Reversible optogenetic control of kinase activity during differentiation and embryonic development

Abstract

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.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and <em>Xenopus</em> Embryonic Development

“…In previous work, it has been determined that the optimal ratio for CIBN-GFP-CaaX:CRY2-mCherry-Raf1 is 2:1 35 . This configuration allows for sufficient membrane recruitment and activation of CRY2-mCherry-Raf1 (Figure 1B).…”

“…The differentiation ratio is calculated by dividing the number of differentiated cells by the number of transfected cells guided by GFP fluorescence. Differentiated cells are defined as those with at least one neurite longer than the size of the cell body 35 . This enhancement in the differentiation ratio arises from: 1) a better delivery of photoactivatable proteins with the bicistronic system and 2) an optimized expression ratio between CIBN and CRY2 35 .…”

“…Shortly after its initial success in controlling neuronal firing 25,26,27 , optogenetics has been extended to control other cellular processes, such as gene transcription, translation, cell migration, differentiation, and apoptosis 28,29,30,31,32,33,34 . A strategy using the photoactivatable protein pair Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN) was recently developed to control Raf1 kinase activity in mammalian cells and Xenopus embryos 35 . CRY2 binds to CIBN upon blue-light stimulation, and the CRY2/CIBN protein complex dissociates spontaneously in the dark 34 .…”

“…It is known that the membrane recruitment of Raf1 enhances its activity 38 . In this system, a tandem CIBN module is anchored to the plasma membrane and CRY2-mCherry is fused to the N-terminal of Raf1 35 . In the absence of blue light, CRY2-mCherry-Raf1 stays in the cytoplasm, and Raf1 is inactive.…”

Drive the Car(go)s—New Modalities to Control Cargo Trafficking in Live Cells

“…The possibility of modulating signaling pathways and cell functions with high spatiotemporal precision offers an entirely new modality to dissect molecular mechanisms governing cell fate determination (Toettcher et al, 2011; Zoltowski and Gardner, 2011; Tucker, 2012; Kim and Lin, 2013; Tischer and Weiner, 2014; Zhang and Cui, 2015). Photoactivatable proteins have been used in multiple model systems including yeast (Shimizu-Sato et al, 2002; Tyszkiewicz and Muir, 2008; Hughes et al, 2012; Strickland et al, 2012), mammalian cells (Levskaya et al, 2009; Wu et al, 2009; Yazawa et al, 2009; Kennedy et al, 2010; Toettcher et al, 2011; Idevall-Hagren et al, 2012; Mills et al, 2012; Zhou et al, 2012; Bugaj et al, 2013; Grusch et al, 2014; Kim et al, 2014; Lee et al, 2014; Taslimi et al, 2014; Zhang et al, 2014; Hughes et al, 2015; Kawano et al, 2015; Yumerefendi et al, 2016), primary neurons (Chen et al, 2013; Kakumoto and Nakata, 2013; Konermann et al, 2013), Drosophila (Boulina et al, 2013), zebrafish embryos (Liu et al, 2012; Motta-Mena et al, 2014; Buckley et al, 2016) and Xenopus embryos (Krishnamurthy et al, 2016). To control cargo trafficking, photoactivatable proteins such as the light, oxygen, voltage-peptide epitope (LOV-pep) and engineered PDZ domain (ePDZ; van Bergeijk et al, 2015) or cryptochrome 2 (CRY2) and cryptochrome 2 interacting basic helix-loop-helix (CIB1; Duan et al, 2015) were fused to cargoes and motor proteins or motor adapters (Figure 1D).…”