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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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Poleward motion of TPX2 is sensitive to inhibition of dynein. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells microinjected with CC1 before photoactivation. Left image is phase contrast, center images show selected images after photoactivation, and far right image shows maximal intensity projection after activation of the entire field of view. Yellow line is at a fixed position. (B) Average rates of poleward motion of TPX2 in control and CC1-injected prometaphase and metaphase cells. *p < 0.01 and **p < 0.05. (C) Distribution of TPX2 and microtubules in uninjected (top) and CC1-injected (bottom) cells. Bars, 10 μm.
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Figure 3: Poleward motion of TPX2 is sensitive to inhibition of dynein. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells microinjected with CC1 before photoactivation. Left image is phase contrast, center images show selected images after photoactivation, and far right image shows maximal intensity projection after activation of the entire field of view. Yellow line is at a fixed position. (B) Average rates of poleward motion of TPX2 in control and CC1-injected prometaphase and metaphase cells. *p < 0.01 and **p < 0.05. (C) Distribution of TPX2 and microtubules in uninjected (top) and CC1-injected (bottom) cells. Bars, 10 μm.

Mentions: TPX2 was originally identified as a factor that was required for the dynein-dependent localization of the kinesin Xklp2 to spindle poles (Wittmann et al., 2000), suggesting that dynein is required for TPX2 transport. To examine this possibility, cells were microinjected with the CC1 fragment of the p150 subunit of dynactin, a treatment that interferes with the dynein–dynactin interaction and blocks dynein-dependent processes in cells (Quintyne et al., 1999; Gaetz and Kapoor, 2004; Ferenz and Wadsworth, 2007). The results show that the rate of poleward motion of TPX2 in prometaphase and metaphase cells is reduced after microinjection of CC1 (Figure 3, A and B, and Supplemental Movie 3). Furthermore, TPX2 was more uniformly distributed along spindle fibers in CC1 injected compared with control cells (Figure 3C). Inhibition of dynein/dynactin by microinjection of CC1 resulted in accumulation of the p150 subunit of dynactin at kinetochores but did not alter the localization of Kif2a at spindle poles (Supplemental Figure 2, A and B), consistent with previous work (Howell et al., 2001; Ferenz and Wadsworth, 2007).


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)

Poleward motion of TPX2 is sensitive to inhibition of dynein. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells microinjected with CC1 before photoactivation. Left image is phase contrast, center images show selected images after photoactivation, and far right image shows maximal intensity projection after activation of the entire field of view. Yellow line is at a fixed position. (B) Average rates of poleward motion of TPX2 in control and CC1-injected prometaphase and metaphase cells. *p < 0.01 and **p < 0.05. (C) Distribution of TPX2 and microtubules in uninjected (top) and CC1-injected (bottom) cells. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Poleward motion of TPX2 is sensitive to inhibition of dynein. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells microinjected with CC1 before photoactivation. Left image is phase contrast, center images show selected images after photoactivation, and far right image shows maximal intensity projection after activation of the entire field of view. Yellow line is at a fixed position. (B) Average rates of poleward motion of TPX2 in control and CC1-injected prometaphase and metaphase cells. *p < 0.01 and **p < 0.05. (C) Distribution of TPX2 and microtubules in uninjected (top) and CC1-injected (bottom) cells. Bars, 10 μm.
Mentions: TPX2 was originally identified as a factor that was required for the dynein-dependent localization of the kinesin Xklp2 to spindle poles (Wittmann et al., 2000), suggesting that dynein is required for TPX2 transport. To examine this possibility, cells were microinjected with the CC1 fragment of the p150 subunit of dynactin, a treatment that interferes with the dynein–dynactin interaction and blocks dynein-dependent processes in cells (Quintyne et al., 1999; Gaetz and Kapoor, 2004; Ferenz and Wadsworth, 2007). The results show that the rate of poleward motion of TPX2 in prometaphase and metaphase cells is reduced after microinjection of CC1 (Figure 3, A and B, and Supplemental Movie 3). Furthermore, TPX2 was more uniformly distributed along spindle fibers in CC1 injected compared with control cells (Figure 3C). Inhibition of dynein/dynactin by microinjection of CC1 resulted in accumulation of the p150 subunit of dynactin at kinetochores but did not alter the localization of Kif2a at spindle poles (Supplemental Figure 2, A and B), consistent with previous work (Howell et al., 2001; Ferenz and Wadsworth, 2007).

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