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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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Depletion of HKIF4A reduces sister chromatid resolution and causes chromosome misalignment. (A) Western blot of HKIF4A in total extracts of MRC-5 cells after 48 h of mock (control) or HKIF4A RNAi transfection. (B) MRC-5 cells were fixed 48 h after transfection with RNAi and stained with DAPI (blue) and anti-HKIF4A (red). The chromosomal signal of HKIF4A was lost due to the RNAi effect. Bar, 5 μm. (C) Quantitation of distribution of mitotic phases in mock- and HKIF4A RNAi-transfected cells 48 h after transfection. Values represent averages from three experiments ± SD.
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fig2: Depletion of HKIF4A reduces sister chromatid resolution and causes chromosome misalignment. (A) Western blot of HKIF4A in total extracts of MRC-5 cells after 48 h of mock (control) or HKIF4A RNAi transfection. (B) MRC-5 cells were fixed 48 h after transfection with RNAi and stained with DAPI (blue) and anti-HKIF4A (red). The chromosomal signal of HKIF4A was lost due to the RNAi effect. Bar, 5 μm. (C) Quantitation of distribution of mitotic phases in mock- and HKIF4A RNAi-transfected cells 48 h after transfection. Values represent averages from three experiments ± SD.

Mentions: To determine the in vivo function of HKIF4A, we depleted the protein from MRC-5 cells by RNA interference. The cellular level of HKIF4A decreased by almost 90% of its initial amount after two consecutive transfections 24 h apart (Fig. 2 A). HKIF4A RNAi did not affect cellular tubulin or lamin A/C levels (Fig. 2 A) and RNAi against lamin A/C did not affect HKIF4A levels (not depicted). HKIF4A depletion resulted in an accumulation of mitotic cells. Although the mitotic index was 0.11 ± 0.06 in mock-transfected control cells, it was 0.2 ± 0.01 in HKIF4A-depleted cells 48 h after transfection (P < 0.05). HKIF4A-depleted mitotic cells showed pronounced defects in various stages of mitosis (Fig. 2 B). 65% of prometaphase cells lacked the typical doughnut shape arrangement of chromosomes, chromosomes were frequently misaligned, and anaphase separation was often incomplete (Fig. 2 B). Similar observations were made upon microinjection of anti-HKIF4A antibody into prometaphase cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200401142.DC1). Quantitation of the distinct mitotic stages of RNAi-transfected cells (Fig. 2 C) indicated that 48 h after transfection the fraction of prometaphase and metaphase cells was dramatically higher (50 ± 5.7%) than in mock-treated cells (29 ± 1.3%). The severity of the observed mitotic defects appeared roughly correlated with the level of HKIF4A depletion (not depicted).


Human chromokinesin KIF4A functions in chromosome condensation and segregation.

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

Depletion of HKIF4A reduces sister chromatid resolution and causes chromosome misalignment. (A) Western blot of HKIF4A in total extracts of MRC-5 cells after 48 h of mock (control) or HKIF4A RNAi transfection. (B) MRC-5 cells were fixed 48 h after transfection with RNAi and stained with DAPI (blue) and anti-HKIF4A (red). The chromosomal signal of HKIF4A was lost due to the RNAi effect. Bar, 5 μm. (C) Quantitation of distribution of mitotic phases in mock- and HKIF4A RNAi-transfected cells 48 h after transfection. Values represent averages from three experiments ± SD.
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Related In: Results  -  Collection

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fig2: Depletion of HKIF4A reduces sister chromatid resolution and causes chromosome misalignment. (A) Western blot of HKIF4A in total extracts of MRC-5 cells after 48 h of mock (control) or HKIF4A RNAi transfection. (B) MRC-5 cells were fixed 48 h after transfection with RNAi and stained with DAPI (blue) and anti-HKIF4A (red). The chromosomal signal of HKIF4A was lost due to the RNAi effect. Bar, 5 μm. (C) Quantitation of distribution of mitotic phases in mock- and HKIF4A RNAi-transfected cells 48 h after transfection. Values represent averages from three experiments ± SD.
Mentions: To determine the in vivo function of HKIF4A, we depleted the protein from MRC-5 cells by RNA interference. The cellular level of HKIF4A decreased by almost 90% of its initial amount after two consecutive transfections 24 h apart (Fig. 2 A). HKIF4A RNAi did not affect cellular tubulin or lamin A/C levels (Fig. 2 A) and RNAi against lamin A/C did not affect HKIF4A levels (not depicted). HKIF4A depletion resulted in an accumulation of mitotic cells. Although the mitotic index was 0.11 ± 0.06 in mock-transfected control cells, it was 0.2 ± 0.01 in HKIF4A-depleted cells 48 h after transfection (P < 0.05). HKIF4A-depleted mitotic cells showed pronounced defects in various stages of mitosis (Fig. 2 B). 65% of prometaphase cells lacked the typical doughnut shape arrangement of chromosomes, chromosomes were frequently misaligned, and anaphase separation was often incomplete (Fig. 2 B). Similar observations were made upon microinjection of anti-HKIF4A antibody into prometaphase cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200401142.DC1). Quantitation of the distinct mitotic stages of RNAi-transfected cells (Fig. 2 C) indicated that 48 h after transfection the fraction of prometaphase and metaphase cells was dramatically higher (50 ± 5.7%) than in mock-treated cells (29 ± 1.3%). The severity of the observed mitotic defects appeared roughly correlated with the level of HKIF4A depletion (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