells stably expressing GFP-CenpA were transfected with siRNAs and imaged for GFP-CenpA by time lapse. Images were acquired every 6 s on a spinning disc confocal microscope using a 63× objective lens with 2 × 2 binning. Interkinetochore distance of individual kinetochore pairs from chromosomes aligned at the metaphase plate was tracked and plotted over 60 s. Error bars show SEM. Bars: (B) 10 μm; (C–F) 5 μm.
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Related In: Results  -  Collection

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fig3: Depletion of DDA3 prolongs metaphase and activates the spindle checkpoint. (A and B) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse starting from 50 h after transfection. Images were captured every 3 min to monitor mitotic progression. The duration from nuclear envelop breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (NEB to unaligned), from the unaligned state to metaphase without unaligned chromosomes (unaligned to metaphase), and from metaphase without unaligned chromosomes to anaphase (metaphase to anaphase) were determined for control and DDA3-depleted cells (n = 30 cells). Still frames from time-lapse movies of representative cells are shown in B. Arrowheads point to unaligned chromosomes. *, P < 0.0013; **, P < 6 × 10−5; ***, P < 0.0026 (two-tailed t test). (C) HeLa cells were transfected with siRNAs. At 50 h after transfection, cells were incubated at 4°C for 10 min, fixed, and stained for kinetochore MTs. Shown are maximum projections from deconvolved z stacks of representative cells stained for CenpA (green), β-tubulin (red), and DNA (blue). Insets show single focal planes of boxed regions. (D and E) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Mad2 (D, red)/ BubR1 (E, red), and DNA (blue). Insets show single focal planes of boxed regions. Mad2 and BubR1 signals on kinetochores were quantified in five control or DDA3-depleted cells at prometaphase (PM) or metaphase (M; n > 100 kinetochores for each quantification). For unaligned chromosomes (UC) in DDA3-depleted metaphase cells, 40 kinetochores on unaligned chromosomes from 10 metaphase cells were quantified. *, P < 6 × 10−32; **, P < 6 × 10−10 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (F and G) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Hec1 (red), and DNA (blue). Images were collected with a 100× objective lens without binning. Interkinetochore (Inter-KT) distance in prometaphase and metaphase cells was quantified from >100 kinetochore pairs in five cells for each quantification. For unaligned chromosomes in DDA3-depleted metaphase cells, 10 kinetochore pairs were quantified. Boxes 1 and 2 in D–F represent aligned and unaligned chromosomes in DDA3-depleted metaphase cells, respectively. *, P < 5 × 10−57; **, P < 1.1−4 (two-tailed t test relative to siControl metaphase cells). (H) HeLa cells stably expressing GFP-CenpA were transfected with siRNAs and imaged for GFP-CenpA by time lapse. Images were acquired every 6 s on a spinning disc confocal microscope using a 63× objective lens with 2 × 2 binning. Interkinetochore distance of individual kinetochore pairs from chromosomes aligned at the metaphase plate was tracked and plotted over 60 s. Error bars show SEM. Bars: (B) 10 μm; (C–F) 5 μm.
Mentions: The cellular function of DDA3 was analyzed by time-lapse imaging of HeLa cells stably expressing GFP-Histone H2B (Fig. 3, A and B). Quantitative analysis of mitotic progression indicated that depletion of DDA3 altered the duration of prometaphase (from nuclear envelope breakdown to initial formation of the metaphase plate) to some extent but more significantly prolonged the duration of metaphase (from the initial formation of the metaphase plate to anaphase onset; Fig. 3 A). This extended metaphase mainly resulted from the persistence of unaligned chromosomes in metaphase cells depleted of DDA3 (Fig. 3 A, unaligned to metaphase). However, this is only a kinetic delay at metaphase. The majority of unaligned chromosomes eventually congressed to the metaphase plate (Fig. 3, A and B), followed by anaphase initiation, even though a certain percentage (20 and 1% for siDDA3-A and siControl metaphase cells, respectively) of transfected cells with a large number of unaligned chromosomes underwent apoptosis after extended arrest at metaphase. Thus, DDA3 is required for efficient chromosome congression to the metaphase plate and for timely onset of anaphase.

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