Limits...
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.

Show MeSH

Related in: MedlinePlus

A BAC transgene containing mTPX2-LAP rescues the phenotype of TPX2-RNAi. (a) Western blot of cell lines with anti-mTPX2 antibody, which recognizes both human TPX2 and mTPX2-LAP, and anti–α-tubulin, after TPX2 or CON RNAi. hTPX2 is efficiently depleted in all lines after TPX2 RNAi, whereas mTPX2-LAP transgenes remain. The asterisk represents the position of the TPX2ΔN protein, which runs faster than the full length, as expected. (b) Immunofluorescence of U2OS cells after CON or TPX2 RNAi and TPX2WT cells after TPX2 RNAi, stained for α-tubulin (green), Cep135 (red), and DNA (blue). U2OS cells without a rescuing transgene show a characteristic phenotype after hTPX2 depletion of collapsed poles and lack of a bipolar spindle, whereas TPX2WT cells containing mTPX2-LAP after hTPX2 depletion have normal spindle morphology. Bar,10 μm. (c) Quantification of the TPX2 depletion phenotype and mTPX2-LAP rescue displayed in b. The percent of mitotic cells (n = >1,000 cells per experiment; three to five experiments for each condition) and percent bipolar spindles (n > 50) mitotic cells per experiment; three to five experiments per condition) after CON or TPX2 RNAi in U2OS or TPX2WT cells were determined. Error bars represent standard deviation. The mTPX2-LAP construct is able to rescue depletion of endogenous TPX2.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: A BAC transgene containing mTPX2-LAP rescues the phenotype of TPX2-RNAi. (a) Western blot of cell lines with anti-mTPX2 antibody, which recognizes both human TPX2 and mTPX2-LAP, and anti–α-tubulin, after TPX2 or CON RNAi. hTPX2 is efficiently depleted in all lines after TPX2 RNAi, whereas mTPX2-LAP transgenes remain. The asterisk represents the position of the TPX2ΔN protein, which runs faster than the full length, as expected. (b) Immunofluorescence of U2OS cells after CON or TPX2 RNAi and TPX2WT cells after TPX2 RNAi, stained for α-tubulin (green), Cep135 (red), and DNA (blue). U2OS cells without a rescuing transgene show a characteristic phenotype after hTPX2 depletion of collapsed poles and lack of a bipolar spindle, whereas TPX2WT cells containing mTPX2-LAP after hTPX2 depletion have normal spindle morphology. Bar,10 μm. (c) Quantification of the TPX2 depletion phenotype and mTPX2-LAP rescue displayed in b. The percent of mitotic cells (n = >1,000 cells per experiment; three to five experiments for each condition) and percent bipolar spindles (n > 50) mitotic cells per experiment; three to five experiments per condition) after CON or TPX2 RNAi in U2OS or TPX2WT cells were determined. Error bars represent standard deviation. The mTPX2-LAP construct is able to rescue depletion of endogenous TPX2.

Mentions: To generate a TPX2 transgene with endogenous expression and regulation, we used a BAC containing the mouse TPX2 gene. The mouse gene is resistant to a siRNA directed against human TPX2 yet has 87% similarity to human TPX2 on the protein level. We generated stable clonal lines in human U2OS cells expressing mouse TPX2 protein tagged with a modified “LAP” construct (Cheeseman and Desai, 2005; Poser et al., 2008) that contained an EGFP tag (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). By generating an antibody to mouse TPX2, we showed that the mouse TPX2-LAP protein appears expressed at similar levels to the endogenous human TPX2 (Fig. 1 a). We call this construct mTPX2WT (see Table I).


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)

A BAC transgene containing mTPX2-LAP rescues the phenotype of TPX2-RNAi. (a) Western blot of cell lines with anti-mTPX2 antibody, which recognizes both human TPX2 and mTPX2-LAP, and anti–α-tubulin, after TPX2 or CON RNAi. hTPX2 is efficiently depleted in all lines after TPX2 RNAi, whereas mTPX2-LAP transgenes remain. The asterisk represents the position of the TPX2ΔN protein, which runs faster than the full length, as expected. (b) Immunofluorescence of U2OS cells after CON or TPX2 RNAi and TPX2WT cells after TPX2 RNAi, stained for α-tubulin (green), Cep135 (red), and DNA (blue). U2OS cells without a rescuing transgene show a characteristic phenotype after hTPX2 depletion of collapsed poles and lack of a bipolar spindle, whereas TPX2WT cells containing mTPX2-LAP after hTPX2 depletion have normal spindle morphology. Bar,10 μm. (c) Quantification of the TPX2 depletion phenotype and mTPX2-LAP rescue displayed in b. The percent of mitotic cells (n = >1,000 cells per experiment; three to five experiments for each condition) and percent bipolar spindles (n > 50) mitotic cells per experiment; three to five experiments per condition) after CON or TPX2 RNAi in U2OS or TPX2WT cells were determined. Error bars represent standard deviation. The mTPX2-LAP construct is able to rescue depletion of endogenous TPX2.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: A BAC transgene containing mTPX2-LAP rescues the phenotype of TPX2-RNAi. (a) Western blot of cell lines with anti-mTPX2 antibody, which recognizes both human TPX2 and mTPX2-LAP, and anti–α-tubulin, after TPX2 or CON RNAi. hTPX2 is efficiently depleted in all lines after TPX2 RNAi, whereas mTPX2-LAP transgenes remain. The asterisk represents the position of the TPX2ΔN protein, which runs faster than the full length, as expected. (b) Immunofluorescence of U2OS cells after CON or TPX2 RNAi and TPX2WT cells after TPX2 RNAi, stained for α-tubulin (green), Cep135 (red), and DNA (blue). U2OS cells without a rescuing transgene show a characteristic phenotype after hTPX2 depletion of collapsed poles and lack of a bipolar spindle, whereas TPX2WT cells containing mTPX2-LAP after hTPX2 depletion have normal spindle morphology. Bar,10 μm. (c) Quantification of the TPX2 depletion phenotype and mTPX2-LAP rescue displayed in b. The percent of mitotic cells (n = >1,000 cells per experiment; three to five experiments for each condition) and percent bipolar spindles (n > 50) mitotic cells per experiment; three to five experiments per condition) after CON or TPX2 RNAi in U2OS or TPX2WT cells were determined. Error bars represent standard deviation. The mTPX2-LAP construct is able to rescue depletion of endogenous TPX2.
Mentions: To generate a TPX2 transgene with endogenous expression and regulation, we used a BAC containing the mouse TPX2 gene. The mouse gene is resistant to a siRNA directed against human TPX2 yet has 87% similarity to human TPX2 on the protein level. We generated stable clonal lines in human U2OS cells expressing mouse TPX2 protein tagged with a modified “LAP” construct (Cheeseman and Desai, 2005; Poser et al., 2008) that contained an EGFP tag (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200802005/DC1). By generating an antibody to mouse TPX2, we showed that the mouse TPX2-LAP protein appears expressed at similar levels to the endogenous human TPX2 (Fig. 1 a). We call this construct mTPX2WT (see Table I).

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