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An optogenetic method for the controlled release of single molecules

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to t...

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Main Authors: Kashyap, Purba (Author) , Bertelli, Sara (Author) , Cao, Fakun (Author) , Kostritskaia, Yulia (Author) , Blank, Fenja (Author) , Srikanth, Niranjan A. (Author) , Schlack-Leigers, Claire (Author) , Saleppico, Roberto (Author) , Bierhuizen, Dolf (Author) , Lu, Xiaocen (Author) , Nickel, Walter (Author) , Campbell, Robert E. (Author) , Plested, Andrew J. R. (Author) , Stauber, Tobias (Author) , Taylor, Marcus J. (Author) , Ewers, Helge (Author)
Format: Article (Journal)
Language:English
Published: 08 March 2024
In: Nature methods
Year: 2024, Volume: 21, Issue: 4, Pages: 666-672
ISSN:1548-7105
DOI:10.1038/s41592-024-02204-x
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1038/s41592-024-02204-x
Verlag, kostenfrei, Volltext: https://www.nature.com/articles/s41592-024-02204-x
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Author Notes:Purba Kashyap, Sara Bertelli, Fakun Cao, Yulia Kostritskaia, Fenja Blank, Niranjan A. Srikanth, Claire Schlack-Leigers, Roberto Saleppico, Dolf Bierhuizen, Xiaocen Lu, Walter Nickel, Robert E. Campbell, Andrew J.R. Plested, Tobias Stauber, Marcus J. Taylor & Helge Ewers
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Summary:We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.
Item Description:Gesehen am 31.07.2024
Physical Description:Online Resource
ISSN:1548-7105
DOI:10.1038/s41592-024-02204-x

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