Regulation of Kif15 localization and motility by the C-terminus of TPX2 and microtubule dynamics

Abstract

Mitotic motor proteins generate force to establish and maintain spindle bipolarity, but how they are temporally and spatially regulated in vivo is unclear. Prior work demonstrated that a microtubule-associated protein, TPX2, targets kinesin-5 and kinesin-12 motors to spindle microtubules. The C-terminal domain of TPX2 contributes to the localization and motility of the kinesin-5, Eg5, but it is not known whether this domain regulates kinesin-12, Kif15. We found that the C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro. Kif15 and Eg5 are partially redundant motors, and overexpressed Kif15 can drive spindle formation in the absence of Eg5 activity. Kif15-dependent bipolar spindle formation in vivo requires the C-terminal domain of TPX2. In the spindle, fluorescent puncta of GFP-Kif15 move toward the equatorial region at a rate equivalent to microtubule growth. Reduction of microtubule growth with paclitaxel suppresses GFP-Kif15 motility, demonstrating that dynamic microtubules contribute to Kif15 behavior. Our results show that the C-terminal region of TPX2 regulates Kif15 in vitro, contributes to motor localization in cells, and is required for Kif15 force generation in vivo and further reveal that dynamic microtubules contribute to Kif15 behavior in vivo.

FIGURE 1:

The C-terminal region of TPX2 contributes to spindle localization of Kif15. (A) Immunofluorescence staining for microtubules (left) and Kif15 (right). Top, parental cells; the remaining three rows show cells depleted of TPX2 and expressing no transgene (parental), transgene encoding full-length TPX2 (middle), or TPX2-710 (bottom). Scale bar, 2 μm. (B) Spindle morphology for parental cells and cells expressing full-length or truncated TPX2; cells on the right were additionally treated with siRNA targeting TPX2. (C) Quantification of fluorescence ratio of Kif15 to tubulin at pole and in the half-spindle. Error bars are SD. Parental cells depleted of TPX2 were only measured at spindle pole due to loss of spindle microtubules.

FIGURE 2:

Inhibition of Kif15 motor stepping requires full-length TPX2. (A) Schematic diagram of experiment. (B) Histograms of GFP-Kif15 velocity (left) and run length (right) for plus end– and minus end–directed motion; n = 261 and 43 motors, respectively. Data from two independent experiments. (C) GFP-Kif15 switches microtubule tracks; arrow marks moving GFP-Kif15 puncta. Time in minutes:seconds. Bar, 1 μm. (D) Histogram of fluorescence intensity of kinesin-1–GFP (top) and GFP-Kif15 (bottom); fluorescence in arbitrary units (A.U.). For kinesin-1–GFP, n = 295, and for GFP-Kif15, n = 652, from two independent experiments. (E) Photobleaching of microtubule-bound GFP-Kif15 from interphase and mitotic extracts. Horizontal pink lines show bleach steps. For interphase, n = 11 particles, five with more than three steps and six with fewer than three steps; data from two independent experiments; for mitotic extracts, n = 15 particles, 10 with more than three steps and five with fewer than three steps. (F) Schematic diagram of constructs used for inhibition experiments (top) and bar graph (bottom) showing ratio of velocity without and with added proteins; error bars, SEM. (G) Kymographs showing motility of GFP-Kif15; added TPX2 construct indicated at the top; vertical axis marker bar, 15 s; horizontal axis marker bar, 1 μm.

FIGURE 3:

TPX2 is required for bipolar spindle formation in cells overexpressing Kif15. (A) LLC-Pk1 cells expressing GFP-Kif15 (left) and parental cells fixed and stained for Kif15 (right). (B) Western blot of extracts from parental and GFP-Kif15–expressing cells; blot stained for Kif15 (top) and tubulin as loading control (bottom). (C) Images of GFP-Kif15–expressing cells treated with siRNA targeting TPX2 (top) or Nuf2 (bottom); GFP-Kif15 (left) and co-nucleofected mCherry-H2B to label chromosomes (right). (D) Bar graphs showing percentage of bipolar, monopolar, and multipolar spindles for each treatment condition. Error bars show SD. (E) Parental cells treated with FCPT, with siRNA targeting TPX2, or with both. Cells were stained for microtubules (bottom) and either Kif15 or Eg5 (top). Bar, 2 μm.

FIGURE 4:

Dynamics of GFP-Kif15 in vivo. (A) Selected frames from a movie of GFP-Kif15–expressing cells; red and yellow arrowheads mark fluorescent particles traveling toward the chromosome region (spindle equator to the right; dark ovals are chromosomes). (B) Live cell expressing GFP-Kif15 progressing from prometaphase (0:00) to metaphase (3:30) and anaphase (9:00); arrows show accumulation of fluorescence near the kinetochores. (C) Kymographs from movie sequences of GFP-Kif15–expressing metaphase and anaphase cells; distance, horizontal axis; time, vertical axis; dark regions are chromosomes; spindle midzone to right. (D) Sequential frames (2-s interval) from movies of GFP-Kif15–expressing control and paclitaxel-treated cells (inverted contrast); motion of fluorescent particles toward the kinetochore region (right) in control but not paclitaxel-treated cells. Green arrowheads mark moving puncta. (E) LLC-Pk1 parental cells fixed and stained for microtubules and Kif15 (top) or TPX2 (bottom); control and paclitaxel as indicated; merged images to the right. (E’) Bar graph showing quantification of images in E. (F) Cartoon showing GFP-Kif15 cells in the presence of STLC; bipolar spindle formation requires TPX2. TPX2 could load Kif15 onto the microtubule, followed by motor motion to the microtubule plus end (a), or TPX2 and Kif15 could both localize to microtubule ends (b). Bars, 2 μm (A–E); time scale, 30 s (C).