Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology

  1. Amal Alex
  2. Valentina Piano
  3. Soumitra Polley
  4. Marchel Stuiver
  5. Stephanie Voss
  6. Giuseppe Ciossani
  7. Katharina Overlack
  8. Beate Voss
  9. Sabine Wohlgemuth
  10. Arsen Petrovic
  11. Yaowen Wu
  12. Philipp Selenko
  13. Andrea Musacchio
  14. Stefano Maffini  Is a corresponding author
  1. Max Planck Institute of Molecular Physiology, Germany
  2. Leibniz Institute of Molecular Pharmacology, Germany
  3. Max Planck Society, Germany

Abstract

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and syntetic biology.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files

Article and author information

Author details

  1. Amal Alex

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Valentina Piano

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Soumitra Polley

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Marchel Stuiver

    In-Cell NMR Laboratory, Leibniz Institute of Molecular Pharmacology, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3437-4468
  5. Stephanie Voss

    Chemical Genomics Centre, Max Planck Society, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Giuseppe Ciossani

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Katharina Overlack

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Beate Voss

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Sabine Wohlgemuth

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Arsen Petrovic

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Yaowen Wu

    Chemical Genomics Centre, Max Planck Society, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2573-8736
  12. Philipp Selenko

    In-Cell NMR Laboratory, Leibniz Institute of Molecular Pharmacology, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  13. Andrea Musacchio

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2362-8784
  14. Stefano Maffini

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    For correspondence
    stefano.maffini@mpi-dortmund.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6380-6560

Funding

Max-Planck-Gesellschaft (Open-access funding)

  • Beate Voss
  • Sabine Wohlgemuth
  • Stefano Maffini

European Research Council (669686)

  • Amal Alex
  • Valentina Piano
  • Soumitra Polley
  • Giuseppe Ciossani
  • Katharina Overlack
  • Beate Voss
  • Sabine Wohlgemuth
  • Arsen Petrovic
  • Andrea Musacchio
  • Stefano Maffini

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

Reviewing Editor

  1. Silke Hauf, Virginia Tech, United States

Publication history

  1. Received: May 8, 2019
  2. Accepted: July 12, 2019
  3. Accepted Manuscript published: July 16, 2019 (version 1)
  4. Version of Record published: July 24, 2019 (version 2)
  5. Version of Record updated: October 10, 2019 (version 3)

Copyright

© 2019, Alex et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Amal Alex
  2. Valentina Piano
  3. Soumitra Polley
  4. Marchel Stuiver
  5. Stephanie Voss
  6. Giuseppe Ciossani
  7. Katharina Overlack
  8. Beate Voss
  9. Sabine Wohlgemuth
  10. Arsen Petrovic
  11. Yaowen Wu
  12. Philipp Selenko
  13. Andrea Musacchio
  14. Stefano Maffini
(2019)
Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology
eLife 8:e48287.
https://doi.org/10.7554/eLife.48287

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