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The assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl) Kif18A. Xenopus Kif18A is expressed during oocyte maturation and its depletion from meiotic egg extract results in severe spindle defects. These defects can be rescued by wild-type Kif18A, but not Kif18A lacking motor activity or the C-terminus. Single-molecule microscopy assays revealed that Xl_Kif18A possesses high processivity, which depends on an additional C-terminal microtubule-binding site. Human tissue culture cells depleted of endogenous Kif18A display mitotic defects, which can be rescued by wild-type, but not tail-less Xl_Kif18A. Thus, Xl_Kif18A is the functional orthologue of human Kif18A whose activity is essential for the correct function of meiotic spindles in Xenopus oocytes.
Fig. 1. Xl_Kif18A is expressed during female meiosis. (A) Domain structure of Xl_Kif18A. (B,C) Immunoblot analyses of MII extract or bead samples after immunodepletion using Ab18Apep (B) or Ab18A-C (C). IgG antibodies were used as a control (Ctrl). (D) Immunoblot analyses of immature stage-VI (S VI) arrested oocytes before and at indicated time points after progesterone treatment using indicated antibodies.
Fig. 2. Xl_Kif18A is a processive kinesin with an additional non-motor MT-binding site. (A) SDS-PAGE analyses of recombinant full length (FL) and δtail (aa 1-845) Kif18A-mGFP-His10. (B) Scheme of TIRF microscopy (top) and exemplary time (y-axis, scale bar: 10 s) versus space (x-axis; scale bar: 5 µm) plots (kymographs) of FL and δtail Kif18A-mGFP-His10. (C) Speed and (D) run length of Kif18A-mGFP-His10 and (E) percentage of all molecules analyzed reaching the microtubule tip (mean±s.d. in red, unpaired t-test: ****P≤0.0001, **P≤0.01). (F) MT pelleting assay with MBP-Kif18ACT-His6 using varying concentrations of MTs (0-10 µM) and KCl (30-200 mM) analyzed by SDS-PAGE (from lane 1, 0 µM MTs, to lane 8, 10 µM MTs) and (G) quantified using ImageJ (mean±s.d., n=3 independent experiments, Kd values derived from one-site-specific binding fit in GraphPad Prism). (H) MT bundling assay using fluorescently labeled, taxol-stabilized MTs and nanomolar concentrations of Kif18A-mGFP-His10.
Fig. 3. Xl_Kif18A is important for meiotic spindle structure. (A) Scheme of the depletion /add-back experiments. (B) Immunoblot of control (IgG)- or Kif18A (Ab18Apep)-depleted extracts supplemented with mRNA encoding wt, Ci or δtail Flag-eGFP-Xl_Kif18A. Right panel shows immunoblot of IgG or Ab18Apep beads. (C) Representative fluorescence images of spindles obtained as described in A. DNA, αβ-tubulin, and Flag-eGFP-Xl_Kif18A are shown in blue, red and green, respectively. Scale bar: 10 µm. (D) Quantification of spindle length to width ratio. (E) Quantification of spindles with multiple/unfocused poles (more than 60 spindles analyzed per condition, mean±s.d., unpaired t-test: ****P≤0.0001).
Fig. 4. Xl_Kif18A can complement the function of human Kif18A. (A) Scheme of the RNAi/rescue experiments using HeLa cells expressing constitutively CENP-A-mCherry and inducibly siRNA resistant human or Xenopus eGFP-Kif18A. (B) Quantification of mitotic timing (timing from NEBD to either commitment to anaphase onset or apoptosis; top) in cell lines treated as described in A using fluorescent time-lapse imaging (time resolution 5 min, more than 200 cells per condition, mean±s.d. in red, n=3 independent experiments) and immunoblot analyses (bottom) of respective cells. Unspecific band is marked with an asterisk. (C) Representative fluorescence images of spindles in mitotic cells expressing eGFP-Kif18A constructs as indicated. HURP, CENP-A-mCherry, and eGFP-Kif18A are shown in blue, red and green, respectively. Insets show magnified view of area marked with white box. Scale bar: 10 µm.
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