Curated Optogenetic Publication Database

Abstract: Serotonin (5-HT) signaling is strictly controlled by the serotonin transporter (SERT). The present study aims to establish optogenetic approaches for the control of SERT localization and function by modulating the interaction between SERT and its regulatory protein, soluble guanylate cyclase (sGC). We generated several cell lines that stably express blue light-inducible optogenetic elements fused to sGC or the fourth internal loop (IL4) motif of SERT. Our results indicated that blue light-induced SERT-sGC interaction by heterodimerizing SsrA embedded in the membrane-associated improved light-induced dimer (iLID) and SspB-sGCβ1 decreased SERT localization in the plasma membrane, thus reducing the maximum transport velocity of SERT without affecting its Km for substrate. The light-induced subcellular redistribution of SERT was shown to be attributable to an interference of the SERT-sGC interaction with SERT trafficking but not PKC-mediated internalization. In addition, the light-induced SERT-sGC interaction was blocked by the IL4 peptide or a mutation in the IL4 motif. Furthermore, light-induced exposure of the IL4 motif in iLID decreased the SERT-sGC interaction by displacing SERT from the SERT-sGC complex, thus increasing SERT localization in the membrane and elevating its ability for substrate uptake. This study achieved light-inducible modulation of the protein-protein interaction that allows for the study of biochemical and cellular processes in live cells.

Abstract: Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Abstract: Intracellular products (e.g., insulin), which are obtained through cell lysis, take up a big share of the biotech industry. It is often time-consuming, laborious, and environment-unfriendly to disrupt bacterial cells with traditional methods. In this study, we developed a molecular device for controlling cell lysis with light. We showed that intracellular expression of a single lysin protein was sufficient for efficient bacterial cell lysis. By placing the lysin-encoding gene under the control of an improved light-controlled system, we successfully controlled cell lysis by switching on/off light: OD600 of the Escherichia coli cell culture was decreased by twofold when the light-controlled system was activated under dark condition. We anticipate that our work would not only pave the way for cell lysis through a convenient biological way in fermentation industry, but also provide a paradigm for applying the light-controlled system in other fields of biotech industry.

Abstract: To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.