Optogenetic control of protein functions in living cells

Abstract

Perturbation of signaling networks by inhibition of target proteins can elucidate complex signaling networks in diverse cellular behaviors. To date, approaches such as knock-out/knock-down and small molecule inhibitors have been widely used to achieve these tasks. However, these techniques suffer from critical limitations, including slow responses, low reversibility, and poor spatiotemporal resolution. A technique that is capable of inactivating target proteins in a rapid, reversible, and highly spatiotemporal manner could crucially facilitate the study of target protein functions in the dynamic intracellular environment. In chapter 1, I developed a versatile optogenetic system for inhibiting protein function in living cells using light, Light-Activated Reversible Inhibition by Assembled Trap (LARIAT). By concerted application of multimeric protein and blue light-mediated interactions between Arabidopsis thaliana cryptochrome 2 and CIB1, I sequestered target proteins into clusters and thereby inactivate target proteins upon light illumination. Using LARIAT, I effectively and reversibly inactivated guanine nucleotide exchange factor, Rho GTPases, PI3kinase and microtubule with high spatiotemporal resolution. I also employed single-domain antibodies to further expand this platform to inhibition of target proteins containing specific epitopes. In chapter 2, I present a single-component optogenetic module that allows rapid and efficient protein clustering to overcome a potential limitation of LARIAT. By conjugating fluorescent proteins and extending a short peptide (termed A9), I achieved superior CRY2 clustering. I demonstrated that quaternary structures of tagged proteins as well as their conjugation site influenced the light-induced CRY2 clustering property. I also identified a veiled role of the C-terminal region of CRY2, showing that extending the C-terminus of CRY2 increased the efficiency of CRY2 clustering. Moreover, this improved module remarkably enhanced the function of a previously reported optogenetic system. This novel optogenetic approach provides an unprecedented opportunity for exquisite perturbation of signaling networks with high spatiotemporal resolution and further expand the optogenetic toolbox.