No abstract

To unravel the complexity of the signaling network and to dissect RTK downstream signaling, chemical approaches were successfully applied. [3][4][5] However, chemical inhibitors, although partially very specific, commonly affect several RTKs at the same time. Other chemical approaches such as chemical dimerizers are limited in their spatiotemporal resolution. Therefore, optogenetic tools play an emerging role to precisely control signaling proteins and their downstream pathways. [6][7][8] Signaling proteins fused to photo receptors or their interaction partners enable reversible lightdependent protein-protein interaction to cluster proteins or to recruit proteins to subcellular structures. Especially the use of non invasive light as activator facilitates the temporal and spatial control of signaling events by optogenetic tools. For simultaneous and orthogonal control of RAF/ERK and AKT signaling, we here compiled a multichromatic system based on two photo receptors derived from Arabidopsis thaliana. This system comprises 1) the blue light photoreceptor cryptochrome 2 (CRY2) with its interaction partner CRY2interacting basic helix-loop-helix protein CIB1 [9] for control of AKT, optoAKT; [10,11] and 2) the red/far-red light photoreceptor phytochrome B (PHYB) with its binding partner phytochrome interaction factor (PIF) [12] for control of ERK via the nucleotide exchange factor and activator of RAS son of sevenless (SOS), optoSOS. [13] Thus, this multichromatic system uses 450 nm blue light to activate optoAKT, and 660 nm red light to activate and 740 nm far-red light to inactivate optoSOS.OptoAKT is based on AKT fused to the photolyase-like domain of the blue light photoreceptor CRY2 (here referred to as CRY2-AKT) and CIBN, the N-terminal 170 amino acids of CIB1, fused to the myristoylation/palmitoylation signal and membrane-anchoring sequence of the tyrosine kinase LCK (m/p-CIBN). [10,11] Upon exposure to blue light, CRY2-AKT forms clusters that are recruited to the plasma membrane through interaction with m/p-CIBN (Figure 1A). AKT activity correlates with its phosphorylation at Thr 308 by PDK1 and Ser 473 by SIN1, a component of mTORC2. [14,15] AKT exists in three isoforms, AKT1, AKT2, and AKT3, that partially vary in their tissue expression and substrate specificity. [16,17] For all three isoforms, light-activatable constructs have been generated, enabling the control of AKT signaling by light (Figure 1B). [18] Here, optoAKT1 was used for further studies. Exposure to blue light activated optoAKT1 as monitored by immunoblotting for phosphorylation at Thr308 (about 26-fold increase in T308Receptor tyrosine kinases (RTKs) transmit the information of growth factors to an intracellular signal transduction machinery that controls diverse cellular processes. Upon activation, most RTKs interact with several signaling proteins and accordingly drive several pathways simultaneously. Depending on the strength and duration of activation, the interplay between these pathways regulates cellular metabolism and gene expression a...

Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Die molekulare Optogenetik hat zum Ziel, molekulare Prozesse in Zellen durch Lichtsignale zu steuern, indem genetisch kodierte Photorezeptoren an zelluläre Proteine fusioniert werden. Dadurch kann die zelluläre Signalleitung mit einer bisher nicht möglich gewesenen zeitlichen und örtlichen Präzision gesteuert werden. Hierzu wurden in den letzten Jahren zahlreiche Methoden entwickelt und neue Einblicke gewonnen. Beispielsweise konnte durch lichtabhängige Aktivierung des T-Zell-Rezeptors das kinetic proofreading Modell bestätigt werden. Außerdem ermöglichen lichtinduzierbare Cre-Rekombinasen eine noch präzisere Kontrolle über Genmanipulationen. Dieser Übersichtsartikel soll als Inspiration dafür dienen, wie die molekulare Optogenetik in der präklinischen Forschung Anwendung finden kann.