Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo

Buckley, Clare E; Moore, Rachel E.; Reade, Anna; Goldberg, Anna R.; Weiner, Orion D.; Clarke, Jon

doi:10.1016/j.devcel.2015.12.011

Cited by 101 publications

(108 citation statements)

References 36 publications

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“…First, this system does not require an exogenous component in order to function because the flavin chromophore that the LOV domain requires to respond to light is endogenous to zebrafish (Motta-Mena et al, 2014;Crosson et al, 2003;Nash et al, 2011). This is not the case in phytochrome-based optogenetics, which requires the addition of phycocyanobilin in order to function (Tischer and Weiner, 2014;Shimizu-Sato et al, 2002;Buckley et al, 2016). Additionally, TAEL is genetically encoded and composed of a single homodimerizing transcriptional activator.…”

Section: Discussionmentioning

confidence: 99%

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

et al. 2016

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Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.

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Section: Discussionmentioning

confidence: 99%

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

et al. 2016

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“…PhyB-PIF6 has been used to control protein translocation within zebrafish embryos through a global light illumination (Beyer et al, 2015). A very recent work used the PhyB-PIF6 to control protein translocation and function within zebrafish through spatially confined light illumination (Buckley et al, 2016). The PhyB-PIF6 system has the highest dynamic range and fastest reversible kinetics (reversible association and dissociation within milliseconds).…”

Section: Discussionmentioning

confidence: 99%

“…One drawback is that a synthetic co-factor, phycocyanobilin or PCB, is needed for a fully functional PhyB-PIF6 system. For in vivo assays, PCB is either supplemented in buffer (Beyer et al, 2015) or injected into the body (Buckley et al, 2016). By contrast, the blue light-sensitive photoactivatable protein CRY2 from Arabidopsis thaliana requires no exogenous co-factors and displays reversible photoactivation kinetics on a time scale of several minutes.…”

Section: Discussionmentioning

confidence: 99%

Reversible optogenetic control of kinase activity during differentiation and embryonic development

et al. 2016

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A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proofof-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves lightinduced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue lightdependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

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“…All phytochromes utilize heme-derived linear tetrapyrrole compounds as their light-sensing chromophores. A red-light-triggered interaction of a plant phytochrome B (PhyB) and the phytochrome-interacting factor 6 (PIF6) from Arabidopsis is successfully applied to transcriptional control 13 , cell signaling 14 and protein localization 15 . Unlike plant phytochromes, which use phytochromobilin or phycocyanobilin tetrapyrroles as a chromophore, a subclass of bacterial phytochrome photoreceptors (BphPs) incorporate biliverdin IXα (BV) tetrapyrrole 16,17 .…”

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confidence: 99%

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo

Cited by 101 publications

References 36 publications

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

Reversible optogenetic control of kinase activity during differentiation and embryonic development

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

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