Figure 1
Schematic representation and in vitro optical activation of Opto-RANK. (a) Schematic representation of the two Opto-RANK constructs, Opto-RANKc and Opto-RANKm. The cytoplasmic domain of the RANK transmembrane protein was used in the constructs. Opto-RANKc consists of the photosensory module CRY2clust, mCherry, and the cytoplasmic domain of RANK. Opto-RANKm contains the self-cleaving P2A peptide, tandem CIBN, and the CAAX motif (at the C-terminus), in addition to the complete Opto-RANKc. (b) Schematic design of RANK activation using Opto-RANK. Opto-RANKc, consisting of CRY2clust, mCherry, and the cytoplasmic domain of RANK, is distributed throughout the cytosol. Upon blue light exposure, Opto-RANKc forms clusters, resulting in the binding of TRAF6 to the oligomerized RANK domain and subsequent activation of downstream signaling cascades. In the case of Opto-RANKm, light-activated Opto-RANKc is recruited to the membrane-anchored CIBN, resulting in membrane localization of the oligomerized RANK. (c) Representative images of HEK293T cells expressing Opto-RANKc and GFP-TRAF6. The cells were illuminated with a 488 nm wavelength laser every 5 s. Images of Opto-RANKc, TRAF6-GFP, and their merge before illumination (dark) and 1 and 3 min after the first illumination. Scale bar: 20 μm. (d) Representative images of HEK293T cells expressing Opto-RANKm and GFP-TRAF6. The cells were illuminated with a 488 nm wavelength laser every 5 s. Images of Opto-RANKc, TRAF6-GFP, and their merge, before illumination (dark) and 5 s and 1 min after the first illumination. Scale bar: 20 μm. Ratio of GFP-TRAF6 positive cells among Opto-RANK positive cells: 95.2% (139 out of 146 cells) in (c) and 94.8% (127 out of 134 cells) in (d) from three independent experiments. In the images, all Opto-RANK positive cells co-express GFP-TRAF6. RANK, receptor activator of nuclear factor-kB; TRAF6, tumor necrosis factor receptor-associated factor 6; GFP, green fluorescent protein.
