Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish

Abstract

Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.

Reversible optogenetic control of kinase activity during differentiation and embryonic development

“…Successful caging of the active protein often requires trial-anderror and computationally targeted protein design. 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).…”

“…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.…”

Photoreceptor engineering

“…As a consequence, chromophore has to be added exogenously, or additional genes for endogenous production of PCB have to be introduced to target tissues as well (Müller et al, 2013c). Despite this limitation, At Phy: At PIF-based systems have been successfully used for regulating by light gene expression of eukaryotic transgenes (Shimizu-Sato et al, 2002; Müller et al, 2013b), protein splicing (Tyszkiewicz and Muir, 2008), GTPase signaling (Levskaya et al, 2009), MAP kinase signaling (Toettcher et al, 2013), nuclear transport (Beyer et al, 2015a), as well as sequestration to subcellular compartments (Yang et al, 2013). Similar to the cryptochrome case above, N-terminal fragments of At PhyA/B (either residues 1–650 or 1–910) and At PIF3/6 (residues 1–100) suffice to elicit red-light-activated, far-red-light-reversible heteroassociation (Levskaya et al, 2009; Müller et al, 2013a,b), with the caveat that a recent study implies that in a yeast transcriptional assay full-length PIF3 supports a higher dynamic range of light activation than the N-terminally truncated version (Pathak et al, 2014).…”

The family of phytochrome-like photoreceptors: diverse, complex and multi-colored, but very useful

“…Alternatively, the light-regulated PhyB-PIF3 interaction can control gene expression, e.g. by splitting the DNA-binding and transactivation domains of GAL4 [44], or co-localize target proteins to subcellular components [45][46][47]. Finally, a DrBphP-phosphodiesterase fusion steers cyclic nucleotide levels by light in vivo [48 ].…”