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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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Depletion of TPX2 from cells results in a spindle defect phenotype distinct from TPX2 mutants or Aurora A depletion. (a) Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 3-min intervals of U2OS cells stably transfected with α-tubulin–mCherry after treatment with CON, TPX2, or AurA siRNA. Thick lines represent means and thin lines represent individual videos. The dashed line represents data from Fig. 4 for reference. (b) Representative still images from time-lapse recordings quantified in a. The cells shown transfected with CON or AurA siRNAs completed mitosis and divided, whereas the cell depleted of TPX2 arrested in mitosis for several hours and displayed fragmented spindle poles. Bar, 10 μm.
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fig6: Depletion of TPX2 from cells results in a spindle defect phenotype distinct from TPX2 mutants or Aurora A depletion. (a) Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 3-min intervals of U2OS cells stably transfected with α-tubulin–mCherry after treatment with CON, TPX2, or AurA siRNA. Thick lines represent means and thin lines represent individual videos. The dashed line represents data from Fig. 4 for reference. (b) Representative still images from time-lapse recordings quantified in a. The cells shown transfected with CON or AurA siRNAs completed mitosis and divided, whereas the cell depleted of TPX2 arrested in mitosis for several hours and displayed fragmented spindle poles. Bar, 10 μm.

Mentions: Although spindles in mTPX2AAA or mTPX2ΔN mutant cells were shorter, they were still bipolar, satisfied the Mad2 checkpoint, and exited mitosis. Longer exposure to RNAi or use of more concentrated siRNA did not result in failure by these cells to form bipolar spindles (unpublished data). This is in contrast to cells depleted of TPX2, which arrest in mitosis, as well as cells depleted of Aurora A, whose phenotypes vary from failure to enter mitosis to defects in cytokinesis (for review see Barr and Gergely, 2007). To compare spindle dynamics of the TPX2 Aurora A binding mutants to that of wild-type cells depleted of TPX2 or Aurora A, we imaged cells containing a fluorescent α-tubulin reporter after RNAi (Videos S4–S6, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). Beginning ∼16 h after TPX2 RNAi, cells entering mitosis generated normal prophase asters, but at NEBD these asters collapsed together, which is similar to the mTPX2ΔN and mTPX2AAA mutants (Fig. 6, a and b; and Video S5). In contrast to the TPX2 mutants, however, cells depleted of TPX2 where unable to separate spindle poles or establish a DNA metaphase plate and the spindles remained collapsed for several hours, after which the spindle poles fragmented and the cells either exited mitosis without chromosome segregation or apoptosed. We found that cells depleted of Aurora A, although also displaying a collapse of spindle poles after NEBD (Fig. 6, a and b; and Video S6), were often able to generate small bipolar spindles and divide and with similar mean pole separation through mitosis as the mTPX2ΔN and mTPX2AAA mutants. Some Aurora A RNAi cells displayed additional defects such as centrosome fragmentation and cytokinesis defects (unpublished data). Thus, it appears that TPX2 has a second Aurora A–independent role in organizing the spindle microtubules into a bipolar array, as cells depleted of Aurora A, although having multiple defects in mitosis, are more often able to assemble bipolar spindles and divide than TPX2 depleted cells.


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)

Depletion of TPX2 from cells results in a spindle defect phenotype distinct from TPX2 mutants or Aurora A depletion. (a) Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 3-min intervals of U2OS cells stably transfected with α-tubulin–mCherry after treatment with CON, TPX2, or AurA siRNA. Thick lines represent means and thin lines represent individual videos. The dashed line represents data from Fig. 4 for reference. (b) Representative still images from time-lapse recordings quantified in a. The cells shown transfected with CON or AurA siRNAs completed mitosis and divided, whereas the cell depleted of TPX2 arrested in mitosis for several hours and displayed fragmented spindle poles. Bar, 10 μm.
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fig6: Depletion of TPX2 from cells results in a spindle defect phenotype distinct from TPX2 mutants or Aurora A depletion. (a) Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 3-min intervals of U2OS cells stably transfected with α-tubulin–mCherry after treatment with CON, TPX2, or AurA siRNA. Thick lines represent means and thin lines represent individual videos. The dashed line represents data from Fig. 4 for reference. (b) Representative still images from time-lapse recordings quantified in a. The cells shown transfected with CON or AurA siRNAs completed mitosis and divided, whereas the cell depleted of TPX2 arrested in mitosis for several hours and displayed fragmented spindle poles. Bar, 10 μm.
Mentions: Although spindles in mTPX2AAA or mTPX2ΔN mutant cells were shorter, they were still bipolar, satisfied the Mad2 checkpoint, and exited mitosis. Longer exposure to RNAi or use of more concentrated siRNA did not result in failure by these cells to form bipolar spindles (unpublished data). This is in contrast to cells depleted of TPX2, which arrest in mitosis, as well as cells depleted of Aurora A, whose phenotypes vary from failure to enter mitosis to defects in cytokinesis (for review see Barr and Gergely, 2007). To compare spindle dynamics of the TPX2 Aurora A binding mutants to that of wild-type cells depleted of TPX2 or Aurora A, we imaged cells containing a fluorescent α-tubulin reporter after RNAi (Videos S4–S6, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). Beginning ∼16 h after TPX2 RNAi, cells entering mitosis generated normal prophase asters, but at NEBD these asters collapsed together, which is similar to the mTPX2ΔN and mTPX2AAA mutants (Fig. 6, a and b; and Video S5). In contrast to the TPX2 mutants, however, cells depleted of TPX2 where unable to separate spindle poles or establish a DNA metaphase plate and the spindles remained collapsed for several hours, after which the spindle poles fragmented and the cells either exited mitosis without chromosome segregation or apoptosed. We found that cells depleted of Aurora A, although also displaying a collapse of spindle poles after NEBD (Fig. 6, a and b; and Video S6), were often able to generate small bipolar spindles and divide and with similar mean pole separation through mitosis as the mTPX2ΔN and mTPX2AAA mutants. Some Aurora A RNAi cells displayed additional defects such as centrosome fragmentation and cytokinesis defects (unpublished data). Thus, it appears that TPX2 has a second Aurora A–independent role in organizing the spindle microtubules into a bipolar array, as cells depleted of Aurora A, although having multiple defects in mitosis, are more often able to assemble bipolar spindles and divide than TPX2 depleted cells.

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