Optogenetic control of biological dynamics in live cell

Abstract

The regulation of cellular functions require precisely tuned intensity, localization and duration of signaling events. Diverse systems has developed to study these studies. Notably, reversibility and spatio-temporal precision of optogenetics are an ideal means to study complex cellular dynamics. Although numerous studies presented optogenetic systems, further development, characterization and applications are needed. Here, specific light-response protein, cryptochrome 2 (CRY2) from Arabidopsis thaliana was engineered and characterized to further expand the optogenetic modules.In chapter 1, optogenetic modules was engineered to allow rapid and efficient protein oligomerization. By tagging different fluorescent proteins or a short peptide, the CRY2 reached a maximal oligomerization to generate a ‘cluster’, named CRY2clust. The veiled role of C-terminus of CRY2 was unraveled and application of CRY2clust on previously reported optogenetic modules remarkably enhanced the efficiency. In chapter 2, optogenetic technique called mRNA-LARIAT was developed to control mRNA localization and translation. The previously reported LARIAT (Light-Activated Reversible Inhibition by Assembled Trap (LARIAT) method was combined with conventional mRNA visualization system. With mRNA-LARIAT, light induces the sequestration of specific mRNAs into large protein clusters, altering mRNA localization and interfering with translation by limiting the ribosome interaction with trapped mRNA. The system is also sensitive and robust platform to visualize endogenous mRNA-protein complexes that have thus far been hardly detectable at the single-cell level due to low signal to noise ratio. With this method, the importance of newly synthesized β-actin molecules in fibroblast cell migration was discovered. This novel optogenetic modules are broadly applicable to study the causal relationships between mRNA or protein dynamics with various biological function and diseases.