Fig. 6: Engineering of light-controlled intrabody for endogenous RAS.

a Schematic representation of a regulation of the intrabody against RAS, designated “iB(RAS),” by NIR light. In darkness, the intrabody shuttles between nucleus and cytoplasm, being predominantly in the nucleus. Under 740 nm light, the iB(RAS)-QPAS1 fusion interacts with BphP1-NES and accumulates in the cytoplasm, due to the summation of NES signals from both interacting partners. As a result, the intrabody can reach endogenous RAS on the plasma membrane. b Cells co-expressing BphP1-NES and iB(RAS)-NES-mCherry-QPAS1-NLS. mCherry fluorescence is observed in the nucleus in darkness and in the cytoplasm under 740 nm light. The process is reversible: in darkness the iB(RAS) moves back to the nucleus. Epifluorescence microscopy; scale bars, 10 µm. c Kinetics of iB(RAS) depletion in the nucleus as detected by mCherry fluorescence, error bars represent SEM, n = 6 cells. Source data are provided as a Source Data file. d Kinetics of the light-driven iB(RAS) accumulation in nucleus (recovery) as a result of BphP1 thermal relaxation, error bars represent SEM, n = 7 cells. Source data are provided as a Source Data file. e HeLa cells co-expressing the optically controlled iB(RAS) (red) and mEGFP-HRAS (green). Zoom ins of white frame-marked regions are shown on the right. White arrows show the membrane structures where iB(RAS) and mEGFP-HRAS co-localize. Epifluorescence microscopy; maximal projection is shown; scale bar, 10 µm. f Relocalization from nucleus to cytoplasm of optically controlled iB(RAS) (red) under 740 nm illumination, with subsequent binding of membrane-associated mEGFP-HRAS (green), white arrow. Confocal microscopy; scale bar, 10 µm. g Mean fluorescence signal on plasma membrane, shown as membrane-to-cytoplasm ratio, quantified for three cells similar to shown in f. Error bars represent SEM. The scheme used for the quantification is presented in Supplementary Fig. 9. Source data are provided as a Source Data file.