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SCFFbxw5 mediates transient degradation of actin remodeller Eps8 to allow proper mitotic progression

Nature Cell Biology volume 15, pages 179–188 (2013)Cite this article

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Abstract

Eps8, a bi-functional actin cytoskeleton remodeller, is a positive regulator of cell proliferation and motility. Here, we describe an unrecognized mechanism regulating Eps8 that is required for proper mitotic progression: whereas Eps8 is stable in G1 and S phase, its half-life drops sharply in G2. This requires G2-specific proteasomal degradation mediated by the ubiquitin E3 ligase SCFFbxw5. Consistent with a short window of degradation, Eps8 disappears from the cell cortex early in mitosis, but reappears at the midzone of dividing cells. Failure to reduce Eps8 levels in G2 prolongs its localization at the cell cortex and markedly delays cell rounding and prometaphase duration. However, during late stages of mitosis and cytokinesis, Eps8 capping activity is required to prevent membrane blebbing and cell-shape deformations. Our findings identify SCFFbxw5-driven fluctuation of Eps8 levels as an important mechanism that contributes to cell-shape changes during entry into—and exit from—mitosis.

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Figure 1: Eps8 is a cell-cycle-regulated protein that undergoes Fbxw5-mediated proteasomal degradation specifically during G2/M.
Figure 2: Eps8 is a substrate of SCFFbxw5in vitro.
Figure 3: Fbxw5 contributes to timely displacement of Eps8 from the cell cortex at the onset of mitosis.
Figure 4: Fbxw5-mediated Eps8 degradation contributes to progression into metaphase.
Figure 5: Fbxw5-mediated Eps8 degradation contributes to timely cell rounding in early mitosis.
Figure 6: Failure to restore Eps8 levels induces membrane blebbing and cell-shape deformation after metaphase in HeLa cells.
Figure 7: MEF E p s 8−/− cells exhibit a post-metaphase membrane blebbing phenotype that can be rescued by re-expression of Eps8 versions with actin capping activity.
Figure 8

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Acknowledgements

We acknowledge J. Ellenberg (EMBL, Heidelberg, Germany), B. Baum (MRC-LMCB, England) and D. Gerlich (IMBA, Austria) for their gifts of stable HeLa cell lines. P. Di Fiore, O. Gruss, B. Mardin and A. Maccario are acknowledged for helpful discussions, F. Milanesi for Eps8 purification, R. Geiß-Friedlander for Fbxw5 construct generation, U. Gern for excellent technical assistance, and B. Mardin, S. Barysch, A. Flotho and U. Winter for critical manuscript reading. This work was supported by the Deutsche Forschungsgemeinschaft (SPP136, ME2279/3-1; F.M and A.W.) and NoE RUBICON, by ALSAC and NIH (2R01GM069530) to B.A.S., by the Associazione Italiana per la Ricerca sul Cancro (AIRC) to G.S and A.D.; by the Italian Ministries of Education-University-Research (MIUR-PRIN), the International Association For Cancer Research, and the European Research Council to G.S.; B.A.S. is an Investigator of the Howard Hughes Medical Institute. M.F.C. is an HHMI fellow of the Damon Runyon Cancer Research Foundation.

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Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany

    Achim Werner, Nina Reifenberger, Gregor Habeck, Janina Becker, Holger Lorenz & Frauke Melchior

  2. IFOM, Fondazione Istituto FIRC di Oncologia Molecolare at IFOM-IEO Campus, 20139 Milan, Italy

    Andrea Disanza & Giorgio Scita

  3. Howard Hughes Medical Institute, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-3678, USA

    Matthew Calabrese & Brenda Schulman

  4. Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

    Henning Urlaub

  5. Department of Clinical Chemistry, University Medical Center, 37075 Göttingen, Germany

    Henning Urlaub

  6. Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di Milano, 20122 Milan, Italy

    Giorgio Scita

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Contributions

A.W. designed and carried out most experiments and wrote the manuscript. A.D. and G.S. carried out analysis of Eps8-knockout MEFs and contributed to experimental design and manuscript writing. N.R. and G.H. carried out some of the experiments. J.B. contributed to the generation of recombinant SCFFbxw5 complexes. M.C. and B.S. provided expert help in the reconstitution of SCF E3 ligases and provided neddylated Cul1/Rbx1 complexes. H.L. provided expert help in time-lapse microscopy and generated the videos. H.U. carried out mass spectrometry analysis. F.M. guided the project, designed experiments and wrote the manuscript.

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Correspondence to Achim Werner or Frauke Melchior.

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Supplementary information

Supplementary Information

Supplementary Information (PDF 963 kb)

Hela cells stably expressing H2B-RFP and Lifeact-GFP were treated with non-targeting siRNA (si-nt; Supplementary Videos S1) or siRNA targeting Fbxw5 (si-Fbxw5; Supplementary Videos S1).

72 h after transfection, z-stack images were acquired with 0.25 μm step width. The images were deconvolved using Wiener filtering. Image processing was performed using Cell Excellence imaging software (Olympus) and ImageJ (NIH, http://rsbweb.nih.gov/ij/). (AVI 2824 kb)

Downregulation of Fbxw5 has no dramatic impact on overall actin distribution.

Hela cells stably expressing H2B-RFP and Lifeact-GFP were treated with non-targeting siRNA (si-nt; Supplementary Videos S1) or siRNA targeting Fbxw5 (si-Fbxw5; Supplementary Videos S2). 72 h after transfection, z-stack images were acquired with 0.25 μm step width. The images were deconvolved using Wiener filtering. Image processing was performed using Cell Excellence imaging software (Olympus) and ImageJ (NIH, http://rsbweb.nih.gov/ij/). (AVI 3294 kb)

Downregulation of Fbxw5 delays progression into metaphase.

HeLa cells stably expressing α-tubulin-GFP (green) and H2B-RFP (red) were synchronized by a double thymidine/release protocol and transfected with non-targeting siRNA or si-Fbxw5 #3. 60 h after siRNA transfection, cells were subjected to fluorescence time-lapse analysis (12 h, 2.5 min intervals). Movies show representative time frames of cells undergoing mitosis. Scale bar, 10 μm. (AVI 27218 kb)

Failure to restore Eps8 levels induces membrane blebbing and cell shape deformation after metaphase in HeLa cells.

HeLa cells were synchronized by a double thymidine/release protocol and transfected with different combinations of si-nt, si-Eps8 and si-Fbxw5 #3. 60 h after siRNA transfection, cells were subjected to DIC time-lapse analysis (14 h, 1.42 min intervals). Videos show representative time frames of cells undergoing mitosis. Scale bar, 10 μm. (MOV 20211 kb)

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Werner, A., Disanza, A., Reifenberger, N. et al. SCFFbxw5 mediates transient degradation of actin remodeller Eps8 to allow proper mitotic progression. Nat Cell Biol 15, 179–188 (2013). https://doi.org/10.1038/ncb2661

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