(J-361) A novel optogenetic split-enzyme system to control protein-protein interactions in the secretory compartment of cells

Optogenetics is a field of study that harnesses light to artificially induce interactions within animal cells. The application of light in developing animals allows manipulation of signaling proteins with high spatial and temporal precision. Developmental signaling proteins are typically processed within the secretory compartment of cells to properly induce tissue formation. We set out to control enzymatic processing of secreted proteins via light-controlled reconstitution and subsequent activation of split enzymes in the endoplasmic reticulum (ER).  First, to assist reconstitution of the split enzyme, we tested various dimerizing coiled-coil helices that were each fused to each half of the enzyme. The coiled-coils dimerize via hydrophobic and electrostatic interactions to elicit a strong binding response. We introduced our two-component split-enzyme system in single-cell zebrafish embryos to test whether enzyme activity can be reconstituted in vivo. We take advantage of Vg1-/- mutants that can be rescued via injection of Vg1 and the enzyme that can process it. Second, to induce split-enzyme reconstitution with light, we employed iLID/SspB proteins, which dimerize under blue-light stimulation. However, light-induced iLID/SspB dimerization has only been observed in the cell cytoplasm; to preserve this interaction, we fused them to the split-enzyme constructs via a transmembrane domain derived from Stim1a (TMstim1a). Last, to test whether blue light induces enzyme reconstitution and function in vivo, we fused iLID/SspB at the cytoplasmic ends of the TMstim1a-fused split enzymes. iLID/SspB-fused split enzymes were injected in zebrafish embryos and were found to colocalize in the ER membrane.

Standard molecular cloning techniques, site-directed mutagenesis and Gibson assembly, were performed to assemble the split-enzyme constructs in pCS2+ plasmid vectors.  Plasmid sequences were confirmed with nanopore DNA sequencing (SNPsaurus LLC). To synthesize mRNAs for each construct, plasmids were linearized and used as templates for transcription and then purified with the E.Z.N.A Total RNA Kit. mRNAs (50 pg/embryo) were injected into Vg1-/- zebrafish embryos at one-cell stage. To determine enzymatic activity, percentage rescue of Vg1-/- mutants was determined at 30 hours post-fertilization (hpf). Embryos were mounted in low-melting agarose at 55 hpf. Confocal fluorescence images were acquired with a Nikon A1R confocal microscope, using 6-hpf embryos that were fixed in 4% paraformaldehyde in 1x phosphate-buffered saline with .1% Tween (PBST).

First, to control enzymatic processing of secreted signaling proteins, we developed a split-enzyme system where dimerizing helices were fused to each split enzyme half (Fig 1A). We report reconstitution of the split enzyme, based on the percentage of Vg1-/- embryos that were rescued with enzyme-processed Vg1. We determined enzyme functionality by observing rescue of Vg1-induced mesoderm and endoderm tissues, such as muscle, two eyes, and a beating heart (Fig. 1B, C). This significant finding implies the possibility that split enzymes can be strongly reconstituted with light stimulation. To develop a light-inducible secreted enzyme, we aim to incorporate iLID/SspB proteins, which can dimerize in the cell cytoplasm upon blue-light stimulation. To eventually position iLID/SspB in the cytoplasm, a transmembrane domain derived from Stim1a (TMStim1a) was fused to each enzyme half. We also attached fluorescent proteins, sfCherry3C and mVenus to determine cellular localization (Fig. 1A). TMStim1a-fused split-enzyme constructs were observed on the ER membrane of early zebrafish embryonic cells, based on the strong overlap of sfCherry3C and mVenus fluorescence in reticulated structures (Fig. 1D). Incorporating coiled-coil helices and iLID/SspB is a promising approach toward promoting protein-protein interactions in secretory compartments with light.

Montague, Tessa G, and Alexander F Schier. “Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm.” eLife vol. 6 e28183. 15 Nov. 2017, doi:10.7554/eLife.2818.

Merljak, Estera, et al. “Coiled Coils as Versatile Modules for Mammalian Cell Regulation.” Synthetic Biology and Engineering, vol. 1, no. 1, 2023, pp. 1–10, doi:10.35534/sbe.2023.10006.

Guntas, Gurkan, et al. “Engineering an Improved Light-Induced Dimer (ILID) for Controlling the Localization and Activity of Signaling Proteins.” Proceedings of the National Academy of Sciences, vol. 112, no. 1, 2014, pp. 112–117, doi:10.1073/pnas.1417910112.