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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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Expression of mutant Ran defective in the GTPase cycle affects the mitotic localization of endogenous hKid in living cells. (A) Synchronized HeLa cells were transfected with expression vectors encoding wt Ran, Q69L Ran, or T24N Ran as described in Materials and methods. 10 h after their release from a second thymidine block, the cells were fixed and stained for endogenous hKid and Ran. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm. (B) Quantification of the results in A. Cells expressing the indicated Ran proteins were fixed and stained. The fluorescent intensity of hKid on the chromosomes (1) and centrosomes (2) was measured using SoftWorx software and their ratios were plotted. The values are expressed as the mean ± standard deviation. Significant differences were detected using t test. *, P < 0.05. Bar, 10 μm.
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fig7: Expression of mutant Ran defective in the GTPase cycle affects the mitotic localization of endogenous hKid in living cells. (A) Synchronized HeLa cells were transfected with expression vectors encoding wt Ran, Q69L Ran, or T24N Ran as described in Materials and methods. 10 h after their release from a second thymidine block, the cells were fixed and stained for endogenous hKid and Ran. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm. (B) Quantification of the results in A. Cells expressing the indicated Ran proteins were fixed and stained. The fluorescent intensity of hKid on the chromosomes (1) and centrosomes (2) was measured using SoftWorx software and their ratios were plotted. The values are expressed as the mean ± standard deviation. Significant differences were detected using t test. *, P < 0.05. Bar, 10 μm.

Mentions: As shown in Fig. 2 A, both Venus–wt hKid and –ΔNLSs hKid were localized predominantly to the mitotic chromosomes and also to the spindle at lower levels, similar to those of endogenous hKid (see Fig. 7). The spindle signal for Venus–ΔNLSs hKid was stronger than that for Venus–wt hKid (Fig. 2 A). The dynamics of these two proteins were next examined by FRAP within a small region of the mitotic chromosomes (Fig. 2 B and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708003/DC1). Chromosome-bound hKid was highly dynamic during prometaphase and metaphase, with the recovery of fluorescence occurring within seconds (Fig. 2 B, left graph). Meanwhile, the mobility of chromosome-bound hKid was greatly reduced during anaphase, as the recovery of the fluorescence required nearly 1 min (Fig. 2 B, right graph). Both in metaphase and anaphase, the recovery of Venus–ΔNLSs hKid was quicker and more efficient than that of Venus–wt hKid, indicating that the turnover, as well as the mobile fraction, of chromosome-bound hKid increases upon the loss of its functional NLSs. Quantitatively, Venus–wt hKid displayed a t1/2 of 2.4 s with 63% recovery in metaphase and a t1/2 of 39 s with 61% recovery in anaphase, whereas Venus–ΔNLSs hKid displayed a t1/2 of 2.0 s with 83% recovery in metaphase and a t1/2 of 22 s with 83% recovery in anaphase (Fig. 2 B and Fig. S2). At present, we do not know exactly how hKid moves in mitotic cells although, conceptually, the hKid in the bleached area could be replaced laterally by hKid from a nonbleached chromosomal area, directly by hKid from the cytosolic pool, or by hKid from the mitotic spindle (Fig. 2 B, diagram).


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)

Expression of mutant Ran defective in the GTPase cycle affects the mitotic localization of endogenous hKid in living cells. (A) Synchronized HeLa cells were transfected with expression vectors encoding wt Ran, Q69L Ran, or T24N Ran as described in Materials and methods. 10 h after their release from a second thymidine block, the cells were fixed and stained for endogenous hKid and Ran. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm. (B) Quantification of the results in A. Cells expressing the indicated Ran proteins were fixed and stained. The fluorescent intensity of hKid on the chromosomes (1) and centrosomes (2) was measured using SoftWorx software and their ratios were plotted. The values are expressed as the mean ± standard deviation. Significant differences were detected using t test. *, P < 0.05. Bar, 10 μm.
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fig7: Expression of mutant Ran defective in the GTPase cycle affects the mitotic localization of endogenous hKid in living cells. (A) Synchronized HeLa cells were transfected with expression vectors encoding wt Ran, Q69L Ran, or T24N Ran as described in Materials and methods. 10 h after their release from a second thymidine block, the cells were fixed and stained for endogenous hKid and Ran. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm. (B) Quantification of the results in A. Cells expressing the indicated Ran proteins were fixed and stained. The fluorescent intensity of hKid on the chromosomes (1) and centrosomes (2) was measured using SoftWorx software and their ratios were plotted. The values are expressed as the mean ± standard deviation. Significant differences were detected using t test. *, P < 0.05. Bar, 10 μm.
Mentions: As shown in Fig. 2 A, both Venus–wt hKid and –ΔNLSs hKid were localized predominantly to the mitotic chromosomes and also to the spindle at lower levels, similar to those of endogenous hKid (see Fig. 7). The spindle signal for Venus–ΔNLSs hKid was stronger than that for Venus–wt hKid (Fig. 2 A). The dynamics of these two proteins were next examined by FRAP within a small region of the mitotic chromosomes (Fig. 2 B and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708003/DC1). Chromosome-bound hKid was highly dynamic during prometaphase and metaphase, with the recovery of fluorescence occurring within seconds (Fig. 2 B, left graph). Meanwhile, the mobility of chromosome-bound hKid was greatly reduced during anaphase, as the recovery of the fluorescence required nearly 1 min (Fig. 2 B, right graph). Both in metaphase and anaphase, the recovery of Venus–ΔNLSs hKid was quicker and more efficient than that of Venus–wt hKid, indicating that the turnover, as well as the mobile fraction, of chromosome-bound hKid increases upon the loss of its functional NLSs. Quantitatively, Venus–wt hKid displayed a t1/2 of 2.4 s with 63% recovery in metaphase and a t1/2 of 39 s with 61% recovery in anaphase, whereas Venus–ΔNLSs hKid displayed a t1/2 of 2.0 s with 83% recovery in metaphase and a t1/2 of 22 s with 83% recovery in anaphase (Fig. 2 B and Fig. S2). At present, we do not know exactly how hKid moves in mitotic cells although, conceptually, the hKid in the bleached area could be replaced laterally by hKid from a nonbleached chromosomal area, directly by hKid from the cytosolic pool, or by hKid from the mitotic spindle (Fig. 2 B, diagram).

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