A bacterial phytochrome-based optogenetic system controllable with near-infrared light

Abstract

Light-mediated control of protein-protein interactions to regulate cellular pathways is an important application of optogenetics. Here, we report an optogenetic system based on the reversible light-induced binding between the bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1-PpsR2 interaction both in vitro and in mammalian cells and then used this interaction to translocate target proteins to specific cellular compartments, such as the plasma membrane and the nucleus. We showed light-inducible control of cell morphology that resulted in a substantial increase of the cell area. We demonstrated light-dependent gene expression with 40-fold contrast in cultured cells, 32-fold in subcutaneous mouse tissue, and 5.7-fold in deep tissues in mice. Characteristics of the BphP1-PpsR2 optogenetic system include its sensitivity to 740- to 780-nm near-infrared light, its ability to utilize an endogenous biliverdin chromophore in eukaryotes (including mammals), and its spectral compatibility with blue-light-driven optogenetic systems.

Figure 1. Spectral properties of BphP1 and characterizations of BphP1–PpsR2 interaction in vitro

(a) Absorbance spectra in Pfr state before (solid line), after photoconversion to Pr state with 740/25 nm (dashed line) and after conversion back to Pfr state with 636/20 nm (dotted line). (b) Action spectrum of Pfr→Pr photoconversion measured as the relative decrease of Pfr absorbance detected at 780 nm upon irradiation with light of specific wavelength. (c) Dependence of half-time of light induced Pfr→Pr photoconversion on intensity of 740/25 nm light (n=3, error bars are s.e.m.). (d) Reversible absorbance BphP1 in Pfr state with 740/25 nm light followed by dark relaxation. (e) Dependence of half-time of light induced BphP1 Pr→Pfr photoconversion on intensity of 636/20 nm light (n=3, error bars are s.e.m.). (f) Reversible absorbance BphP1 in Pfr state turned off with 740/25 nm light and then turned on with 636/20 nm light. Absorbance in (b), (d) and (f) was measured at 780 nm. (g) Time-course of FRET changes during BphP1 photoswitching between the Pr and Pfr states either together with PpsR2-mRuby2 (sold line) or together with mRuby2 control (dashed line). Solid arrows correspond to 740/25 nm illumination. Dashed arrows correspond to 636/20 nm illumination. (h) Half-time of dark relaxation of BphP1 (Pr→Pfr transition) in presence of various quantities of PpsR2-mRuby2 (n=3, error bars are s.e.m.). (i) Reversible dark relaxation cycles from the Pr to the Pfr state of the BphP1 and PpsR2 mixture with 8:1 ratio. Arrows correspond to 740/25 nm illumination.

Figure 2. Re-localization of BphP1 to plasma membrane induced by light

(a) Chart depicting a light-induced interaction between cytoplasmic BphP1-mCherry and membrane-bound PpsR2-mVenus-CAAX. (b) Fluorescence images of the representative HeLa cell co-expressing PpsR2-mVenus-CAAX (green) and BphP1-mCherry (red) before illumination (left), after 3 min of 740/40 nm illumination with 0.9 mW cm⁻² (middle), and after 20 min of dark relaxation (right). Bars, 10 μm. (c) Intensity profile of BphP1-mCherry fluorescence of the cell in (b) marked with a white line before (solid line) and after (dashed line) 3 min of 740/40 nm illumination. (d) Intensity profile of BphP1-mCherry fluorescence of the cell in (b) marked with a white line after 3 min of 740/40 nm illumination (dashed line) and after subsequent 24 min in darkness (dotted line). (e) Time-course of BphP1-mCherry fluorescence intensity in cytoplasm during three cycles of 3 min of 740/40 nm irradiation with 0.2 mW cm⁻² followed by 30 min in darkness (n=5; white lines represent mean ±s.e.m.). mCherry fluorescence was measured every 15 s during 740/40 nm light illumination and every 180 s during dark relaxation. (f) Time-course of the BphP1-mCherry fluorescence intensity in cytoplasm during three cycles of 3 min of 740/40 nm irradiation with 0.2 mW cm⁻² followed by 3 min of 650/10 nm irradiation with 0.35 mW cm⁻² (n=5; white lines represent mean ±s.e.m.). mCherry fluorescence was measured every 15 s. All imaging was performed at 37°C using an epifluorescence microscope. Light intensities were measured at the back focal plane of a 60×1.35 NA objective lens.

Figure 3. Light-induction of cellular cytoskeletal re-arrangements

(a) Chart depicting a light-induced recruitment of cytoplasmic BphP1-mCherry-DHPH to membrane-bound PpsR2-mVenus-CAAX that results in cytoskeletal rearrangements of mammalian cells. (b, c) Fluorescence images of the representative HeLa cells co-expressing either (b) BphP1-mCherry-DHPH (red) and PpsR2-mVenus-CAAX (green) or (c) BphP1-mCherry-DHPH (red) and mVenus-CAAX control (green) before (top) and after (bottom) 30 min irradiation with 740/40 nm light of 0.2 mW cm⁻² (first 3 min continuous irradiation with 740/40 with following 27 min of pulse illumination 15 s On 45 s Off). Bars, 10 μm. (d) Time dependent changes in cellular areas of HeLa cells, which co-express either BphP1-mCherry-DHPH and PpsR2-mVenus-CAAX (n=5; error bars are s.e.m.), or BphP1-mCherry-DHPH and mVenus-CAAX control (n=5; error bars are s.e.m.), during irradiation with 740/40 nm light of 0.2 mW cm⁻² (first 3 min continuous irradiation with 740/40 with following 27 min of pulsed illumination 15 s On and 45 s Off). Fluorescent images were taken every 15 s during continuous and every 60 s during pulsed irradiation with 740/40 nm. All imaging was performed at 37°C using an epifluorescence microscope. The light power densities were measured at the back focal plane of a 60×1.35 NA objective lens.

Figure 4. Recruitment of BphP1 to nucleus and light-induced transcription activation

(a) Fluorescence images of representative HeLa cells co-expressing NLS-PpsR2-mVenus and BphP1-mCherry incubated either in darkness or under irradiation with 740/25 nm pulsed light (30 s On and 180 s Off) of 0.2 mW cm⁻². Images were acquired at 37°C using an epifluorescence microscope. Bars, 10 μm. (b) Chart depicting the proposed systems for light-inducible transcription activation. NIR light converts BphP1 into Pr state and induces heterodimerization with PpsR2. Nuclear localization signal (NLS) fused with PpsR2 facilitates translocation of the heterodimer to nucleus where BphP1 fusions interact with tetO DNA repeats via fused TetR. VP16 fused with PpsR2 recruits transcription initiation complex and triggers transcription of a reporter gene. (c) Kinetics of a light-to-dark ratio of SEAP signal detected in culture media of HeLa cells bearing BphP1-mCherry-TetR co-transfected with NLS-PpsR2-VP16 producing plasmid and pTRE-Tight-SEAP (7×tetO) reporter plasmid after 48 h (n=3; error bars are s.e.m.). (d) Termination of SEAP transcription in HeLa cells with the same constructs as in (c) illuminated with 740/25 nm followed by 60 h of darkness or followed by 12 h of 636/25 nm illumination and then by 48 h of darkness. Data were normalized to SEAP signal of cells irradiated with 740/25 nm for 72 h; the signal from non-irradiated cells was subtracted (n=3; error bars are s.e.m.).

Figure 5. Light-induced transcription activation in mice

(a) Rluc8 bioluminescence detected in mice with subcutaneously injected HeLa cells stably expressing BphP1-mCherry-TetR and co-transfected with the NLS-PpsR2-VP16 producing plasmid and pTRE-Tight-Rluc8 reporter plasmid kept either in darkness (top) or illuminated with 740/25 nm light of 1 mW cm⁻² (bottom) for 48 h. (b) Rluc8 signals detected in dark treated animals and in illuminated animals shown in (a) (n=3; error bars are s.e.m.). (c) Rluc8 signals in mice with subcutaneously injected HeLa cells co-transfected with pGAVPO plasmid encoding GAL4(65)-VVD-VP16 and pU5-Rluc8 reporter plasmid kept in darkness (top) or illuminated with 470/15 nm light of 1 mW cm⁻² (bottom) for 48 h. (d) Rluc8 signals detected in the dark treated and illuminated animals shown in (c) (n=3; error bars are s.e.m.). (e) Rluc8 signals detected in mice after hydrodynamic co-transfection with pKA-207I10 (encoding NLS-PpsR2-VP16-IRESv10-BphP1-mCherry-VP16) (50 μg) and pTRE-Tight-Rluc8 (5 μg) plasmids. Mice kept in darkness (top) or illuminated with 740/25 nm light of 5 mW cm⁻² (bottom) for 48 h. (f) Kinetics of the Rluc8 expression in mice shown in (e) kept in darkness or illuminated for 72 h (n=3; error bars are s.e.m.). (g) Rluc8 signals detected in mice after hydrodynamic co-transfection with pGAVPO (50 μg) and pU5-Rluc8 (5 μg) plasmids. Mice kept in darkness (top) or illuminated with 470/15 nm light of 5 mW cm⁻² (bottom) for 24 h. (h) Kinetics of Rluc8 expression in mice shown in (g) kept in darkness or illuminated for 72 h (n=3; error bars are s.e.m.).