1. Cell Biology
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Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells

  1. Lorena Benedetti
  2. Jonathan S Marvin
  3. Hanieh Falahati
  4. Andres Guillén-Samander
  5. Loren L Looger  Is a corresponding author
  6. Pietro De Camilli  Is a corresponding author
  1. Howard Hughes Medical Institute, United States
  2. Howard Hughes Medical Institute, Yale School of Medicine, United States
  3. Howard Hughes Medical Institute, Yale University School of Medicine, United States
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Cite this article as: eLife 2020;9:e63230 doi: 10.7554/eLife.63230

Abstract

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

Data availability

The constructs generated in this study will be available in Addgene (#162243-162255). All data generated in the mutagenesis screen can be found in Supplementary File 3. The complete list of primers used for the mutagenesis can be found in Supplementary Files 4, 5. Primers used for cloning are reported in the Key Resources Table. The sequences of the enhanced Magnets mutants generated have been deposited in Genebank: eMagAF (GenBank accession number: MW203024), eMagBF (GenBank accession number: MW203025), eMagA (GenBank accession number: MW203026), eMagB (GenBank accession number: MW203027). All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4 and associated supplements.

Article and author information

Author details

  1. Lorena Benedetti

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Jonathan S Marvin

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Hanieh Falahati

    Neuroscience, Howard Hughes Medical Institute, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Andres Guillén-Samander

    Department of Neuroscience and Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Loren L Looger

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    For correspondence
    loogerl@janelia.hhmi.org
    Competing interests
    The authors declare that no competing interests exist.
  6. Pietro De Camilli

    Departments of Neuroscience and Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    For correspondence
    pietro.decamilli@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9045-0723

Funding

National Institutes of Health (NS36251)

  • Pietro De Camilli

National Institutes of Health (P30DK045735)

  • Pietro De Camilli

National Institutes of Health (DA018343)

  • Pietro De Camilli

Jung Foundation for Science and Research

  • Andres Guillén-Samander

HHMI Life Sciences Associate

  • Hanieh Falahati

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

Ethics

Animal experimentation: All experimental procedures involving the use of mice were performed in agreement with the Yale University Institutional Animal Care and Use Committee (IACUC) (protocol number 2018-07422), and with the Janelia Farm Research Campus Institutional Animal Care and Use Committee and Institutional Biosafety Committee (protocol number 18-173).

Reviewing Editor

  1. Melike Lakadamyali, University of Pennsylvania, United States

Publication history

  1. Received: September 18, 2020
  2. Accepted: November 10, 2020
  3. Accepted Manuscript published: November 11, 2020 (version 1)
  4. Version of Record published: December 14, 2020 (version 2)

Copyright

© 2020, Benedetti 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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