Vector construction
SuperNova Green was generated from SuperNova/pRSETB. To introduce point mutations at Y66 and V44A, direct mutagenesis was performed using the inverse polymerase chain reaction (PCR) method. V44A-KillerRed and mKillerOrange were established by introducing V44A and Y66W to the KillerRed-pRSETB and the SuperNova/pRSETB plasmid respectively using the same method. miniSOG was cloned from a miniSOG-C1 (Addgene #54821) plasmid into pRSETB using the BamHI-EcoRI restriction site.
For mammalian cell expression, PCR-amplified miniSOG, SNG, mCherry and EGFP were cloned into 2xCOXVIII-SNR/pcDNA3.1 by BamHI and EcoRI restriction enzyme digestion. Fusion with vimentin was performed by replacing Vimentin-Kohinoor/pcDNA3.1 (Addgene #67772) with SNG, SNR, KillerRed and KillerRed V44A using BamHI and EcoRI restriction enzyme digestion. The fibrillarin construct was made by replacing SNR in SNRΔ11-Fibrillarin/pcDNA3.1 with KillerRed, KillerRed V44A and SNG using the HindIII/BamHI restriction site. Lyn-SNG/pcDNA3, SNG-H2B/pcDNA3.1 and LifeAct-SNG/pcDNA3 were made by replacing OeNL in Lyn-OeNL/pcDNA3.1 (Addgene #89528), Nano-lantern-H2B/pcDNA3 (Addgene #51971) and Kohinoor-Actin/pcDNA3.1 (Addgene #67776) with SNG at the BamHI-EcoRI restriction site.
For selective CALI constructs, the PHdomain sequence was obtained from GFP-C1-PLCdelta-PH (Addgene #21179). Fusion proteins were generated using the restriction sites AgeI-Venus-BglII-PHdomain-XbaI-SNG-EcoRI and AgeI-mNeptune-BglII-PHdomain-XbaI-SNR-EcoRI and then cloned to C1 plasmids. All oligonucleotides used in this experiment are listed in Additional file 1: Table S1.
Plasmids were transformed into XL-10 Gold Escherichia coli cells (Agilent Technologies, Santa Clara, CA, USA) using the heat shock method. A single colony was picked and cultured in 1.5 LB medium containing 0.1 mg/mL carbenicillin and then processed for plasmid purification. The DNA sequences of mutants were confirmed by dye terminator sequencing using a Big Dye Terminator v1.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
Protein purification
pRSETB containing a gene encoding protein tagged with N-terminal polyhistidine tags was transformed into JM109 (DE3) (Promega, Madison, WI, USA) by heat shock transformation at 42 oC for 45 s. The transformants were then plated onto agar plates containing 0.1 mg/mL carbenicillin. Colonies were cultured in 200 mL LB media containing 0.1 mg/mL carbenicillin at 23 °C with gentle shaking at 80 rpm for 4 days. Polyhistidine-tagged proteins were purified by Ni-NTA agarose (Qiagen, Hilden, Germany) chromatography, then eluted using 200 mM imidazole in TN buffer (10 mM Tris-HCl pH 8, 150 mM NaCl). The eluted proteins were processed with buffer exchange chromatography using a PD-10 column (GE Healthcare, Chicago, IL, USA). The final elution was diluted in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-KOH (pH 7.4).
Spectroscopy
Protein concentrations were measured using an alkaline denaturation method. Protein purity was confirmed using sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) analysis. Absorption spectra were measured on a V630-Bio spectrophotometer (JASCO, Easton, MD, USA). The absorbance peak was used for the molar extinction measurement. The molar extinction coefficient was defined by the equation ε = A/c, where ε is the molar extinction coefficient at the absorbance peak, A is the absorption at the peak wavelength and c is the protein concentration.
For the fluorescence spectrum measurement, the protein was diluted until absorption at the peak wavelength was 0.05. The fluorescence spectrum was measured using an F7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The emission spectrum was measured using 380, 400, 420, 440, 480 and 510 nm as excitation wavelengths. Meanwhile 490, 510, 540, 560, 580 and 610 nm were used for the emission wavelengths.
To measure the quantum yield, the protein was diluted to 5 μM. The absolute quantum yield of the protein was measured using a Hamamatsu Photonics C9920-01 spectrometer (Hamamatsu Photonics) at 610 and 510 nm for SNR and SNG respectively.
Size exclusion chromatography
Size exclusion chromatography was performed with a Superdex75 100/300GL column (GE Healthcare) with ÄKTA explorer 10S (GE Healthcare). We injected 1 mL of 10 μM protein into the column and then eluted it with 10 mM HEPES and 100 mM NaCl, pH 7.2. Elution was performed at 1 mL/min.
Photobleaching assay
An SNG and EGFP 10 μM protein solution was placed in a silicone microwell (1–2 mm in diameter) and topped with a cover glass. Protein solutions were exposed to 17 W/cm2 of 447/60-25 nm (Brightline) and 475/42-25 nm (Brightline) excitation light for SNG and EGFP respectively using a mercury arc lamp as the light source. Images were taken every 10 min for 8 h. The fluorescence intensity from the images was measured using Metamorph software (Molecular Devices, San Jose, CA, USA). Curve fitting and determination of t1/2 were done using Origin Software (OriginLab, Northampton, MA, USA).
Cell culture, transfection and localization imaging
HeLa cells (RIKEN BRC, Ibaraki, Japan) and HEK293T cells (ATCC, Manassas, VA, USA) were cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 with phenol red (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Riverside, MO, USA). Cells were incubated at 37 °C with 5% CO2. For subculture, cells were washed with sterile phosphate-buffered saline (PBS) and dissociated with trypsin. Cells subjected to plasmid transfection were seeded on 3-mm glass bottom dishes, and the DNA was transfected using either the calcium phosphate method or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For all live imaging experiments performed here, after 48 h of transfection, the medium was changed to DMEM/F12 without phenol red (ThermoFisher Scientific) added with 100 μg/mL penicillin/streptomycin (ThermoFisher Scientific). Imaging of subcellular localization was done using a confocal microscope (FV1000, Olympus) with a 60× NA 1.4 oil immersion objective. Images were taken using a 450 nm and a 580 nm multi-argon ion laser.
Singlet oxygen and superoxide measurement
In vitro 1O2 generation was measured using ADPA (anthracene-9,10-dipropionic acid) (Molecular Probes). We added 10 μL of 1 mM ADPA to 50 μL of 50 μM protein diluted in 50 mM HEPES-KOH, pH 7.4. Then 15 μL of the mixture was diluted in 300 μL PBS (the final concentrations of ADPA and protein were 7.9 μM and 2 μM respectively) and placed in a cuvette. The solutions were irradiated with 47 mW/cm2 excitation light (438/24, 475/28 and 542/27 nm) from light engine spectra (Lumencor, Beaverton, OR, USA). The fluorescence intensity of ADPA (ex/em = 350/430 nm) was measured every 1 min using an F7000 fluorescence spectrophotometer (Hitachi).
To assess 1O2 generation of SNG and miniSOG, pcDNA3.1 plasmids encoding sensitizer protein with mitochondria translocalization signal were transfected to HeLa cells using the calcium phosphate method. Then 48 h after transfection, the cells were incubated with 25 nM Si-DMA (Dojindo, Kumamoto, Japan) in DMEM/F12 without phenol red (ThermoFisher Scientific) for 45 min at 37 °C. The cells then were irradiated with 4 W/cm2 447/60-25 nm (Brightline) excitation light from a mercury arc lamp for 10 s. Images before and after irradiation were taken at the Cy5 channel (633 nm laser) with a Nikon A1 confocal system (Nikon, Tokyo, Japan). For O2•- generation, HeLa cells expressing SNG or miniSOG were incubated with 1–2 μM MitoSOX (ThermoFisher Scientific) in DMEM/F12 without phenol red for 10 min and then irradiated with excitation light, as in the Si-DMA experiment, for 30 s. Images before and after irradiation were taken at the mCherry channel (with a 543 nm excitation laser). The fluorescence increase was measured in the same region of interest within the cells using NIS Elements Software (Nikon).
Quenching experiments were done on HeLa cells expressing SNG and miniSOG plated on 96-well plates. Cells were treated with 200 U/mL polyethylene glycol (PEG)-SOD (Sigma Aldrich, St. Louis, MO, USA), 1000 U/mL catalase from bovine liver (Wako) or 60 mM mannitol (Wako, Tokyo, Japan) in DMEM/F12 without phenol red for 1 h and then irradiated under a fluorescence microscope (Nikon Eclipse TE2000, oil immersion 40× Plan Apo objective lens, NA 1.4) with 2 W/cm2 447/60-25 nm (Brightline) excitation light from a light engine (Lumencor) for 2 min. Differential interference contrast (DIC) images were taken using an ORCA-Flash 4.0 Camera (Hamamatsu Photonics, Hamamatsu, Japan). At least two images were taken from two independent dishes, and then cell death was enumerated 6 h post-irradiation.
Phototoxicity in mammalian cells
To assess phototoxicity in mammalian cells, pcDNA3.1 plasmids encoding SNR, SNG, miniSOG, mCherry and EGFP were transfected to HeLa cells (plated on a 35-mm glass bottom dish) using the calcium phosphate transfection method. At 48 h post-transfection, cells were irradiated on a fluorescence microscope (Nikon Eclipse TE2000-E, oil immersion 60× Plan Apo objective lens, NA 1.4) with 2 W/cm2 excitation light from an Intensilight C-HGFIE (Nikon). The filters used were 447/60-25 (Brightline), 475/42-25 (Brightline) and 562/40 (Brightline) for SNG/miniSOG, EGFP and SNR/mCherry respectively. DIC images were taken using an ORCA-Flash 4.0 (Hamamatsu Photonics). At least four different images were taken from each dish, and at least two dishes per construct were analysed for this experiment. The percentage of cell death was calculated every 1 h for 6 h post-irradiation.
Selective cell ablation
To perform selective cell ablation, stable cell lines expressing SNR and SNG independently inside a mitochondrial matrix were generated by transfection of SNR/SNG-pcDNA3 to HeLa cells using Lipofectamine 2000. Selection was then performed using 400 μg/mL Geneticin (Invitrogen). Cells expressing SNR and SNG were then co-cultured in 30-mm glass bottom dishes and subjected to light irradiation of 447/60-25 nm and 562/40 nm at ~ 4 W/cm2 (mercury arc lamp). Cells were irradiated for 2 min, then DIC images were taken at 0, 3 and 5 h post-irradiation using an ORCA-Flash 4.0 (Hamamatsu Photonics).
Selective CALI
HEK293 cells were co-transfected with Venus-PHdomain-SNG/C1 and mNeptune-PHdomain-SNR/C1 using Lipofectamine 2000. At 48 h post-transfection, imaging was performed with a confocal microscope (A1 Nikon Confocal, Nikon Eclipse Ti). Light irradiation was performed using an Intensilight (Nikon) with ~ 3 W/cm2 447/60-25 and 562/40 nm excitation under a 60× oil immersion objective lens NA 1.4 (Nikon) for 10 s. Images of Venus and mNeptune fluorescence were taken using a 488 nm laser and a 633 nm laser. Image analysis was performed using the QuimP plugin [50] in Fiji Software [51] to measure the fluorescence intensity in the cytoplasm and plasma membrane of cells. The ratio increase presented in Fig. 3c was calculated as:
$$ \Delta Ratio=\frac{Icytoplasm{t}_x}{Imembrane{t}_x}-\frac{Icytoplasm{t}_0}{Imembrane{t}_0} $$
where tx refers to the time point after light irradiation and t0 is the time point before light irradiation.
Statistical analysis
Data fitting and statistical analysis were performed using Origin 8 software (OriginLab) and SPSS statistics (IBM). Statistical values including the exact N and statistical significance are reported in the figure captions.