Berlin 2024 – scientific programme
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BP: Fachverband Biologische Physik
BP 27: Single Molecule Biophysics
BP 27.4: Talk
Thursday, March 21, 2024, 10:15–10:30, H 0112
An optogenetic method for the controlled release of single molecules — P Kashyap1, S Bertelli2, F Cao3, Y Kostritskaia4, F Blank1, N Srikanth3, C Schlack1, R Saleppico5, D Bierhuizen1, X Liu6, W Nickel5, RE Campbell6, A Plested2, T Stauber4, MJ Taylor3, and •H Ewers1 — 1Freie Universität Berlin — 2Humboldt-Universität zu Berlin — 3MPI Infection Biology — 4Medical School Hamburg — 5Heidelberg University — 6University of Alberta
We developed a system for optogenetic release of single molecules in live 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 the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.
Keywords: optogenetics; single molecule imaging; single ion channel