An optogenetic method for the controlled release of single molecules

Kashyap, P.; Bertelli, S.; Cao, F.; Kostritskaia, Y.; Blank, F.; Srikanth, N.; Saleppico, R.; Bierhuizen, D.; Lu, X.; Nickel, W.; Campbell, R. E.; Plested, A.; Stauber, T.; Taylor, M. J.; Ewers, H.

doi:10.1101/2023.09.16.557871

bioRxiv · 10.1101/2023.09.16.557871

An optogenetic method for the controlled release of single molecules

Kashyap, P.·Bertelli, S.·Cao, F.·Kostritskaia, Y.·Blank, F.·Srikanth, N.·Saleppico, R.·Bierhuizen, D.·Lu, X.·Nickel, W.·Campbell, R. E.·Plested, A.·Stauber, T.·Taylor, M. J.·Ewers, H.

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Abstract

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.

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Kashyap, P., Bertelli, S., Cao, F., Kostritskaia, Y., Blank, F., Srikanth, N., Saleppico, R., Bierhuizen, D., Lu, X., Nickel, W., Campbell, R. E., Plested, A., Stauber, T., Taylor, M. J., Ewers, H.. 2023-09-17. An optogenetic method for the controlled release of single molecules. https://doi.org/10.1101/2023.09.16.557871

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