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Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A.

Bird AW, Hyman AA - J. Cell Biol. (2008)

Bottom Line: Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes.Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present.A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany. bird@mpi-cbg.de

ABSTRACT
To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

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TPX2 localizes to kinetochore fibers independent of interaction with Aurora A. Immunofluorescence analysis of TPX2 localization. (a) U2OS cells in metaphase (top row) and anaphase (bottom row) stained for DNA, CREST, hTPX2 (green), and α-tubulin (black and white). Insets are enlarged to show TPX2 localization to microtubules that end at kinetochores (kinetochore fibers). (b) mTPX2WT cells after hTPX2(RNAi) with same staining as in a, except with mTPX2 instead of hTPX2 to recognize the transgene. (c) mTPX2AAA cells after hTPX2(RNAi) with same staining as in b. mTPX2 mutated to abolish interaction with Aurora A is still able to localize to kinetochore-fibers. Bar, 10 μm.
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fig3: TPX2 localizes to kinetochore fibers independent of interaction with Aurora A. Immunofluorescence analysis of TPX2 localization. (a) U2OS cells in metaphase (top row) and anaphase (bottom row) stained for DNA, CREST, hTPX2 (green), and α-tubulin (black and white). Insets are enlarged to show TPX2 localization to microtubules that end at kinetochores (kinetochore fibers). (b) mTPX2WT cells after hTPX2(RNAi) with same staining as in a, except with mTPX2 instead of hTPX2 to recognize the transgene. (c) mTPX2AAA cells after hTPX2(RNAi) with same staining as in b. mTPX2 mutated to abolish interaction with Aurora A is still able to localize to kinetochore-fibers. Bar, 10 μm.

Mentions: We next asked whether the interaction with Aurora A is required for TPX2 interaction with spindles. Time-lapse videos showed that mTPX2WT was recruited to asters immediately after NEBD and spread onto the spindle as it formed. (Video S1, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). Analysis of fixed samples immunostained for human TPX2 or mouse TPX2 in metaphase and anaphase showed that TPX2 localizes preferentially to kinetochore fibers (Fig. 3). Indeed, cells with anaphase lagging chromosomes maintained TPX2 staining along microtubules connecting these lagging chromosomes but not on all microtubules (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). mTPX2WT cells, as well as both the mutant mTPX2AAA and mTPX2ΔN cells, after hTPX2(RNAi) displayed the same dynamic localization of TPX2-LAP through mitosis (Videos S2 and S3) and also retained kinetochore fiber–specific localization upon immunofluorescence with anti–mouse TPX2 antibodies (Fig. 3). Thus, we show that TPX2 localizes to kinetochore fibers and that the interaction between TPX2 and Aurora A is not required for this localization.


Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A.

Bird AW, Hyman AA - J. Cell Biol. (2008)

TPX2 localizes to kinetochore fibers independent of interaction with Aurora A. Immunofluorescence analysis of TPX2 localization. (a) U2OS cells in metaphase (top row) and anaphase (bottom row) stained for DNA, CREST, hTPX2 (green), and α-tubulin (black and white). Insets are enlarged to show TPX2 localization to microtubules that end at kinetochores (kinetochore fibers). (b) mTPX2WT cells after hTPX2(RNAi) with same staining as in a, except with mTPX2 instead of hTPX2 to recognize the transgene. (c) mTPX2AAA cells after hTPX2(RNAi) with same staining as in b. mTPX2 mutated to abolish interaction with Aurora A is still able to localize to kinetochore-fibers. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483532&req=5

fig3: TPX2 localizes to kinetochore fibers independent of interaction with Aurora A. Immunofluorescence analysis of TPX2 localization. (a) U2OS cells in metaphase (top row) and anaphase (bottom row) stained for DNA, CREST, hTPX2 (green), and α-tubulin (black and white). Insets are enlarged to show TPX2 localization to microtubules that end at kinetochores (kinetochore fibers). (b) mTPX2WT cells after hTPX2(RNAi) with same staining as in a, except with mTPX2 instead of hTPX2 to recognize the transgene. (c) mTPX2AAA cells after hTPX2(RNAi) with same staining as in b. mTPX2 mutated to abolish interaction with Aurora A is still able to localize to kinetochore-fibers. Bar, 10 μm.
Mentions: We next asked whether the interaction with Aurora A is required for TPX2 interaction with spindles. Time-lapse videos showed that mTPX2WT was recruited to asters immediately after NEBD and spread onto the spindle as it formed. (Video S1, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). Analysis of fixed samples immunostained for human TPX2 or mouse TPX2 in metaphase and anaphase showed that TPX2 localizes preferentially to kinetochore fibers (Fig. 3). Indeed, cells with anaphase lagging chromosomes maintained TPX2 staining along microtubules connecting these lagging chromosomes but not on all microtubules (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). mTPX2WT cells, as well as both the mutant mTPX2AAA and mTPX2ΔN cells, after hTPX2(RNAi) displayed the same dynamic localization of TPX2-LAP through mitosis (Videos S2 and S3) and also retained kinetochore fiber–specific localization upon immunofluorescence with anti–mouse TPX2 antibodies (Fig. 3). Thus, we show that TPX2 localizes to kinetochore fibers and that the interaction between TPX2 and Aurora A is not required for this localization.

Bottom Line: Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes.Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present.A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany. bird@mpi-cbg.de

ABSTRACT
To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

Show MeSH
Related in: MedlinePlus