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Poleward transport of TPX2 in the mammalian mitotic spindle requires dynein, Eg5, and microtubule flux.

Ma N, Tulu US, Ferenz NP, Fagerstrom C, Wilde A, Wadsworth P - Mol. Biol. Cell (2010)

Bottom Line: Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5.Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression.Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT
TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

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Microtubule bundle formation near chromatin in cells expressing TPX2 and TPX2-710. (A) Microtubule formation after release from nocodazole in live cells expressing GFP-tubulin. Selected frames from time-lapse sequences of control cells (top row) or cells transfected with full-length mCherry-TPX2 (middle row) or mCherry-TPX2-710 (bottom row). Time is in minutes:seconds after release from nocodazole. Arrowheads mark kinetochore-fiber-like bundles of microtubules. Bars, 10 μm. (B) Model for TPX2 behavior. Kinetochore fiber formation in control cells (a–c)and in cells overexpressing full-length TPX2 (d) or TPX2-710 (e).
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Figure 6: Microtubule bundle formation near chromatin in cells expressing TPX2 and TPX2-710. (A) Microtubule formation after release from nocodazole in live cells expressing GFP-tubulin. Selected frames from time-lapse sequences of control cells (top row) or cells transfected with full-length mCherry-TPX2 (middle row) or mCherry-TPX2-710 (bottom row). Time is in minutes:seconds after release from nocodazole. Arrowheads mark kinetochore-fiber-like bundles of microtubules. Bars, 10 μm. (B) Model for TPX2 behavior. Kinetochore fiber formation in control cells (a–c)and in cells overexpressing full-length TPX2 (d) or TPX2-710 (e).

Mentions: To determine whether the TPX2–Eg5 interaction impacts microtubule formation near chromosomes, we used a nocodazole washout assay (Tulu et al., 2006). When control LLC-Pk1 cells expressing GFP-tubulin are released from nocodazole, microtubules assemble near chromosomes and at centrosomes and subsequently coalesce to form a bipolar spindle (Figure 6A and Supplemental Movie 7). In contrast, when cells expressing full-length or TPX2-710 were released from nocodazole, microtubule formation in the chromosome region was greatly enhanced and coalescence of microtubule foci into a bipolar spindle failed (Figure 6A and Supplemental Movies 8 and 9). In some cases, discrete puncta of fluorescence were observed in the chromosome region, probably corresponding to microtubules forming at kinetochores (Figure 6A, bottom, 36 min). Because the level of overexpression of each protein varies in the transfected cells, we could not quantitatively compare the extent of microtubule formation in these experiments. However, although both full-length and truncated TPX2 promoted microtubule formation near chromosomes in this assay, only full-length TPX2 accumulated at the center of microtubule foci, whereas the truncated protein was mostly diffuse (Figure 6A, right), consistent with its behavior after photoactivation.


Poleward transport of TPX2 in the mammalian mitotic spindle requires dynein, Eg5, and microtubule flux.

Ma N, Tulu US, Ferenz NP, Fagerstrom C, Wilde A, Wadsworth P - Mol. Biol. Cell (2010)

Microtubule bundle formation near chromatin in cells expressing TPX2 and TPX2-710. (A) Microtubule formation after release from nocodazole in live cells expressing GFP-tubulin. Selected frames from time-lapse sequences of control cells (top row) or cells transfected with full-length mCherry-TPX2 (middle row) or mCherry-TPX2-710 (bottom row). Time is in minutes:seconds after release from nocodazole. Arrowheads mark kinetochore-fiber-like bundles of microtubules. Bars, 10 μm. (B) Model for TPX2 behavior. Kinetochore fiber formation in control cells (a–c)and in cells overexpressing full-length TPX2 (d) or TPX2-710 (e).
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Related In: Results  -  Collection

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Figure 6: Microtubule bundle formation near chromatin in cells expressing TPX2 and TPX2-710. (A) Microtubule formation after release from nocodazole in live cells expressing GFP-tubulin. Selected frames from time-lapse sequences of control cells (top row) or cells transfected with full-length mCherry-TPX2 (middle row) or mCherry-TPX2-710 (bottom row). Time is in minutes:seconds after release from nocodazole. Arrowheads mark kinetochore-fiber-like bundles of microtubules. Bars, 10 μm. (B) Model for TPX2 behavior. Kinetochore fiber formation in control cells (a–c)and in cells overexpressing full-length TPX2 (d) or TPX2-710 (e).
Mentions: To determine whether the TPX2–Eg5 interaction impacts microtubule formation near chromosomes, we used a nocodazole washout assay (Tulu et al., 2006). When control LLC-Pk1 cells expressing GFP-tubulin are released from nocodazole, microtubules assemble near chromosomes and at centrosomes and subsequently coalesce to form a bipolar spindle (Figure 6A and Supplemental Movie 7). In contrast, when cells expressing full-length or TPX2-710 were released from nocodazole, microtubule formation in the chromosome region was greatly enhanced and coalescence of microtubule foci into a bipolar spindle failed (Figure 6A and Supplemental Movies 8 and 9). In some cases, discrete puncta of fluorescence were observed in the chromosome region, probably corresponding to microtubules forming at kinetochores (Figure 6A, bottom, 36 min). Because the level of overexpression of each protein varies in the transfected cells, we could not quantitatively compare the extent of microtubule formation in these experiments. However, although both full-length and truncated TPX2 promoted microtubule formation near chromosomes in this assay, only full-length TPX2 accumulated at the center of microtubule foci, whereas the truncated protein was mostly diffuse (Figure 6A, right), consistent with its behavior after photoactivation.

Bottom Line: Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5.Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression.Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT
TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

Show MeSH
Related in: MedlinePlus