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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 mitotic spindle integrity and cellular ploidy. (A) Mitotic MRC-5 cells were stained with anti-tubulin, anti-HKIF4A antibodies, and DAPI to reveal the spindle and the chromosomal states in mock-transfected (control) or RNAi-transfected MRC-5 cells 48 h after transfection. The HKIF4A-depleted cells showed dramatic defects in spindle structure accompanied by chromosome mis-segregation. Anaphase and late telophase cells showed lagging chromosomes or lagging chromosomes forming a bridge (forked arrowheads). Bar, 5 μm. (B) Mitotic phenotypes 48 h after transfection with HKIF4A RNAi were scored against the total number of mitotic cells in a particular phase of the cell cycle. The frequency of disorganized prometaphase, mis-aligned metaphase chromosomes, and anaphase bridges was increased compared with the control cells. Values represent averages from 220 cells ± SD from three experiments. (C) Quantitation of different spindle phenotypes obtained 48 h after RNAi treatment of cells. The numbers of disorganized and defocused spindles in the mitotic figures of HKIF4-depleted cells is increased compared with mock-transfected cells. Values represent averages from three experiments ± SD. (D) Quantitation of aneuploidy caused by chromosome mis-segregation in HKIF4A-depleted MRC-5 cells. The percentage of chromosome spreads containing the indicated number of chromosomes was determined. Values represent data from four independent experiments (n= 30).
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fig3: HKIF4A is required for maintenance of mitotic spindle integrity and cellular ploidy. (A) Mitotic MRC-5 cells were stained with anti-tubulin, anti-HKIF4A antibodies, and DAPI to reveal the spindle and the chromosomal states in mock-transfected (control) or RNAi-transfected MRC-5 cells 48 h after transfection. The HKIF4A-depleted cells showed dramatic defects in spindle structure accompanied by chromosome mis-segregation. Anaphase and late telophase cells showed lagging chromosomes or lagging chromosomes forming a bridge (forked arrowheads). Bar, 5 μm. (B) Mitotic phenotypes 48 h after transfection with HKIF4A RNAi were scored against the total number of mitotic cells in a particular phase of the cell cycle. The frequency of disorganized prometaphase, mis-aligned metaphase chromosomes, and anaphase bridges was increased compared with the control cells. Values represent averages from 220 cells ± SD from three experiments. (C) Quantitation of different spindle phenotypes obtained 48 h after RNAi treatment of cells. The numbers of disorganized and defocused spindles in the mitotic figures of HKIF4-depleted cells is increased compared with mock-transfected cells. Values represent averages from three experiments ± SD. (D) Quantitation of aneuploidy caused by chromosome mis-segregation in HKIF4A-depleted MRC-5 cells. The percentage of chromosome spreads containing the indicated number of chromosomes was determined. Values represent data from four independent experiments (n= 30).

Mentions: To determine the role of HKIF4A in mitotic spindle function, we analyzed MTs and chromosomes from mock-transfected and HKIF4A-depleted MRC-5 cells 48 h after transfection with RNAi (Fig. 3). Compared with mock-transfected cells, immunofluorescence microscopy showed dramatic mitotic spindle defects (Fig. 3 A). MT organization of both prometaphase and metaphase spindles was abnormal and was accompanied by chromosome alignment defects (Fig. 3 A, arrow). In a number of cases spindle poles appeared less focused, and in extreme cases chromosomes scattered out of the spindle axis and the spindle completely lost its integrity (Fig. 3 A, solid arrowheads). In addition to prometaphase and metaphase defects, HKIF4A depletion also caused defective cytokinesis (Fig. 3 A). Cells lacking HKIF4A frequently exhibited lagging chromosomes in anaphase and after anaphase, and although the cells started to constrict, the cleavage furrow did not ingress completely (Fig. 3 A, forked arrow). Almost 50% of anaphase cells exhibited lagging chromosomes or chromatin bridges. Some anaphase bridges were observed to persist into telophase, resulting in formation of a large nucleus, binucleate cells, and multiple micronuclei (not depicted).


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 mitotic spindle integrity and cellular ploidy. (A) Mitotic MRC-5 cells were stained with anti-tubulin, anti-HKIF4A antibodies, and DAPI to reveal the spindle and the chromosomal states in mock-transfected (control) or RNAi-transfected MRC-5 cells 48 h after transfection. The HKIF4A-depleted cells showed dramatic defects in spindle structure accompanied by chromosome mis-segregation. Anaphase and late telophase cells showed lagging chromosomes or lagging chromosomes forming a bridge (forked arrowheads). Bar, 5 μm. (B) Mitotic phenotypes 48 h after transfection with HKIF4A RNAi were scored against the total number of mitotic cells in a particular phase of the cell cycle. The frequency of disorganized prometaphase, mis-aligned metaphase chromosomes, and anaphase bridges was increased compared with the control cells. Values represent averages from 220 cells ± SD from three experiments. (C) Quantitation of different spindle phenotypes obtained 48 h after RNAi treatment of cells. The numbers of disorganized and defocused spindles in the mitotic figures of HKIF4-depleted cells is increased compared with mock-transfected cells. Values represent averages from three experiments ± SD. (D) Quantitation of aneuploidy caused by chromosome mis-segregation in HKIF4A-depleted MRC-5 cells. The percentage of chromosome spreads containing the indicated number of chromosomes was determined. Values represent data from four independent experiments (n= 30).
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Related In: Results  -  Collection

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fig3: HKIF4A is required for maintenance of mitotic spindle integrity and cellular ploidy. (A) Mitotic MRC-5 cells were stained with anti-tubulin, anti-HKIF4A antibodies, and DAPI to reveal the spindle and the chromosomal states in mock-transfected (control) or RNAi-transfected MRC-5 cells 48 h after transfection. The HKIF4A-depleted cells showed dramatic defects in spindle structure accompanied by chromosome mis-segregation. Anaphase and late telophase cells showed lagging chromosomes or lagging chromosomes forming a bridge (forked arrowheads). Bar, 5 μm. (B) Mitotic phenotypes 48 h after transfection with HKIF4A RNAi were scored against the total number of mitotic cells in a particular phase of the cell cycle. The frequency of disorganized prometaphase, mis-aligned metaphase chromosomes, and anaphase bridges was increased compared with the control cells. Values represent averages from 220 cells ± SD from three experiments. (C) Quantitation of different spindle phenotypes obtained 48 h after RNAi treatment of cells. The numbers of disorganized and defocused spindles in the mitotic figures of HKIF4-depleted cells is increased compared with mock-transfected cells. Values represent averages from three experiments ± SD. (D) Quantitation of aneuploidy caused by chromosome mis-segregation in HKIF4A-depleted MRC-5 cells. The percentage of chromosome spreads containing the indicated number of chromosomes was determined. Values represent data from four independent experiments (n= 30).
Mentions: To determine the role of HKIF4A in mitotic spindle function, we analyzed MTs and chromosomes from mock-transfected and HKIF4A-depleted MRC-5 cells 48 h after transfection with RNAi (Fig. 3). Compared with mock-transfected cells, immunofluorescence microscopy showed dramatic mitotic spindle defects (Fig. 3 A). MT organization of both prometaphase and metaphase spindles was abnormal and was accompanied by chromosome alignment defects (Fig. 3 A, arrow). In a number of cases spindle poles appeared less focused, and in extreme cases chromosomes scattered out of the spindle axis and the spindle completely lost its integrity (Fig. 3 A, solid arrowheads). In addition to prometaphase and metaphase defects, HKIF4A depletion also caused defective cytokinesis (Fig. 3 A). Cells lacking HKIF4A frequently exhibited lagging chromosomes in anaphase and after anaphase, and although the cells started to constrict, the cleavage furrow did not ingress completely (Fig. 3 A, forked arrow). Almost 50% of anaphase cells exhibited lagging chromosomes or chromatin bridges. Some anaphase bridges were observed to persist into telophase, resulting in formation of a large nucleus, binucleate cells, and multiple micronuclei (not depicted).

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