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Human chromokinesin KIF4A functions in chromosome condensation and segregation.

Mazumdar M, Sundareshan S, Misteli T - J. Cell Biol. (2004)

Bottom Line: Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation.HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture.Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.

View Article: PubMed Central - PubMed

Affiliation: National Cancer Institute, National Institutes of Health, Bldg. 41, Rm. B 507, 41 Library Dr., Bethesda, MD 20892, USA. mazumdam@mail.nih.gov

ABSTRACT
Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome arm--associated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interference--mediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.

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HKIF4A is required for maintenance of normal metaphase chromosome morphology. (A) Chromosome spreads from control and HKIF4A-depleted MRC-5 cells were stained with DAPI. Metaphase chromosomes from RNAi-treated cells show longitudinal shortening and widening compared with mock-transfected control cells. Bars, 5 μm. (B) Projected images of chromosome spreads were analyzed and the width (x axis) and length (y axis) of all chromosomes in a spread measured. Note that the large variance in length in the control sample is due to the differences in size amongst human chromosomes. Values represent typical spreads as in A. (C) Time-lapse imaging of HeLa cells stably expressing GFP-histone 2B. Cells were either mock transfected or transfected with HKIF4A RNAi for 48 h and were imaged every 5 min by acquiring a complete z-stack of 10 sections each 1 μm apart. All images were acquired and processed under identical conditions. Bar, 5 μm.
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fig4: HKIF4A is required for maintenance of normal metaphase chromosome morphology. (A) Chromosome spreads from control and HKIF4A-depleted MRC-5 cells were stained with DAPI. Metaphase chromosomes from RNAi-treated cells show longitudinal shortening and widening compared with mock-transfected control cells. Bars, 5 μm. (B) Projected images of chromosome spreads were analyzed and the width (x axis) and length (y axis) of all chromosomes in a spread measured. Note that the large variance in length in the control sample is due to the differences in size amongst human chromosomes. Values represent typical spreads as in A. (C) Time-lapse imaging of HeLa cells stably expressing GFP-histone 2B. Cells were either mock transfected or transfected with HKIF4A RNAi for 48 h and were imaged every 5 min by acquiring a complete z-stack of 10 sections each 1 μm apart. All images were acquired and processed under identical conditions. Bar, 5 μm.

Mentions: Because HKIF4A is localized all along the condensed chromosome arms, we examined the consequences of HKIF4A depletion on the structural integrity of mitotic chromosomes. Metaphase chromosome spreads from mock-transfected or HKIF4A RNAi-transfected cells were prepared after 2 h of colcemid block and stained with DAPI (Fig. 4). RNAi-mediated depletion of HKIF4A induced significant hypercondensation and chromosomes from HKIF4A-depleted cells were dramatically shorter than chromosomes from mock-transfected cells (Fig. 4 A). The average length of control chromosomes was 4.88 μm with a range between 1.5 and 10 μm, reflecting the variable sizes of human chromosomes. The width of the chromosomes was on average 0.68 μm with a range of 0.5–0.8 μm (Fig. 4 B). In contrast, chromosomes from depleted cells were on average 3-μm long and 1.2-μm wide, with a range of 1.1–5.5 μm in length and 0.8–1.5 μm in width (Fig. 5 B). These differences were statistically significant at the P < 0.001 level. To rule out that the observed hypercondensation of chromosomes was caused by artifacts of chromosome spread preparation, and more importantly, to exclude the possibility that hypercondensation was caused by prolonged presence of chromosomes in mitosis, we analyzed chromosomes in intact cells. We followed progression of mitosis from nuclear envelope breakdown to telophase in single living HeLa cells stably expressing histone H2B-GFP that are either mock-transfected or RNAi-transfected (Fig. 4 C). In the majority of cells in the RNAi-treated population, chromosomes were more condensed compared with mock-transfected cells even before prometaphase (Fig. 4 C). As expected, cells containing hypercondensed chromosomes experienced a delay in mitotic progression (Fig. 4 C). Furthermore, chromosomes were already significantly more condensed in HKIF4A-depleted cells compared with control cells even before breakdown of the nuclear envelope in early prophase (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200401142/DC1).


Human chromokinesin KIF4A functions in chromosome condensation and segregation.

Mazumdar M, Sundareshan S, Misteli T - J. Cell Biol. (2004)

HKIF4A is required for maintenance of normal metaphase chromosome morphology. (A) Chromosome spreads from control and HKIF4A-depleted MRC-5 cells were stained with DAPI. Metaphase chromosomes from RNAi-treated cells show longitudinal shortening and widening compared with mock-transfected control cells. Bars, 5 μm. (B) Projected images of chromosome spreads were analyzed and the width (x axis) and length (y axis) of all chromosomes in a spread measured. Note that the large variance in length in the control sample is due to the differences in size amongst human chromosomes. Values represent typical spreads as in A. (C) Time-lapse imaging of HeLa cells stably expressing GFP-histone 2B. Cells were either mock transfected or transfected with HKIF4A RNAi for 48 h and were imaged every 5 min by acquiring a complete z-stack of 10 sections each 1 μm apart. All images were acquired and processed under identical conditions. Bar, 5 μm.
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fig4: HKIF4A is required for maintenance of normal metaphase chromosome morphology. (A) Chromosome spreads from control and HKIF4A-depleted MRC-5 cells were stained with DAPI. Metaphase chromosomes from RNAi-treated cells show longitudinal shortening and widening compared with mock-transfected control cells. Bars, 5 μm. (B) Projected images of chromosome spreads were analyzed and the width (x axis) and length (y axis) of all chromosomes in a spread measured. Note that the large variance in length in the control sample is due to the differences in size amongst human chromosomes. Values represent typical spreads as in A. (C) Time-lapse imaging of HeLa cells stably expressing GFP-histone 2B. Cells were either mock transfected or transfected with HKIF4A RNAi for 48 h and were imaged every 5 min by acquiring a complete z-stack of 10 sections each 1 μm apart. All images were acquired and processed under identical conditions. Bar, 5 μm.
Mentions: Because HKIF4A is localized all along the condensed chromosome arms, we examined the consequences of HKIF4A depletion on the structural integrity of mitotic chromosomes. Metaphase chromosome spreads from mock-transfected or HKIF4A RNAi-transfected cells were prepared after 2 h of colcemid block and stained with DAPI (Fig. 4). RNAi-mediated depletion of HKIF4A induced significant hypercondensation and chromosomes from HKIF4A-depleted cells were dramatically shorter than chromosomes from mock-transfected cells (Fig. 4 A). The average length of control chromosomes was 4.88 μm with a range between 1.5 and 10 μm, reflecting the variable sizes of human chromosomes. The width of the chromosomes was on average 0.68 μm with a range of 0.5–0.8 μm (Fig. 4 B). In contrast, chromosomes from depleted cells were on average 3-μm long and 1.2-μm wide, with a range of 1.1–5.5 μm in length and 0.8–1.5 μm in width (Fig. 5 B). These differences were statistically significant at the P < 0.001 level. To rule out that the observed hypercondensation of chromosomes was caused by artifacts of chromosome spread preparation, and more importantly, to exclude the possibility that hypercondensation was caused by prolonged presence of chromosomes in mitosis, we analyzed chromosomes in intact cells. We followed progression of mitosis from nuclear envelope breakdown to telophase in single living HeLa cells stably expressing histone H2B-GFP that are either mock-transfected or RNAi-transfected (Fig. 4 C). In the majority of cells in the RNAi-treated population, chromosomes were more condensed compared with mock-transfected cells even before prometaphase (Fig. 4 C). As expected, cells containing hypercondensed chromosomes experienced a delay in mitotic progression (Fig. 4 C). Furthermore, chromosomes were already significantly more condensed in HKIF4A-depleted cells compared with control cells even before breakdown of the nuclear envelope in early prophase (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200401142/DC1).

Bottom Line: Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation.HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture.Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.

View Article: PubMed Central - PubMed

Affiliation: National Cancer Institute, National Institutes of Health, Bldg. 41, Rm. B 507, 41 Library Dr., Bethesda, MD 20892, USA. mazumdam@mail.nih.gov

ABSTRACT
Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome arm--associated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interference--mediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.

Show MeSH
Related in: MedlinePlus