Curated Optogenetic Publication Database

Abstract: Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

Abstract: An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Abstract: Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Abstract: Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

Abstract: Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.