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Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Tahara K, Takagi M, Ohsugi M, Sone T, Nishiumi F, Maeshima K, Horiuchi Y, Tokai-Nishizumi N, Imamoto F, Yamamoto T, Kose S, Imamoto N - J. Cell Biol. (2008)

Bottom Line: Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells.Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes.Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, Institute of Physical and Chemical Research, Wako, Saitama, 351-0198, Japan.

ABSTRACT
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

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Loss of the functional NLSs in hKid reduces its interaction with mitotic chromosomes in living cells. (A) Representative images of Venus–wt hKid and Venus–ΔNLSs hKid in stably transformed live HeLa cells. Cells in metaphase (left) and anaphase (right) are shown. To compare the localization signals in the cells, the fluorescent intensities were digitally compensated. The actual fluorescent intensity of Venus–ΔNLSs hKid was much higher than that of Venus–wt hKid. Bar, 10 μm. (B) FRAP analysis of Venus–wt hKid or Venus–ΔNLSs hKid on chromosomes. The mean intensity of a bleached chromosomal spot (a), a nonbleached chromosomal spot (b, control), and a nonchromosomal spot (c, background) were measured over time (micrographs). All FRAP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from six to nine cells are plotted. The diagram below depicts our experimental design. All possibilities for the replacement and turnover of hKid between each compartment are indicated by arrows. Bar, 10 μm. (C) FLIP analysis of Venus–wt hKid or Venus–ΔNLSs hKid in the presence (−nocodazole) or absence (+nocodazole) of a mitotic spindle. A spot outside the chromosomes and mitotic spindle (a) was irradiated, and the mean intensities of a chromosome spot (b) and a spot outside the cells (c, background) were measured before and during irradiation (micrographs). All FLIP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from 37–42 cells are plotted. The diagrams below depict our experimental design. Arrows indicate putative replacements and the turnover of hKid between each compartment. Each t1/2 value was calculated from the best-fit curve as described in Materials and methods. Bar, 10 μm.
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fig2: Loss of the functional NLSs in hKid reduces its interaction with mitotic chromosomes in living cells. (A) Representative images of Venus–wt hKid and Venus–ΔNLSs hKid in stably transformed live HeLa cells. Cells in metaphase (left) and anaphase (right) are shown. To compare the localization signals in the cells, the fluorescent intensities were digitally compensated. The actual fluorescent intensity of Venus–ΔNLSs hKid was much higher than that of Venus–wt hKid. Bar, 10 μm. (B) FRAP analysis of Venus–wt hKid or Venus–ΔNLSs hKid on chromosomes. The mean intensity of a bleached chromosomal spot (a), a nonbleached chromosomal spot (b, control), and a nonchromosomal spot (c, background) were measured over time (micrographs). All FRAP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from six to nine cells are plotted. The diagram below depicts our experimental design. All possibilities for the replacement and turnover of hKid between each compartment are indicated by arrows. Bar, 10 μm. (C) FLIP analysis of Venus–wt hKid or Venus–ΔNLSs hKid in the presence (−nocodazole) or absence (+nocodazole) of a mitotic spindle. A spot outside the chromosomes and mitotic spindle (a) was irradiated, and the mean intensities of a chromosome spot (b) and a spot outside the cells (c, background) were measured before and during irradiation (micrographs). All FLIP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from 37–42 cells are plotted. The diagrams below depict our experimental design. Arrows indicate putative replacements and the turnover of hKid between each compartment. Each t1/2 value was calculated from the best-fit curve as described in Materials and methods. Bar, 10 μm.

Mentions: To determine whether mitotic function of hKid is regulated by nucleocytoplasmic transport factors, we examined the contribution of each NLS to the mitotic behavior of hKid in living cells. For this purpose, cell lines stably expressing fluorescently tagged wild-type (wt) hKid (Venus–wt hKid) or mutant hKid defective in both its NLSs (Venus–ΔNLSs hKid) were established. Because the overexpression of hKid is highly cytotoxic and induces mitotic abnormalities (Ohsugi et al., 2003; this study), an Flp–FRT recombination system was used that allows for the selection of cells carrying only one or a few copies of an objective gene integrated into defined genomic loci (Yahata et al., 2005; see Materials and methods). The cell lines expressed Venus–wt hKid and –ΔNLSs hKid at a level below or nearly equal to that of endogenous hKid (Fig. 2 B), respectively, without affecting the cell cycle/mitotic profile. We were unable to establish lines expressing higher levels of Venus–wt hKid, probably because overexpression of wt hKid is more toxic for cells than overexpression of mutant hKid defective in its NLSs.


Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Tahara K, Takagi M, Ohsugi M, Sone T, Nishiumi F, Maeshima K, Horiuchi Y, Tokai-Nishizumi N, Imamoto F, Yamamoto T, Kose S, Imamoto N - J. Cell Biol. (2008)

Loss of the functional NLSs in hKid reduces its interaction with mitotic chromosomes in living cells. (A) Representative images of Venus–wt hKid and Venus–ΔNLSs hKid in stably transformed live HeLa cells. Cells in metaphase (left) and anaphase (right) are shown. To compare the localization signals in the cells, the fluorescent intensities were digitally compensated. The actual fluorescent intensity of Venus–ΔNLSs hKid was much higher than that of Venus–wt hKid. Bar, 10 μm. (B) FRAP analysis of Venus–wt hKid or Venus–ΔNLSs hKid on chromosomes. The mean intensity of a bleached chromosomal spot (a), a nonbleached chromosomal spot (b, control), and a nonchromosomal spot (c, background) were measured over time (micrographs). All FRAP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from six to nine cells are plotted. The diagram below depicts our experimental design. All possibilities for the replacement and turnover of hKid between each compartment are indicated by arrows. Bar, 10 μm. (C) FLIP analysis of Venus–wt hKid or Venus–ΔNLSs hKid in the presence (−nocodazole) or absence (+nocodazole) of a mitotic spindle. A spot outside the chromosomes and mitotic spindle (a) was irradiated, and the mean intensities of a chromosome spot (b) and a spot outside the cells (c, background) were measured before and during irradiation (micrographs). All FLIP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from 37–42 cells are plotted. The diagrams below depict our experimental design. Arrows indicate putative replacements and the turnover of hKid between each compartment. Each t1/2 value was calculated from the best-fit curve as described in Materials and methods. Bar, 10 μm.
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Related In: Results  -  Collection

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fig2: Loss of the functional NLSs in hKid reduces its interaction with mitotic chromosomes in living cells. (A) Representative images of Venus–wt hKid and Venus–ΔNLSs hKid in stably transformed live HeLa cells. Cells in metaphase (left) and anaphase (right) are shown. To compare the localization signals in the cells, the fluorescent intensities were digitally compensated. The actual fluorescent intensity of Venus–ΔNLSs hKid was much higher than that of Venus–wt hKid. Bar, 10 μm. (B) FRAP analysis of Venus–wt hKid or Venus–ΔNLSs hKid on chromosomes. The mean intensity of a bleached chromosomal spot (a), a nonbleached chromosomal spot (b, control), and a nonchromosomal spot (c, background) were measured over time (micrographs). All FRAP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from six to nine cells are plotted. The diagram below depicts our experimental design. All possibilities for the replacement and turnover of hKid between each compartment are indicated by arrows. Bar, 10 μm. (C) FLIP analysis of Venus–wt hKid or Venus–ΔNLSs hKid in the presence (−nocodazole) or absence (+nocodazole) of a mitotic spindle. A spot outside the chromosomes and mitotic spindle (a) was irradiated, and the mean intensities of a chromosome spot (b) and a spot outside the cells (c, background) were measured before and during irradiation (micrographs). All FLIP measurements were plotted as a function of time (graphs). The mean values and their standard deviations from 37–42 cells are plotted. The diagrams below depict our experimental design. Arrows indicate putative replacements and the turnover of hKid between each compartment. Each t1/2 value was calculated from the best-fit curve as described in Materials and methods. Bar, 10 μm.
Mentions: To determine whether mitotic function of hKid is regulated by nucleocytoplasmic transport factors, we examined the contribution of each NLS to the mitotic behavior of hKid in living cells. For this purpose, cell lines stably expressing fluorescently tagged wild-type (wt) hKid (Venus–wt hKid) or mutant hKid defective in both its NLSs (Venus–ΔNLSs hKid) were established. Because the overexpression of hKid is highly cytotoxic and induces mitotic abnormalities (Ohsugi et al., 2003; this study), an Flp–FRT recombination system was used that allows for the selection of cells carrying only one or a few copies of an objective gene integrated into defined genomic loci (Yahata et al., 2005; see Materials and methods). The cell lines expressed Venus–wt hKid and –ΔNLSs hKid at a level below or nearly equal to that of endogenous hKid (Fig. 2 B), respectively, without affecting the cell cycle/mitotic profile. We were unable to establish lines expressing higher levels of Venus–wt hKid, probably because overexpression of wt hKid is more toxic for cells than overexpression of mutant hKid defective in its NLSs.

Bottom Line: Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells.Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes.Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, Institute of Physical and Chemical Research, Wako, Saitama, 351-0198, Japan.

ABSTRACT
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

Show MeSH
Related in: MedlinePlus