iLID-antiGFP-nanobody is a flexible targeting strategy for recruitment to GFP-tagged proteins

Abstract

Optogenetics is a fast-growing field, that applies light-sensitive proteins to manipulate cellular processes. A popular optogenetics tool is the improved light-induced dimer (iLID). It comprises two components, iLID and SspB, which heterodimerize upon illumination with blue light. This system is often used to recruit proteins to a specific subcellular location, e.g. by targeting the iLID to the plasma membrane. The targeting requires modification of the iLID with a targeting sequence. To skip the modification of the iLID and use existing GFP fusion as targets, we fuse an antiGFP nanobody to the iLID. We show that the antiGFP nanobody is able to locate iLID to GFP-tagged proteins. Plus, the light-dependent recruitment of SspB to iLID, localized by the antiGFP nanobody to a GFP-tagged protein, is still functioning efficiently. This approach increases flexibility, enabling the recruitment of any GFP-tagged protein, without the necessity of protein engineering.

Activation of NF-κB signaling by optogenetic clustering of IKKα and β

“…Therefore, we fused the photoreceptor Cry2 to mCherry (mCh) for visualization, a nanobody that can specifically bind to the fluorescent protein eGFP (NbGFP) and a nuclear export sequence (NES) to direct the fusion protein to the cytoplasm (Cry2-mCh-NES-NbGFP). The protein of interest is fused to eGFP, which is a usually well-tolerated fusion partner [37][38][39] . From this basic design, we went on to mutate Cry2 to Cry2olig to increase clustering (Cry2olig-mCh-NES-NbGFP) and in the next step, inspired by the optoDroplet tool, incorporated FUSN.…”