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Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control

Nature Cell Biology volume 15, pages 948–957 (2013)Cite this article

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Abstract

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide–disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

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Figure 1: Kip3-mediated microtubule-sliding activity.
Figure 2: Kip3-mediated sliding of dynamic microtubules.
Figure 3: Kip3-CC is a sliding-proficient but depolymerizing-deficient Kip3 variant.
Figure 4: Kip3-CC retains comparable motility to Kip3.
Figure 5: Counterbalancing effects between the sliding and destabilizing activity of Kip3 in the control of spindle length.
Figure 6: The sliding activity of Kip3 promotes spindle assembly.
Figure 7: A slide–disassemble model to explain the complex effects of Kip3 on regulating spindle length and stability.

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Acknowledgements

We are grateful to the Reck-Peterson Laboratory at Harvard Medical School for sharing the usage of their TIRF microscope. We appreciate H. Li’s help with FACS analysis. We thank M. Gupta and R. Ohi for suggestions and comments on the manuscript. D. Pellman is supported by Howard Hughes Medical Institute and a National Institute of Health grant (GM61345). M. Thery is supported by the Human Frontier Scientific Program (RGY0088/201). H. Arellano-Santoyo is an international fellow of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA

    Xiaolei Su, Hugo Arellano-Santoyo & David Pellman

  2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, and Pediatric Hematology/Oncology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    Xiaolei Su, Hugo Arellano-Santoyo & David Pellman

  3. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    Xiaolei Su, Hugo Arellano-Santoyo & David Pellman

  4. Laboratoire de Physiologie Cellulaire et Végétale, Institut de Recherches en Technologies et Sciences pour le Vivant, UMR5168, CNRS/CEA/INRA/Université Joseph Fourier 38054, Grenoble, France

    Didier Portran, Jeremie Gaillard, Marylin Vantard & Manuel Thery

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Contributions

X.S. and D. Pellman conceived the project and wrote the manuscript. H.A-S. performed the assay measuring microtubule dynamics and analysed the data. D. Portran, J.G., M.V. and M.T. performed the micropattering assay and analysed the data. X.S. performed the remainder of the experiments and analysed the data.

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Correspondence to David Pellman.

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Integrated supplementary information

Supplementary Figure 1 Protein purification panel and demonstration of Kip3-CC dimer formation.

Coomassie staining after SDS-PAGE was used to visualize Kip3, Kip3ΔT-LZ (Kip3 without its tail domain), Kip3-CC (Kip3 with its neck replaced with leucine zipper), and Kip3-M (Kip3 motor domain) with or without EGS [ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)] cross-linking. Note that after cross-linking Kip3-CC migrated at approximately the predicted size of a dimer, as Kip3 did. In contrast, Kip3-M remains at the similar position before and after cross-linking. Protein input: 1 μg.

Supplementary Figure 2 Comparable amounts of Kip3 and Kip3ΔT-LZ on track microtubules, related to the sliding assay in Fig. 1b.

(LEFT) TMR-labeled Kip3 or Kip3ΔT-LZ decorated track microtubules in the sliding assay. Scale bar: 5 μm. (RIGHT) Quantification of fluorescence intensity of Kip3 and Kip3ΔT-LZ along track microtubules at the beginning of sliding assays. Shown is mean±s.e.m. (N = 30 microtubules). Protein input: 150 nM.

Supplementary Figure 3 Categories of possible microtubule movements mediated by Kip3.

In each of the six situations, the bottom microtubule is immobilized to coverslip (blue bar). Kip3 motors, upright or inverted, slide the top cargo microtubules. The sliding direction was described as “ +”, “ −”, or “ +/−”, which indicates plus end-leading, minus end-leading, or tug-of-war, respectively. The microtubule orientation is indicated as being ‘p’ (parallel) or ‘a’ (anti-parallel). The observed frequencies for each type of movements was listed (N = 110 microtubule pairs).

Supplementary Figure 4 Kip3 slides anti-parallel microtubules and occasionally parallel microtubules.

Polarity-labeled microtubules (bright red for the plus end of cargo microtubules and bright green for the plus end of track microtubules) show an anti-parallel orientation (#1–#3) and a parallel orientation (#4). “ +” and “ −” indicate the microtubule plus end and minus end, respectively. Scale bar: 5 μm. Kymographs at the bottom show sliding movement of the numbered cargo microtubules. Scale bar: 2 μm (horizontal) and 2 min (vertical). Kip3 input: 150 nM.

Supplementary Figure 5 Kip3 induces the formation of curvy microtubule bundles.

Image panels show examples of curvy microtubule bundles in the presence of 10 μM tubulin together with indicated proteins (50 nM of Kip3 or/and 33 nM of Ase1). The images were taken 30 min after the reaction was started. Note that this panel does not include the substantial amount of asters pairs that were either non-connected or connected by straight bundles. The quantification of overall bundle curvature (including straight bundles) is shown in Fig. 2c. Scale bar: 10 μm.

Supplementary Figure 6 Comparable fluorescence intensities of motors along microtubules in the dynamic microtubule assay in Fig. 3.

(a) Kymographs show microtubule dynamics in the presence of Kip3 or Kip3-CC. (TOP) HiLyte 647-labeled tubulin. (BOTTOM) TMR-labeled motor. Input: 150 nM Kip3, 6 nM Kip3-CC. Scale bar: 5 μm (horizontal) and 1 min (vertical). b) Quantification of fluorescence intensity of Kip3 and Kip3-CC along microtubules. Shown is mean±s.e.m. (N = 47 and 50 microtubules for Kip3 and Kip3-CC, respectively). c) Quantification of the growth and shrinkage rates, related to Fig. 3e. Shown is mean±s.e.m. (N = 71, 46, and 63 growth events for Kip3, Kip3-CC, and control, respectively; N = 164, 25, and 23 shrinkage events for Kip3, Kip3-CC, and control, respectively). P value was obtained from a two-tailed Student’s T test.

Supplementary Figure 7 The spindle localization of Ase1 and Stu2 in cells overexpressing Kip3-CC.

(a) Representative images of a control cell and a cell overexpressing Kip3-CC. These cells were synchronized in S phase by hydroxyurea. Microtubules were labeled with CFP-Tub1. Ase1 was visualized with a 3xYFP tag. Scale bar: 2 μm. b) Comparison of spindle-associated Ase1 and spindle length between control cells and cells overexpressing Kip3-CC. Shown is mean+s.e.m. (N = 50 cells). c) Representative images of a control cell and a cell overexpressing Kip3-CC. Microtubules were labeled with CFP-Tub1. Stu2 was tagged with EYFP. Scale bar: 2 μm. d) Comparisons of the fluorescence intensity of Stu2 at mid region of pre-anaphase spindles (arrows in Supplementary Fig. S7c) between control cells and cells overexpressing Kip3-CC. Shown is mean+s.e.m. (N = 64 cells).

Supplementary Table 1 Dynamic instability of yeast cytoplasmic microtubules.
Full size table
Supplementary Table 2 Strains used in this study.
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Supplementary Information (PDF 664 kb)

Kip3-driven sliding and ‘tug-of-war’ movements of stabilized microtubules.

Cargo microtubules (red) move along track microtubules (green) in the presence of 150 nM Kip3. Videos play at 300× speed. Field view: 27×60 μm. (MOV 3247 kb)

Rare flipping of cargo microtubules in the sliding assay.

Cargo microtubules (red) move along track microtubules (green) in the presence of 150 nM Kip3. Videos play at 300× speed. Shown is sliding followed by flipping over and sliding in the opposite direction. This is a rare event, which was found in 4 out of 300 microtubule pairs. Scale bar: 1 μm. (MOV 77 kb)

A control for micropatterned sliding assay.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites. Scale bar: 10 μm. (MOV 130 kb)

Kip3-mediated buckling of dynamic microtubules.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 50 nM Kip3. One microtubule bundle started to buckle around 13:00. Scale bar: 10 μm. (MOV 127 kb)

Ase1-induced microtubule bundles.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 33 nM Ase1. Straight and stable bundles were formed. Scale bar: 10 μm. (MOV 124 kb)

Kip3-mediated buckling of Ase1 bundled microtubules.

Dynamic microtubules (green) were growing from seeds (red) adsorbed on micropatterned sites in the presence of 50 nM Kip3 and 33 nM Ase1. A stable microtubule bundle steadily buckled and elongated. Scale bar: 10 μm. (MOV 103 kb)

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Su, X., Arellano-Santoyo, H., Portran, D. et al. Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control. Nat Cell Biol 15, 948–957 (2013). https://doi.org/10.1038/ncb2801

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