Light-induced nuclear transport – Employing phytochromes for nuclear trafficking of transcription factorsView all posters
University of Freiburg, Germany
Light-induced nuclear transport Employing phytochromes for nuclear trafficking of transcription factors Abstract In plants, phytochromes are key in light perception and play an important regulative role for red/far-red-light controlled responses. The nuclear translocation of the excited photoreceptor is nowadays considered as an essential step in signal processing. In this work, we employ mammalian cells to demonstrate the red-light-induced nuclear transport of a genetically engineered phytochrome B (phyB). Upon red-light exposure, the photoreceptor gains tight interaction capabilities to its dedicated NLS-harboring Phytochrome Interaction Factor 3 (PIF3) leading to the formation of a nuclear import complex thus driving the nuclear traffic. A red-light responsive reporter-gene expression system was developed based on this principle by the fusion of the phytochrome moiety (i) directly to a transcriptional activation domain (VP16) and (ii) indirectly in a biotin-dependent coupling to the DNA-binding domain TetR. The expression system shows a tunable spectral response with the highest activity at 660 nm. Illumination with light of a longer wavelength leads to a step-wise decrease in reporter expression and to a complete shutoff at 740nm. Additionally, fusions to fluorescent proteins were generated for the visualization of the nuclear transport by confocal imaging. Further development employing a nuclear export signal (NES) led to a red-light/far-red-light switchable nuclear shuttling of the phytochrome allowing a spatiotemporal control of the allocation and exclusion of phyB fusion-proteins to the nucleus. The principle at hand may reveal further information for basic research on plant phytochrome-singalling, as the formation of so called ‘nuclear speckles’ was observed – structures, which function is hitherto unknown and under heavy investigation mainly in Arabidopsis. On the other hand, the employment of the orthogonal Phytochrome B-based signalling module in mammalian cells allows for a highly valuable de-novo design of robust and sustainable biomolecular circuits and synthetic pathways at a spatiotemporal resolution.