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

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.

Figure 1.

Movement of PA-GFP-TPX2 in the mitotic spindle. (A) Photoactivation of PA-GFP-TPX2 in LLC-Pk1 cells. Top row, phase contrast images of each cell before photoactivation; bottom rows, fluorescence images of PA-GFP-TPX2 for each cell. Time after photoactivation is shown in bottom right. Asterisks in the anaphase cell show the constant position of the spindle pole. (B) Kymographs of photoactivated marks from the cells shown in A. (C) Average rates of poleward motion of TPX2 near the equator and pole. (D) Average rates of poleward motion of TPX2 and PA-GFP-tubulin. For PA-GFP-tubulin, 14, 15, 9, and 10 cells were used to analyze poleward motion during prometaphase, metaphase, anaphase, and telophase, respectively. *p < 0.01 and **p < 0.05. Bar, 10 μm.

Figure 2.

Poleward motion of TPX2 is sensitive to inhibition of microtubule flux. (A) Images of PA-GFP-TPX2-LLC-Pk1 cells treated with 10 nM nocodazole (top) or microinjected with anti-Kif2a and MCAK antibodies (bottom) and photoactivated. 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 for control, nocodazole treated and anti-Kif2a/MCAK injected cells. Bars, 10 μm. *p < 0.01 and **p < 0.05. (C) Average rates of microtubule poleward motion (cells expressing PA-GFP-tubulin; n = 11) and TPX2 motion (cells expressing PA-GFP-TPX2; n = 11) in cells treated with low nocodazole. **p < 0.05.

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.

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.

Figure 5.

Organization of kinetochore fiber microtubules requires the C terminus of TPX2. (A) LLC-Pk1-α cells expressing full-length (top) or mCherryTPX2–710 (bottom); merged images on the right. (B) Quantification of spindle phenotypes. Values represent the mean of three independent experiments ± SD; at least 100 cells were counted for each experiment. (C) Spindle morphology in control, untransfected cells (top), and cells transfected with full-length (middle), or TPX2-710 (bottom). Panels show DNA, Hec1/mCherryTPX2, microtubules, and merged. Insets show higher magnification of microtubule bundles and Hec1 staining. (D) Microtubules in control (top) and cells transfected withTPX2 (middle) or TPX2-710 after treatment at 4°C. (E) Interkinetochore tension in nocodazole treated cells, nonaligned and aligned chromosomes in control untransfected cells, aligned chromosomes in cells expressing full-length mCherryTPX2, and aligned and nonaligned chromosomes in cells expressing TPX2-710. *p < 0.01.

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).