Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells
- Howard Hughes Medical Institute, United States
- Howard Hughes Medical Institute, Yale School of Medicine, United States
- Howard Hughes Medical Institute, Yale University School of Medicine, United States
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
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).
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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Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells
eLife 9:e63230.
https://doi.org/10.7554/eLife.63230
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