The chromokinesin Kid is required for maintenance of proper metaphase spindle size

Abstract

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

Figure 1.

Effects of Kid RNAi on HeLa cells. (A) Reduction of Kid expression after siRNA-Kid transfection. HeLa cell lysates prepared 48 h after transfection with either siRNA-Kid (Kid RNAi) or siRNA-control (con RNAi) were analyzed by Western blotting with antibodies against Kid, dynein, NuMA, and α-tubulin (internal control). (B) DNA contents histograms for control (1) and Kid-depleted (2) cells analyzed by LSC at 72 h after transfection. The green box in histogram 2 indicates cells with a DNA content <2n. Subpanel 3 shows a micrograph of typical cells that fall within the green box in histogram 2. (C) Bar graph. Quantification of mitotic states in control and Kid-depleted cells. One hundred fifty mitotic cells were scored from each of four independent experiments. Error bars represent the SD. (D and E) Immunofluorescence images of control (D) and Kid-depleted (E) cells. Forty-eight hours after transfection, cells were fixed and stained to reveal the distribution of Kid (green), α-tubulin (red), and DNA (blue). Bars, 5 μm.

Figure 2.

Suppression of Kid expression decreases spindle size. (A) Bar graph shows the average distance between two centrosomes in siRNA-control (con RNAi)-treated cells, siRNA-Kid (Kid RNAi)-treated cells, and siRNA-Kid-treated HeLa cells transfected with RNAi-resistant Kid-WT construct (Kid). Cells were fixed and stained with antibodies against Myc-tag and γ-tubulin, and the pole-to-pole distance was measured. Twenty metaphase cells were scored in each of five independent experiments. Error bars represent the SD. ^*p < 0.0005. (B) immunofluorescence images of monopolar HeLa cell. Kid-depleted (Kid RNAi) and control (con RNAi) HeLa cells were treated with 100 μM monastrol for 2 h, fixed, and processed for immunofluorescence staining with antibodies against γ-tubulin (green) and kinetochore-specific CREST (red). DNA was visualized with Hoechst33342 stain (blue in the right panels). (C) Histograms of the average distances between centrosomes and kinetochores monopolar spindles obtained from siRNA-Kid- (Kid RNAi) and siRNA-control (con RNAi)-transfected monopolar cells. The average value of 10 centrosome-to-kinetochore distances was calculated for each spindle. Each bar value, in turn, reflects the average of five spindle values. Error bars represent the SD. ^*p < 0.0005. (D) Immunofluorescence analysis of Kid-depleted HeLa cells demonstrating that NuMA localization is unaffected. Cells were fixed, and stained with antibodies against Kid (green) and NuMA (red). DNA (blue) was visualized with Hoechst stain.

Figure 3.

Less robust kinetochore fibers in Kid-depleted cells. (A) siRNA-Kid-treated cells were incubated on ice for 10 min to induce complete disassembly of all nonbundled microtubules and then fixed and processed for immunofluorescence using Kid (green in merge panel) and α-tubulin (left and red in merge panel) antibodies and Hoechst33342 for DNA staining (blue in merge panel). (B) Spindle fluorescence intensities in Kid-depleted or -nondepleted mitotic cells were measured using NIH Image software. The average of spindle fluorescence in nondepleted cells has been normalized to 1. The ratio of spindle fluorescence intensities in Kid-depleted cells to nondepleted cells is shown.

Figure 4.

DNA binding region and motor activity of Kid were not required to rescue the short spindle phenotype caused by Kid depletion. (A) Schematic representation of truncated and mutated Kid constructs. Kid has a kinesin-like motor domain (red box), a coiled-coil region (gray box), a helix-hairpin-helix domain (blue box), and the second microtubule-binding region (green box). Amino acid positions are indicated to the right. (B) Immunofluorescence analysis of Kid-depleted HeLa cells transfected with RNAi-refractory Kid mutant constructs. Cells were fixed and stained with antibodies against Myc-tag (green in merge) and α-tubulin (red in merge). DNA (blue in merge panel) was visualized with Hoechst33342 stain. (C) Intercentrosome distances in siRNA-Kid-treated HeLa cells transfected with RNAi-refractory Kid mutant constructs. Cells were fixed and stained with antibodies against Myc-tag and γ-tubulin and measured the pole-to-pole distance. Twenty metaphase cells were scored in each of five independent experiments. Error bars represent the SD. ^*p < 0.0005.

Figure 5.

Domain requirements for Kid-induced MT bundling in vitro and in cells. (A) Schematic representation of truncated Kid constructs. Kid has a kinesin-like motor domain (red box), a coiled-coil region (gray box), a helix-hairpin-helix domain (blue box), and a second microtubule-binding region (green box). Amino acid positions are indicated to the right. (B) Images observed by fluorescent microscopy when Kid fragments were mixed with rhodamine-labeled MTs in vitro. (C) immunofluorescence images of Kid-depleted cells expressing Kid mutant fragments at high levels. The Kid-depleted HeLa cells were transfected with Myc-Kidr-delDB (top), Myc-Kidr-delC (second panels), Myc-Kidr-delM (third panels), or Myc-Kidr-motor (bottom) constructs and then fixed and stained for mutant Kid protein, α-tubulin, and DNA with antibodies against Myc-tag and α-tubulin, and Hoechst33342, respectively.

Figure 6.

Both the coiled-coil region and the second MT-binding region were required to rescue the short spindle phenotype caused by Kid depletion. (A) Immunofluorescence analysis of Kid-depleted HeLa cells transfected with RNAi-refractory Kid mutant constructs. Cells were fixed and stained with antibodies against Myc-tag (green in merge panel) and α-tubulin (red in merge panel). DNA (blue in merge panel) was visualized with Hoechst33342 stain. (B) Intercentrosome distances in siRNA-Kid-treated HeLa cells transfected with RNAi-refractory Kid mutant constructs. The pole-to-pole distance of the cells from experiments shown in A was measured. Twenty metaphase cells were scored in each of five independent experiments. Error bars represent the SD. ^*p < 0.0005.

Figure 7.

Proposed model for Kid's roles during prometaphase/metaphase. Role of chromosomes-bound Kid (left): Kid localized on or around prometaphase/metaphase chromosome arms exert a force to transport the arms toward the plus end of the spindle MTs. Role of spindle-bound Kid (right): Kid may induce MT bundles to enhance the integrity of the spindle. Here, two possible mechanisms are shown. 1) MT cross-linking: Kid may directly cross-link parallel MTs such as kinetochore MTs or cross-link centrosome-nucleated MTs with chromosome-emanating MTs. 2) Factor recruitment: Kid may recruit other factors to stabilize the spindle MTs bundled by Kid.