Clathrin is required for the function of the mitotic spindle

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Abstract

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell1,2,3,4. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells5, but is shut down during mitosis6, when clathrin concentrates at the spindle apparatus7,8. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

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Figure 1: Clathrin was targeted to the mitotic spindle of NRK cells.
Figure 2: Clathrin was targeted to the mitotic spindle via the N-terminal domain of the heavy chain.
Figure 3: Inhibition of clathrin-mediated endocytosis did not disrupt mitosis.
Figure 4: Depletion of clathrin results in destabilized kinetochore fibres, defective congression of chromosomes and prolonged activation of the spindle checkpoint.
Figure 5: Full-length CHC, but not CHC N-terminal domain, is sufficient to rescue the mitotic defects found in cells depleted of endogenous CHC.

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Acknowledgements

We thank W. C. Earnshaw, G. Fang, G. Ihrke, A. P. Jackson and M. S. Robinson for their gifts of antibodies, plasmids and cells. We also thank J. W. Raff and M. S. Robinson for useful discussion. This work was supported by the MRC and the Human Frontiers Science Program (grant to L.L.). N.A.B. was funded by the MRC.

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Correspondence to Stephen J. Royle.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Notes

Contains Supplementary Results, Supplementary Figure Legends, Supplementary Tables and Supplementary Methods. (DOC 83 kb)

Supplementary Figure S1

Clathrin was targeted to the mitotic spindle of NRK cells. (JPG 523 kb)

Supplementary Figure S2

Clathrin light chains were targeted to the mitotic spindle. (JPG 149 kb)

Supplementary Figure S3

Clathrin remains associated with the spindle apparatus after extraction of soluble proteins. (JPG 264 kb)

Supplementary Figure S4

The association of clathrin with microtubules was not via coated membranes. (JPG 427 kb)

Supplementary Figure S5

Knockdown of clathrin heavy chain using RNAi. (JPG 435 kb)

Supplementary Figure S6

Clathrin depletion did not significantly alter spindle morphology. (JPG 170 kb)

Supplementary Figure S7

Mitotic defects in clathrin-depleted cells. (JPG 302 kb)

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Royle, S., Bright, N. & Lagnado, L. Clathrin is required for the function of the mitotic spindle. Nature 434, 1152–1157 (2005) doi:10.1038/nature03502

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