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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 interacts with components of condensin I and II. (A) Western blot of anti-HKIF4A pull-down from nuclear or mitotic extract. hCAP-E, -G, and -G2 physically associate with HKIF4A. Protein G beads without antibody or mouse IgG were used as controls. Input loading was one fifth of total. (B–D) Colocalization of HKIF4A with condensin I and II complex. HKIF4A (green) partially colocalized with condensin components hCAP-E, hCAP-G, and hCAP-G2 (red) in punctate regions along the length of the chromosome arms. Far right panels show higher magnifications of individual chromosomes from corresponding merged panels. Bar, 5 μm. (E) Western blot of cell extracts from mock- and HKIF4A RNAi-transfected MRC-5 nonsynchronized and mitotic cells with hCAP-E antibody showed that the condensin level remained unchanged after depletion of HKIF4A. (F) Loss of chromokinesin HKIF4A results in condensin components failing to localize along the chromosome axis in a defined pattern. hCAP-E, G, and G2 (green) were diffusely distributed over the condensed mitotic chromatin mass (DNA, blue). Bar, 5 μm.
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fig5: HKIF4A interacts with components of condensin I and II. (A) Western blot of anti-HKIF4A pull-down from nuclear or mitotic extract. hCAP-E, -G, and -G2 physically associate with HKIF4A. Protein G beads without antibody or mouse IgG were used as controls. Input loading was one fifth of total. (B–D) Colocalization of HKIF4A with condensin I and II complex. HKIF4A (green) partially colocalized with condensin components hCAP-E, hCAP-G, and hCAP-G2 (red) in punctate regions along the length of the chromosome arms. Far right panels show higher magnifications of individual chromosomes from corresponding merged panels. Bar, 5 μm. (E) Western blot of cell extracts from mock- and HKIF4A RNAi-transfected MRC-5 nonsynchronized and mitotic cells with hCAP-E antibody showed that the condensin level remained unchanged after depletion of HKIF4A. (F) Loss of chromokinesin HKIF4A results in condensin components failing to localize along the chromosome axis in a defined pattern. hCAP-E, G, and G2 (green) were diffusely distributed over the condensed mitotic chromatin mass (DNA, blue). Bar, 5 μm.

Mentions: To ask whether HKIF4A depletion leads to hypercondensation of chromosomes via the condensation machinery, we tested whether HKIF4A physically interacts in vivo with condensin. Two distinct condensin complexes, condensin I and II, which share the SMC subunits hCAP-C and -E, but differ in their non-SMC components, hCAP-D, -G, and -H, have been described previously (Ono et al., 2003). Immunoprecipitation with anti-HKIF4A antibody from nuclear extract of nonsynchronized MRC-5 or HeLa cells or from mitotic HeLa extracts specifically pulled down hCAP-E, -G, and -G2 (Fig. 5 A; see Fig. S3 for controls). The physical association of HKIF4A with both condensin complexes I and II was corroborated by immunofluorescence microscopy of chromosomes in intact mitotic cells (Fig. 5, B–D). HKIF4A partially colocalized with hCAP-E, -G, and -G2 along the length of the chromosomes in what appeared as overlapping punctate regions, possibly indicating that only a subpopulation of HKIF4A interacts with condensin subunits (Fig. 5, B–D).


Human chromokinesin KIF4A functions in chromosome condensation and segregation.

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

HKIF4A interacts with components of condensin I and II. (A) Western blot of anti-HKIF4A pull-down from nuclear or mitotic extract. hCAP-E, -G, and -G2 physically associate with HKIF4A. Protein G beads without antibody or mouse IgG were used as controls. Input loading was one fifth of total. (B–D) Colocalization of HKIF4A with condensin I and II complex. HKIF4A (green) partially colocalized with condensin components hCAP-E, hCAP-G, and hCAP-G2 (red) in punctate regions along the length of the chromosome arms. Far right panels show higher magnifications of individual chromosomes from corresponding merged panels. Bar, 5 μm. (E) Western blot of cell extracts from mock- and HKIF4A RNAi-transfected MRC-5 nonsynchronized and mitotic cells with hCAP-E antibody showed that the condensin level remained unchanged after depletion of HKIF4A. (F) Loss of chromokinesin HKIF4A results in condensin components failing to localize along the chromosome axis in a defined pattern. hCAP-E, G, and G2 (green) were diffusely distributed over the condensed mitotic chromatin mass (DNA, blue). Bar, 5 μm.
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fig5: HKIF4A interacts with components of condensin I and II. (A) Western blot of anti-HKIF4A pull-down from nuclear or mitotic extract. hCAP-E, -G, and -G2 physically associate with HKIF4A. Protein G beads without antibody or mouse IgG were used as controls. Input loading was one fifth of total. (B–D) Colocalization of HKIF4A with condensin I and II complex. HKIF4A (green) partially colocalized with condensin components hCAP-E, hCAP-G, and hCAP-G2 (red) in punctate regions along the length of the chromosome arms. Far right panels show higher magnifications of individual chromosomes from corresponding merged panels. Bar, 5 μm. (E) Western blot of cell extracts from mock- and HKIF4A RNAi-transfected MRC-5 nonsynchronized and mitotic cells with hCAP-E antibody showed that the condensin level remained unchanged after depletion of HKIF4A. (F) Loss of chromokinesin HKIF4A results in condensin components failing to localize along the chromosome axis in a defined pattern. hCAP-E, G, and G2 (green) were diffusely distributed over the condensed mitotic chromatin mass (DNA, blue). Bar, 5 μm.
Mentions: To ask whether HKIF4A depletion leads to hypercondensation of chromosomes via the condensation machinery, we tested whether HKIF4A physically interacts in vivo with condensin. Two distinct condensin complexes, condensin I and II, which share the SMC subunits hCAP-C and -E, but differ in their non-SMC components, hCAP-D, -G, and -H, have been described previously (Ono et al., 2003). Immunoprecipitation with anti-HKIF4A antibody from nuclear extract of nonsynchronized MRC-5 or HeLa cells or from mitotic HeLa extracts specifically pulled down hCAP-E, -G, and -G2 (Fig. 5 A; see Fig. S3 for controls). The physical association of HKIF4A with both condensin complexes I and II was corroborated by immunofluorescence microscopy of chromosomes in intact mitotic cells (Fig. 5, B–D). HKIF4A partially colocalized with hCAP-E, -G, and -G2 along the length of the chromosomes in what appeared as overlapping punctate regions, possibly indicating that only a subpopulation of HKIF4A interacts with condensin subunits (Fig. 5, B–D).

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