Kinesin-14 family proteins HSET/XCTK2 control spindle length by cross-linking and sliding microtubules

Abstract

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin alpha/beta, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling. HSET overexpression in HeLa cells resulted in longer spindles, similar to what was seen with NLS mutants of XCTK2 in extracts, suggesting that Kinesin-14 proteins play similar roles in extracts and in somatic cells. Conversely, HSET knockdown by RNAi resulted in shorter spindles but did not affect pole formation. The change in spindle length was not dependent on K-fibers, as elimination of the K-fiber by Nuf2 RNAi resulted in an increase in spindle length that was partially rescued by co-RNAi of HSET. However, these changes in spindle length did require microtubule sliding, as overexpression of an HSET mutant that had its sliding activity uncoupled from its ATPase activity resulted in cells with spindle lengths shorter than cells overexpressing wild-type HSET. Our results are consistent with a model in which Ran regulates the association of Kinesin-14s with importin alpha/beta to prevent aberrant cross-linking and bundling of microtubules by sequestering Kinesin-14s in the nucleus during interphase. Kinesin-14s act during mitosis to cross-link and slide between parallel microtubules to regulate spindle length.

Figure 1.

HSET perturbation affects spindle morphology. (A) Control or GFP-HSET–overexpressing cells were processed for immunofluorescence to visualize MTs (magenta), HSET (green), and DNA (blue). Control cells were stained with anti-HSET, whereas transfected GFP-HSET was visualized directly by GFP fluorescence. (B) The average pole-to-pole distances and spindle widths of control and GFP-HSET–overexpressing cells are reported as mean ± SEM from three independent experiments. Average spindle lengths: control 13.9 ± 0.3 μm, overexpression 16.1 ± 0.6 μm; average spindle widths: control 9.34 ± 0.02 μm, overexpression 7.8 ± 0.4 μm (C) Cells transfected with either a control Luciferase or an HSET-specific siRNA were processed for immunofluorescence to visualize MTs (magenta), HSET (green), and DNA (blue). (D) The average pole-to-pole distances and spindle widths of control and HSET RNAi cells are reported as mean ± SEM from three independent experiments. Average spindle lengths: control 14.3 ± 0.5 μm, HSET RNAi 11.7 ± 0.4 μm; average spindle widths: control, 9.8 ± 0.6 μm; and HSET RNAi, 10.8 ± 0.3 μm. Scale bar, 10 μm.

Figure 2.

HSET affects spindle morphology in spindles lacking K-fibers. (A) Control HeLa cells (top three rows) or GFP-HSET overexpressing HeLa cells (bottom row) were transfected with either control Luciferase, hNuf2, or hNuf2+HSET siRNAs, synchronized, released, and then processed for immunofluorescence. In each panel, the Hec1 (magenta), HSET (green,) and DNA (blue) staining are shown. Scale bar, 10 μm. (B) Histograms of pole-to-pole distances quantified from a total of 60 spindles in three independent experiments for each knockdown condition. The best fit Gaussian distribution is shown superimposed in magenta. Average spindle lengths: control, 14.0 ± 0.4 μm; hNuf2 RNAi, 18.3 ± 0.5 μm; hNuf2 + HSET RNAi, 15.5 ± 0.6 μm; and hNuf2 RNAi + HSET overexpression, 19.7 ± 0.4 μm.

Figure 3.

Sliding activity is required for HSET/XCTK2-dependent spindle length change. (A) Cells expressing GFP-HSET or GFP-HSET N593K were synchronized, released, and then processed for immunofluorescence. In each panel, the MTs (magenta), HSET or HSET N593K mutant (green), and DNA (blue) staining are shown. (B) Histogram of spindle lengths quantified from a total of 60 spindles in three independent experiments for each expression condition. The Gaussian distribution is superimposed in magenta. Average spindle lengths: HSET, 17.5 ± 0.2 μm and HSET N593K, 15.7 ± 0.6 μm. (C) The fluorescence intensity of GFP-HSET (magenta) or GFP-HSET N593K (green) is plotted relative to the pole-to-pole distance from a total of ∼60 cells from three independent experiments. (D–F) GFP, GFP-HSET, or GFP-HSET N593K were added to Xenopus extracts at 2.5-fold the endogenous XCTK2 concentration before spindle assembly. (D) Representative images of spindles showing MTs (magenta) and DNA (blue). (E) Western blot of reactions probed with either anti-GFP antibodies (top panel) or anti-tubulin antibodies (bottom panel) to show that equivalent amounts of each protein were added to the extract. Note that 50 nM control GFP protein added to the extracts is below the detection limit of the anti-GFP antibody. (F) Quantification of the spindle length in extracts containing 2.5-fold excess GFP, GFP-HSET, or GFP-HSET N593K. A total of 60 structures were measured in three independent extracts, and the mean ± SEM is reported. Average spindle lengths: control GFP, 31.2 ± 1.7 μm; HSET, 37.6 ± 1.7 μm; and HSET 593K, 24.8 ± 1.1 μm. Scale bar, 10 μm.

Figure 4.

The spatial distribution of HSET and XCTK2 is dependent on the NLS. (A) Bipartite NLS sequences within the tail domains of XCTK2 and HSET showing the canonical positively charged residues highlighted in red. The corresponding mutant sequences are shown below the wt sequence with the mutations highlighted in red. (B) GFP-XCTK2, GFP-XCTK2-NLSa, or GFP-XCTK2-NLSb, at 20 nM, was added to interphase Xenopus egg extracts and incubated for 60 min. Representative structures are shown with the GFP fusion protein (green) and DNA (blue). Scale bar, 10 μm. (C) Western blot of GFP fusion proteins (XCTK2 or the NLS mutants) from Xenopus egg extracts that were immunoprecipitated with a control IgG or an anti-GFP antibody and then probed sequentially with antibodies to GFP and importin β and α. (D) HeLa cells were transiently transfected with pEGFP-HSET, pEGFP-HSET NLSa, or pEGFP-HSET-NLSb and then processed for immunofluorescence to visualize the GFP fusion protein (green) and the DNA (blue). Scale bar, 10 μm. (E) Western blot of GFP-HSET that was immunoprecipitated from HeLa cells stably expressing GFP-HSET with either a control IgG or an anti-GFP antibody and then probed with antibodies to GFP and importin β.

Figure 5.

Ran regulates the ability of XCTK2 to control spindle length. (A–C) GFP, GFP-XCTK2, GFP-XCTK2 NLSa, and GFP-XCTK2 NLSb proteins were added to cycled Xenopus egg extracts at a fivefold molar excess relative to the endogenous XCTK2 concentration at the time of the second CSF addition. (A) Representative fields of view showing MTs (magenta), the GFP fusion protein (green), and DNA (blue). (B) Western blot of samples from A that were probed with both anti-GFP antibodies (top panel) and anti-tubulin antibodies (bottom panel) to show that equivalent amounts of each protein were added to the extract. The GFP control concentration is below the detection limit of the antibody. (C) Quantification of the spindle length in extracts containing 10-fold excess exogenous GFP, GFP-XCTK2 (XCTK2), GFP-XCTK2 NLSa (NLSa), and GFP-XCTK2 NLSb (NLSb) proteins. A total of ∼80 structures were measured in each of three independent extracts, and the mean ± SEM is reported. Average spindle lengths: control GFP, 31.4 ± 1.7 μm; XCTK2, 34.6 ± 3.3 μm; NLSa, 69.2 ± 3.3 μm; and NLSb, 85.2 ± 11.0 μm. Scale bar, 50 μm.

Figure 6.

Model for regulation of Kinesin-14 function in spindle organization. During interphase, Kinesin-14s are transported into the nucleus through association of the NLS in its tail with importin α/β, which prevents the bundling of cytoplasmic MTs. After nuclear envelope breakdown, a Ran-GTP gradient is formed in the vicinity of chromosomes, which results in the dissociation of Kinesin-14s from importin α/β. Once Kinesin-14s are activated by Ran, they likely cross-link MTs and transport MTs to the spindle pole through the minus-end movement of the motor domain. This activity is mediated through parallel MTs within a half spindle.