Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons.

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

An inducible system for in vitro and in vivo Fas activation using FKBP-FRB-rapamycin complex.

The inducible activation system is valuable for investigating spatiotemporal roles of molecules. A chemically inducible activation system for Fas (CD95/APO-1), which works efficiently to induce apoptosis and leads non-apoptotic pathways, has not yet been developed. Here, we engineered a rapamycin-induced dimerization system of Fas consisting of FKBP and FRB proteins. Treatment of rapamycin specifically induces cellular apoptosis. In neurons and cells with high c-FLIP expression, rapamycin-induced Fas activation triggered the activation of the non-apoptotic pathway components instead of cell death. Intracranial delivery of the system could be utilized to induce apoptosis of tumor cells upon rapamycin treatment. Our results demonstrate a novel inducible Fas activation system which operates with high efficiency and temporal precision in vitro and in vivo promising a potential therapeutic strategy.

Locally Activating TrkB Receptor Generates Actin Waves and Specifies Axonal Fate.

Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.

Engineering of bacterial exotoxins for highly efficient and receptor-specific intracellular delivery of diverse cargos.

The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.

Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling.

Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.