1.
Optogenetic control of YAP cellular localisation and function.
Abstract:
YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.
2.
A photosensitive degron enables acute light-induced protein degradation in the nervous system.
Abstract:
Acutely inducing degradation enables studying the function of essential proteins. Available techniques target proteins post-translationally, via ubiquitin or by fusing destabilizing domains (degrons), and in some cases degradation is controllable by small molecules. Yet, they are comparably slow, possibly inducing compensatory changes, and do not allow localized protein depletion. The photosensitizer miniature singlet oxygen generator (miniSOG), fused to proteins of interest, provides fast light-induced protein destruction, e.g. affecting neurotransmission within minutes, but the reactive oxygen species (ROS) generated also affect proteins nearby, causing multifaceted phenotypes. A photosensitive degron (psd), recently developed and characterized in yeast, only targets the protein it is fused to, acting quickly as it is ubiquitin-independent, and the B-LID light-inducible degron was similarly shown to affect protein abundance in zebrafish. We implemented the psd in Caenorhabditis elegans and compared it to miniSOG. The psd effectively caused protein degradation within one hour of low intensity blue light (30 μW/mm(2)). Targeting synaptotagmin (SNT-1::tagRFP::psd), required for efficient neurotransmission, reduced locomotion within 15 minutes of illumination and within one hour behavior and miniature postsynaptic currents (mPSCs) were affected almost to the same degree seen in snt-1 mutants. Thus, psd effectively photo-degrades specific proteins, quickly inducing loss-of-function effects without affecting bystander proteins.
