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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 spindle pole separation and spindle length establishment. (a) Immunofluorescence of mTPX2WT, mTPX2AAA, and mTPX2ΔN mitotic cells after hTPX2(RNAi), stained for CREST, Cep135, DNA, and α-tubulin. Centrosomes are collapsed to chromatin when the TPX2–Aurora A interaction is abolished. Bar, 10 μm. (b) Centrosome-kinetochore distances are shorter in mTPX2AAA and mTPX2ΔN mutants. The centrosome-kinetochore distance from cells stained as in a was determined by measuring individual centrosome-kinetochore distances from metaphase cells in three dimensions (see Materials and methods). For each condition, measurements were taken from four independent cells (eight centrosomes). Red dots show the data points measured and the bars show the means. (c) In TPX2 mutants unable to bind or activate Aurora A, spindle poles collapse immediately after NEBD and only slightly elongate to form shorter metaphase spindles. Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 1-min intervals of mTPX2WT, mTPX2AAA, and mTPX2ΔN (mTPX2-GFP) cells after hTPX2(RNAi). Thick lines represent means and thin lines represent individual videos. (d) Representative still images from time-lapse recordings quantified in b.
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fig4: The TPX2–Aurora A interaction is required for spindle pole separation and spindle length establishment. (a) Immunofluorescence of mTPX2WT, mTPX2AAA, and mTPX2ΔN mitotic cells after hTPX2(RNAi), stained for CREST, Cep135, DNA, and α-tubulin. Centrosomes are collapsed to chromatin when the TPX2–Aurora A interaction is abolished. Bar, 10 μm. (b) Centrosome-kinetochore distances are shorter in mTPX2AAA and mTPX2ΔN mutants. The centrosome-kinetochore distance from cells stained as in a was determined by measuring individual centrosome-kinetochore distances from metaphase cells in three dimensions (see Materials and methods). For each condition, measurements were taken from four independent cells (eight centrosomes). Red dots show the data points measured and the bars show the means. (c) In TPX2 mutants unable to bind or activate Aurora A, spindle poles collapse immediately after NEBD and only slightly elongate to form shorter metaphase spindles. Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 1-min intervals of mTPX2WT, mTPX2AAA, and mTPX2ΔN (mTPX2-GFP) cells after hTPX2(RNAi). Thick lines represent means and thin lines represent individual videos. (d) Representative still images from time-lapse recordings quantified in b.

Mentions: Analysis of fixed cells after hTPX2(RNAi) in mTPX2AAA or mTPX2ΔN mutant cells showed that spindles were smaller than normal (Fig. 2, bottom left; and Figs. 3 c, and 4 a). To quantify the defect, cells were fixed and stained with DAPI as well as with antibodies against α-tubulin, Cep135 (to mark centrosomes), and CREST (to mark kinetochores). Centrosome-to-kinetochore distances were measured in three-dimensional space. Cells harboring TPX2 mutants had shorter mean centrosome-to-kinetochore distances compared with those of wild type (Fig. 4 b, 2.58 ± 0.48 μm for mTPX2AAA and 2.43 ± 0.62 μm for mTPX2ΔN, compared with 4.35 ± 0.52 μm in wild type).


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 spindle pole separation and spindle length establishment. (a) Immunofluorescence of mTPX2WT, mTPX2AAA, and mTPX2ΔN mitotic cells after hTPX2(RNAi), stained for CREST, Cep135, DNA, and α-tubulin. Centrosomes are collapsed to chromatin when the TPX2–Aurora A interaction is abolished. Bar, 10 μm. (b) Centrosome-kinetochore distances are shorter in mTPX2AAA and mTPX2ΔN mutants. The centrosome-kinetochore distance from cells stained as in a was determined by measuring individual centrosome-kinetochore distances from metaphase cells in three dimensions (see Materials and methods). For each condition, measurements were taken from four independent cells (eight centrosomes). Red dots show the data points measured and the bars show the means. (c) In TPX2 mutants unable to bind or activate Aurora A, spindle poles collapse immediately after NEBD and only slightly elongate to form shorter metaphase spindles. Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 1-min intervals of mTPX2WT, mTPX2AAA, and mTPX2ΔN (mTPX2-GFP) cells after hTPX2(RNAi). Thick lines represent means and thin lines represent individual videos. (d) Representative still images from time-lapse recordings quantified in b.
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fig4: The TPX2–Aurora A interaction is required for spindle pole separation and spindle length establishment. (a) Immunofluorescence of mTPX2WT, mTPX2AAA, and mTPX2ΔN mitotic cells after hTPX2(RNAi), stained for CREST, Cep135, DNA, and α-tubulin. Centrosomes are collapsed to chromatin when the TPX2–Aurora A interaction is abolished. Bar, 10 μm. (b) Centrosome-kinetochore distances are shorter in mTPX2AAA and mTPX2ΔN mutants. The centrosome-kinetochore distance from cells stained as in a was determined by measuring individual centrosome-kinetochore distances from metaphase cells in three dimensions (see Materials and methods). For each condition, measurements were taken from four independent cells (eight centrosomes). Red dots show the data points measured and the bars show the means. (c) In TPX2 mutants unable to bind or activate Aurora A, spindle poles collapse immediately after NEBD and only slightly elongate to form shorter metaphase spindles. Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 1-min intervals of mTPX2WT, mTPX2AAA, and mTPX2ΔN (mTPX2-GFP) cells after hTPX2(RNAi). Thick lines represent means and thin lines represent individual videos. (d) Representative still images from time-lapse recordings quantified in b.
Mentions: Analysis of fixed cells after hTPX2(RNAi) in mTPX2AAA or mTPX2ΔN mutant cells showed that spindles were smaller than normal (Fig. 2, bottom left; and Figs. 3 c, and 4 a). To quantify the defect, cells were fixed and stained with DAPI as well as with antibodies against α-tubulin, Cep135 (to mark centrosomes), and CREST (to mark kinetochores). Centrosome-to-kinetochore distances were measured in three-dimensional space. Cells harboring TPX2 mutants had shorter mean centrosome-to-kinetochore distances compared with those of wild type (Fig. 4 b, 2.58 ± 0.48 μm for mTPX2AAA and 2.43 ± 0.62 μm for mTPX2ΔN, compared with 4.35 ± 0.52 μm in wild type).

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