Spatiotemporal control of cell signalling using a light-switchable protein interaction

Abstract

Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to precisely control cellular behavior has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors1 and naturally-occurring channel rhodopsins have been used to directly perturb neuronal networks2,3. The difficulty of engineering light sensitive proteins remains a significant impediment to the optical control to most cell-biological processes. Here we demonstrate the use of a new genetically-encoded light-control system based on an optimized reversible protein-protein interaction from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principal be generically used to control diverse functions. Here we show that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. We show that light-gated translocation of the upstream activators of rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein-protein interaction that has been optimized in this work should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.

Phytochromes and Phytochrome Interacting Factors

“…2); however, the functional importance of this interaction has yet to be determined (Fujimori et al, 2004). Interestingly, the APB domain of PIF6 and PIF3 along with the N-terminal domain of phyB have been used to develop a light-switchable gene regulatory module for studying cellular signaling pathways in metazoans (Shimizu-Sato et al, 2002;Levskaya et al, 2009;Toettcher et al, 2011aToettcher et al, , 2011bToettcher et al, , 2013.…”

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

“…Perturbations using small chemicals 4 or lightsensitive molecules 5,6 are now exploited to understand biological functions 7 . For instance, gating activity by light-switchable interactions (allostery, scaffolding) using optogenetic tools has become a promising approach for localizing signalling pathways 8,9 . Furthermore, engineering essential functional units such as switches, oscillators and logic gates 10 offers the possibility to design specific cellular tasks and create novel functionalities 11,12 .…”

Engineered temperature compensation in a synthetic genetic clock

“…Synthetic biologists have constructed a wide variety of synthetic gene circuits, including switches (2-4), logic gates (5)(6)(7)(8), oscillators (9)(10)(11), and light-sensitive networks (12). Often, these circuits are built from genetic parts that regulate protein production, either at the transcriptional or translational level (13,14).…”