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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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The TPX2–Aurora A interaction is required for chromatin/kinetochore-mediated microtubule nucleation. U2OS, mTPX2WT, mTPX2AAA, and mTPX2ΔN cells transfected with TPX2 or CON siRNA were treated on ice to completely depolymerize microtubules, transferred to 37°C for 90 s to allow repolymerization, and immediately fixed. Immunofluorescence images are shown of cells stained with α-tubulin and CREST (top rows) and Cep135 and DNA (bottom rows). In addition to aster microtubule polymerization associated with centrosomes, microtubule polymerization broadly associated with chromatin and often specifically associated with kinetochores was evident in all cell lines after CON siRNA and in mTPX2WT cells after TPX2 RNAi. In U2OS, mTPX2AAA, and mTPX2ΔN cells after TPX2 RNAi, microtubule repolymerization was no longer seen associated with chromatin or kinetochores, but centrosome-associated microtubule aster polymerization was still evident. Bar, 10 μm.
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fig8: The TPX2–Aurora A interaction is required for chromatin/kinetochore-mediated microtubule nucleation. U2OS, mTPX2WT, mTPX2AAA, and mTPX2ΔN cells transfected with TPX2 or CON siRNA were treated on ice to completely depolymerize microtubules, transferred to 37°C for 90 s to allow repolymerization, and immediately fixed. Immunofluorescence images are shown of cells stained with α-tubulin and CREST (top rows) and Cep135 and DNA (bottom rows). In addition to aster microtubule polymerization associated with centrosomes, microtubule polymerization broadly associated with chromatin and often specifically associated with kinetochores was evident in all cell lines after CON siRNA and in mTPX2WT cells after TPX2 RNAi. In U2OS, mTPX2AAA, and mTPX2ΔN cells after TPX2 RNAi, microtubule repolymerization was no longer seen associated with chromatin or kinetochores, but centrosome-associated microtubule aster polymerization was still evident. Bar, 10 μm.

Mentions: The microtubules that contribute to the forming prometaphase spindle have been proposed to originate from both centrosomes and chromosomes. TPX2 has previously been shown to be required for kinetochore/chromatin-dependent microtubule nucleation in human cells (Tulu et al., 2006). To test whether TPX2-dependent Aurora A activation was required for kinetochore/chromatin nucleation, we used a microtubule repolymerization assay to distinguish nucleation of microtubules from centrosomes and chromatin. In this assay, U2OS cells were transferred to an ice bath for 30 min to completely depolymerize microtubules. Cells were then transferred in brief to 37°C media to allow for repolymerization and immediately fixed and stained for tubulin, DNA, Cep135, and CREST. After 90 s of incubation at 37°C, wild-type U2OS cells displayed characteristic microtubule polymerization from centrosomes, as well as polymerization clearly associated with chromatin and kinetochores and away from centrosomes (Fig. 8). After hTPX2(RNAi) in U2OS cells, microtubule polymerization around chromatin and kinetochores was abolished, whereas centrosome microtubule nucleation remained, confirming previous observations (Tulu et al., 2006). After hTPX2(RNAi) in mTPX2WT cells, microtubule polymerization was again evident around both centrosomes and chromatin/kinetochores in a majority of cells. mTPX2 protein also colocalized with both microtubule populations (unpublished data). In contrast, polymerization was rarely seen around chromatin and kinetochores after hTPX2(RNAi) in mTPX2ΔN or mTPX2AAA mutant cells (Fig. 8). Thus, the TPX2–Aurora A interaction is required for chromatin/kinetochore-dependent microtubule nucleation.


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)

The TPX2–Aurora A interaction is required for chromatin/kinetochore-mediated microtubule nucleation. U2OS, mTPX2WT, mTPX2AAA, and mTPX2ΔN cells transfected with TPX2 or CON siRNA were treated on ice to completely depolymerize microtubules, transferred to 37°C for 90 s to allow repolymerization, and immediately fixed. Immunofluorescence images are shown of cells stained with α-tubulin and CREST (top rows) and Cep135 and DNA (bottom rows). In addition to aster microtubule polymerization associated with centrosomes, microtubule polymerization broadly associated with chromatin and often specifically associated with kinetochores was evident in all cell lines after CON siRNA and in mTPX2WT cells after TPX2 RNAi. In U2OS, mTPX2AAA, and mTPX2ΔN cells after TPX2 RNAi, microtubule repolymerization was no longer seen associated with chromatin or kinetochores, but centrosome-associated microtubule aster polymerization was still evident. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: The TPX2–Aurora A interaction is required for chromatin/kinetochore-mediated microtubule nucleation. U2OS, mTPX2WT, mTPX2AAA, and mTPX2ΔN cells transfected with TPX2 or CON siRNA were treated on ice to completely depolymerize microtubules, transferred to 37°C for 90 s to allow repolymerization, and immediately fixed. Immunofluorescence images are shown of cells stained with α-tubulin and CREST (top rows) and Cep135 and DNA (bottom rows). In addition to aster microtubule polymerization associated with centrosomes, microtubule polymerization broadly associated with chromatin and often specifically associated with kinetochores was evident in all cell lines after CON siRNA and in mTPX2WT cells after TPX2 RNAi. In U2OS, mTPX2AAA, and mTPX2ΔN cells after TPX2 RNAi, microtubule repolymerization was no longer seen associated with chromatin or kinetochores, but centrosome-associated microtubule aster polymerization was still evident. Bar, 10 μm.
Mentions: The microtubules that contribute to the forming prometaphase spindle have been proposed to originate from both centrosomes and chromosomes. TPX2 has previously been shown to be required for kinetochore/chromatin-dependent microtubule nucleation in human cells (Tulu et al., 2006). To test whether TPX2-dependent Aurora A activation was required for kinetochore/chromatin nucleation, we used a microtubule repolymerization assay to distinguish nucleation of microtubules from centrosomes and chromatin. In this assay, U2OS cells were transferred to an ice bath for 30 min to completely depolymerize microtubules. Cells were then transferred in brief to 37°C media to allow for repolymerization and immediately fixed and stained for tubulin, DNA, Cep135, and CREST. After 90 s of incubation at 37°C, wild-type U2OS cells displayed characteristic microtubule polymerization from centrosomes, as well as polymerization clearly associated with chromatin and kinetochores and away from centrosomes (Fig. 8). After hTPX2(RNAi) in U2OS cells, microtubule polymerization around chromatin and kinetochores was abolished, whereas centrosome microtubule nucleation remained, confirming previous observations (Tulu et al., 2006). After hTPX2(RNAi) in mTPX2WT cells, microtubule polymerization was again evident around both centrosomes and chromatin/kinetochores in a majority of cells. mTPX2 protein also colocalized with both microtubule populations (unpublished data). In contrast, polymerization was rarely seen around chromatin and kinetochores after hTPX2(RNAi) in mTPX2ΔN or mTPX2AAA mutant cells (Fig. 8). Thus, the TPX2–Aurora A interaction is required for chromatin/kinetochore-dependent microtubule nucleation.

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