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

Abstract

A large percentage of proteins form higher-order structures in order to fulfill their func33 tion. These structures are crucial for the precise spatial and temporal regulation of the 34 cellular signaling network. Investigation of this network requires sophisticated research 35 tools, such as optogenetic tools, that allow dynamic control over the signaling mole36 cules. Cryptochrome 2 and its variations are the best-characterized oligomerizing pho37 toreceptors the optogenetics toolbox has to offer. Therefore, we utilized this switch and 38 combined it with an eGFP-binding nanobody, to build a toolbox of optogenetic con39 structs that enables the oligomerization of any eGFP-tagged protein of interest. We 40 further introduced the higher clustering variant Cry2olig and an intrinsically disordered 41 region to create higher-order oligomers or phase-separated assemblies to investigate 42 the impact of different oligomerization states on eGFP-tagged signaling molecules. We 43 apply these constructs to cluster IKKα and IKKβ, which resemble the central signaling 44 integrator of the NF-κB pathway, thereby engineer a potent, blue-light-inducible acti45 vator of NF-κB signaling.