Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

Abstract

The focal adhesion (FA) proteins talin and vinculin connect integrin to actomyosin networks, acting as the core mechanosensitive FA machinery. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we performed in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. Neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, they link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files would be provided for Figures 2,3,4,6 and 7.

Article and author information

Author details

  1. Charlotte F Kelley

    Cellular and Membrane Trafficking, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7684-9049
  2. Thomas Litschel

    Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7123-8364
  3. Stephanie Schumacher

    Cellular and Membrane Trafficking, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Dirk Dedden

    Cellular and Membrane Trafficking, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3630-1270
  5. Petra Schwille

    Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6106-4847
  6. Naoko Mizuno

    Cellular and Membrane Trafficking, Max Planck Institute of Biochemistry, Martinsried, Germany
    For correspondence
    mizuno@biochem.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1594-2821

Funding

European Molecular Biology Organization (EMBO Long-term Fellowship Award)

  • Charlotte F Kelley

Alexander von Humboldt-Stiftung (Research Fellowship for Postdoctoral Researchers)

  • Charlotte F Kelley

Horizon 2020 Framework Programme (Marie Sklodowska-Curie Action)

  • Charlotte F Kelley

Boehringer Ingelheim Stiftung (Plus 3)

  • Naoko Mizuno

H2020 European Research Council (FocAd)

  • Naoko Mizuno

European Molecular Biology Organization (Young Investigator Award)

  • Naoko Mizuno

Max-Planck-Gesellschaft

  • Naoko Mizuno

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Pekka Lappalainen, University of Helsinki, Finland

Version history

  1. Received: February 18, 2020
  2. Accepted: July 10, 2020
  3. Accepted Manuscript published: July 13, 2020 (version 1)
  4. Version of Record published: July 27, 2020 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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  1. Charlotte F Kelley
  2. Thomas Litschel
  3. Stephanie Schumacher
  4. Dirk Dedden
  5. Petra Schwille
  6. Naoko Mizuno
(2020)
Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin
eLife 9:e56110.
https://doi.org/10.7554/eLife.56110

Share this article

https://doi.org/10.7554/eLife.56110

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