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Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography

Nature Cell Biology volume 17, pages 605614 (2015) | Download Citation

Abstract

The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate ‘roof tile’-like overlaps. These shorten to produce the force, ‘zipping’ the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.

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Acknowledgements

We are particularly grateful to the EMBL electron microscopy facility and S. Pruggnaller, A. Habermann, V. Rybin, Y. Belyaev and R. Gibeaux for help with embryo processing, image acquisition and image analysis. We are grateful to J. Ellenberg, M. Vabulas and C. Pohl for critical reading and discussion of the manuscript. We thank I. Simeonova, A. Schmidkunz, N. Wollf, S. Wali and V. Eltsova for tracing. M.E. was supported by an EIPOD fellowship; N.D. was supported in part by a Fellowship from the Canadian Cancer Society Research Institute (Terry Fox Foundation, award no. 018608). Core funding (EMBL, University of Zurich, CEF and CEFII) and an ERC starting grant to A.S.F. provided further support.

Author information

Affiliations

  1. Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15 60438 Frankfurt am Main, Germany

    • Mikhail Eltsov
    • , Zhou Yu
    •  & Achilleas S. Frangakis
  2. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany

    • Mikhail Eltsov
    • , Nadia Dubé
    •  & Damian Brunner
  3. Electron Microscopy Core Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany

    • Mikhail Eltsov
    •  & Uta Haselmann-Weiss
  4. University of Zurich, Institute of Molecular Life Sciences, Winterthurerstr. 190, 8057 Zurich, Switzerland

    • Nadia Dubé
    • , Laurynas Pasakarnis
    •  & Damian Brunner
  5. Institut de recherches cliniques de Montréal (IRCM), 110 avenue des Pins Ouest, Montréal (Québec), H2W 1R7, Canada

    • Nadia Dubé

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Contributions

M.E. designed and carried out experiments, analysed data and wrote the manuscript. N.D., Z.Y., L.P. and U.H-W. carried out experiments and analysed data. D.B. and A.S.F. designed experiments, analysed data and wrote the manuscript. All authors have proofread the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Damian Brunner or Achilleas S. Frangakis.

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Videos

  1. 1.

    Surface representation of LE and AS cells in the zipping area as shown in Fig. 2B and C.

    The model is tilting around the central vertical axis. LE cells are sequentially removed to reveal the underlying AS cell surfaces.

  2. 2.

    Surface representation of the LE cells shown in Fig. 3B.

    The model is tilting around the central horizontal axis. The LE cells LE#1 and LE#2 from one epidermis front (red/brown) are subsequently removed to reveal the contact areas (dark red and purple spheres) with the opposing LE cell (green).

  3. 3.

    Live fluorescence imaging of α-catenin-GFP in a zipping embryo.

    α-catenin-GFP (expressed in paired-expressing cell stripes) marks LE/LE adherens junctions and visualizes amongst others the formation of nascent AJs between opposing LE cells as shown in Fig. 3F. Lateral views (Z-stack maximal projections) were acquired at 1 min intervals.

  4. 4.

    Live fluorescence imaging of EB1-GFP in LE cells visualizing growing MT plus ends.

    Whereas LE cell bodies show bidirectional EB1 movement, in cell protrusions, it is predominantly (and almost exclusively) moving towards the protrusion tips at all zipping stages, revealing that MTs are mainly oriented such that their plus ends are growing into LE cell protrusions.

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DOI

https://doi.org/10.1038/ncb3159