Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany.
Humboldt-Universität zu Berlin and Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany.
Nat Methods. 2024 Apr;21(4):666-672. doi: 10.1038/s41592-024-02204-x. Epub 2024 Mar 8.
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.
我们开发了一种在细胞中光遗传学释放单分子的系统。我们通过光可裂解蛋白将可溶性和跨膜蛋白限制在高尔基体中，并通过短脉冲光将其释放。我们的方法允许用光剂量依赖性地将功能性蛋白质递送到细胞质和质膜中，其数量与单分子成像兼容，极大地简化了对任何活细胞中蛋白质的单分子显微镜研究。我们能够通过将 BK 和 LRRC8/体积调节阴离子通道递送到质膜来重新构建离子电导。最后，我们能够通过用光控制释放已敲除的信号蛋白来诱导 T 淋巴细胞中由白细胞介素 1 刺激的 NF-kB 信号转导。我们观察到光诱导形成的功能性炎症信号复合物仅在激活的细胞中触发核因子 kappa-B 激酶抑制剂的磷酸化。因此，我们开发了一种光遗传学方法，用于在单分子水平上重建和研究细胞功能。
