1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

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
Tools and Resources
  • Cited 4
  • Views 4,685
  • Annotations
Cite this article as: eLife 2019;8:e48287 doi: 10.7554/eLife.48287

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.

Metrics

  • 4,685
    Page views
  • 652
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    Qiang Liu et al.
    Research Article Updated

    The multimodal sensory channel transient receptor potential vanilloid-3 (TRPV3) is expressed in epidermal keratinocytes and implicated in chronic pruritus, allergy, and inflammation-related skin disorders. Gain-of-function mutations of TRPV3 cause hair growth disorders in mice and Olmsted syndrome in humans. Nevertheless, whether and how TRPV3 could be therapeutically targeted remains to be elucidated. We here report that mouse and human TRPV3 channel is targeted by the clinical medication dyclonine that exerts a potent inhibitory effect. Accordingly, dyclonine rescued cell death caused by gain-of-function TRPV3 mutations and suppressed pruritus symptoms in vivo in mouse model. At the single-channel level, dyclonine inhibited TRPV3 open probability but not the unitary conductance. By molecular simulations and mutagenesis, we further uncovered key residues in TRPV3 pore region that could toggle the inhibitory efficiency of dyclonine. The functional and mechanistic insights obtained on dyclonine-TRPV3 interaction will help to conceive therapeutics for skin inflammation.

    1. Biochemistry and Chemical Biology
    Molly C Sutherland et al.
    Research Article

    Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.