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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 requires an interaction with Eg5. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells treated with monastrol (top) or with siRNA targeting Eg5 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) GST pull-down. Top, Coomassie Blue-stained gel (CBB); asterisks mark the major bands of protein in each lane. Bottom, Western blot probed with Eg5 antibodies; the supernatant and pelleted fractions of the pull-down are shown. (C) Average rates of motion for cells treated with the indicated inhibitors. *p < 0.01. (D) Photoactivation of PA-GFP-TPX2 (top) and PA-GFP-TPX2-710 (bottom); kymographs are on the right. Note that in the cell expressing PA-GFP-TPX2-710 fluorescence is distributed along the length of the microtubules within 1 min of photoactivation. Bars, 10 μm.
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Figure 4: Poleward motion of TPX2 requires an interaction with Eg5. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells treated with monastrol (top) or with siRNA targeting Eg5 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) GST pull-down. Top, Coomassie Blue-stained gel (CBB); asterisks mark the major bands of protein in each lane. Bottom, Western blot probed with Eg5 antibodies; the supernatant and pelleted fractions of the pull-down are shown. (C) Average rates of motion for cells treated with the indicated inhibitors. *p < 0.01. (D) Photoactivation of PA-GFP-TPX2 (top) and PA-GFP-TPX2-710 (bottom); kymographs are on the right. Note that in the cell expressing PA-GFP-TPX2-710 fluorescence is distributed along the length of the microtubules within 1 min of photoactivation. Bars, 10 μm.

Mentions: For glutathione transferase (GST) pull-down experiments, the N-terminal half of TPX2 (TNT, 1-364), the C-terminal half of TPX2 (TCT, 365-745), and TCT lacking the C-terminal 35 amino acids (TCTΔ35) were subcloned into pGEX-4T-1 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and purified from bacteria (BL-21). His-tagged Eg5 (amino acids 1-685, corresponding to the head-stalk region) was expressed in bacteria. GST pull-down was performed as described previously (Ma et al., 2009). In the experiment shown in Figure 4, 1/70 and one half the volume of supernatants and pellets were applied to the gel, respectively.


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 requires an interaction with Eg5. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells treated with monastrol (top) or with siRNA targeting Eg5 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) GST pull-down. Top, Coomassie Blue-stained gel (CBB); asterisks mark the major bands of protein in each lane. Bottom, Western blot probed with Eg5 antibodies; the supernatant and pelleted fractions of the pull-down are shown. (C) Average rates of motion for cells treated with the indicated inhibitors. *p < 0.01. (D) Photoactivation of PA-GFP-TPX2 (top) and PA-GFP-TPX2-710 (bottom); kymographs are on the right. Note that in the cell expressing PA-GFP-TPX2-710 fluorescence is distributed along the length of the microtubules within 1 min of photoactivation. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Poleward motion of TPX2 requires an interaction with Eg5. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells treated with monastrol (top) or with siRNA targeting Eg5 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) GST pull-down. Top, Coomassie Blue-stained gel (CBB); asterisks mark the major bands of protein in each lane. Bottom, Western blot probed with Eg5 antibodies; the supernatant and pelleted fractions of the pull-down are shown. (C) Average rates of motion for cells treated with the indicated inhibitors. *p < 0.01. (D) Photoactivation of PA-GFP-TPX2 (top) and PA-GFP-TPX2-710 (bottom); kymographs are on the right. Note that in the cell expressing PA-GFP-TPX2-710 fluorescence is distributed along the length of the microtubules within 1 min of photoactivation. Bars, 10 μm.
Mentions: For glutathione transferase (GST) pull-down experiments, the N-terminal half of TPX2 (TNT, 1-364), the C-terminal half of TPX2 (TCT, 365-745), and TCT lacking the C-terminal 35 amino acids (TCTΔ35) were subcloned into pGEX-4T-1 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and purified from bacteria (BL-21). His-tagged Eg5 (amino acids 1-685, corresponding to the head-stalk region) was expressed in bacteria. GST pull-down was performed as described previously (Ma et al., 2009). In the experiment shown in Figure 4, 1/70 and one half the volume of supernatants and pellets were applied to the gel, respectively.

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