StayGold variants for molecular fusion and membrane-targeting applications

Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. We applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.

Supplementary Fig. 3 | Amino acid sequence alignment of StayGold and its variants.Adaptors and linkers are shaded in the same colors as in Extended Data Fig. 3. StayGold is abbreviated as SG.Either (n1)SG or (n1)oxSG was used as the starting material.Their N-terminus had a mutation A2V relative to that of SG.Supplementary Fig. 3 Supplementary Fig. 4 | Pseudonative SDS-PAGE of purified bacterially expressed proteins.
A panel (an uncropped photo of the gel) showing the final stage of the revolution reaching QC2-6 FIQ (lane 18).QC2-6 (lane 3) and its derivatives were analyzed in reference to EGFP (lane 1) and StayGold (lane 2).QC2-6 FIQ (= QC2-6 Y187F/R144I/T155Q) exhibited a slightly greater mobility than QC2-6.Lanes 4-12: nine bright colonies were selected from a mutant library on the basis of products of error-prone PCR on QC2-6 Y187F/R144I.ran: random mutagenesis.Shown is a representative of n = 4 independent experiments that detected different electrophoretic mobility between QC2-6 and QC2-6 FIQ.Supplementary Fig. 4 Supplementary Fig. 5 | Spectral assessment of inherent qualities of FP chromophores.Absorption spectra of FPs before (black line) and after (red line) denaturation with 0.1 M NaOH.The FP concentration was 10 µM, and the path length was 1 cm.All these FPs carry X-Tyr-Gly, a chromophore-forming tripeptide, and their alkali-denatured chromophores contain a dehydrotyrosine residue conjugated to the imidazolone group and absorb light maximally at 447 nm with a molar extinction coefficient of 44,000 M −1 cm −1 .This value was used for the determination of their absolute molar extinction coefficients (Table 1).StayGold is abbreviated as SG.Intensity-normalized curves.FPs were expressed as fusions to histone 2B (H2B) in HeLa cells.Illumination intensity, 8.66 W cm −2 .The curves shown are representative of three repetitions (n = 3 independent experiments).StayGold is abbreviated as SG.Curves are colored as follows.QC2-6 FIQ (mSG) and QC2-6(PT) (mSG2), green; SG and its tandem dimers, dark green; EGFP, dark blue; mEGFP, blue; mNeonGreen, red; mClover3, black; mGreenLantern, magenta.See Fig. 2.    G/R ratio: The fluorescence of a green-emitting FP was divided by the mCherry fluorescence on each pixel and averaged over regions.Normalized G/R ratio: The above ratio value was normalized to that of StayGold.Correction factor: The product of the excitation and emission detection efficiencies of a green-emitting FP in the imaging system (Supplementary Fig. 7) was normalized to that of SG to give a correction factor.Cellular brightness: The above Normalized G/R ratio was divided by the correction factor to give cellular brightness (Fig. 3b, Table 1

1 | 1 SupplementaryFig. 2 |
Fluoppi assay using PB1 tag for assessment of FP monomericity.The p62 PB1 domain is fused to a green-emitting FP (green), which is dimeric (a) or monomeric (b).left, PB1 self-associates via an equilibrium in a front-to-back topology to form a high-molecular-weight homo-oligomer.The conserved acidic/hydrophobic and lysine/arginine residues of PB1 are indicated by red and blue bars, respectively.a, Schematic showing that, as the FP homo-dimerizes, multiple PB1-FP molecules build crosslinks, resulting in the cytosolic formation of fluorescent puncta (green shading).The dimer interfaces of the FP are indicated by thick black bars.b, Schematic showing that, due to the monomericity of the FP, PB1-FP and its oligomers diffuse freely throughout the cytosol.right, WF images of cells with (a) and without (b) puncta; the images are representative of 3 repetitions (n = 3 independent transfections).Each gray scale indicates that the lowest and highest fluorescence intensities.Approximately 40% of PB1-Venus-expressing HeLa cells exhibited multiple punctate signals in the cytoplasmic compartment (a), whereas nearly 100% of the PB1-mVenus-expressing HeLa cells showed homogenous signal distribution (b) (see Extended Data Fig. 2).Scale bars, 20 µm.Supplementary Fig.Comparison between OSER and Fluoppi for the assessment of Venus monomericity/dispersibility.a, OSER; three typical images of HeLa cells that showed whorls of CytERM-Venus signals.b, Fluoppi; three typical images of HeLa cells that showed puncta of PB1-Venus signals.PB1-Venus appeared to yield more efficient signal concentration with less background than CytERM-Venus.Each gray scale indicates that the lowest and highest fluorescence intensities.Scale bars, 20 µm.Representative of n = 3 transfections for OSER and Fluoppi each.

6 SupplementaryFig. 7 |
Spectral throughputs of green-emitting FPs in the imaging system for determination of cellular brightness.Normalized excitation (dotted line) and emission (solid line) spectra of individual FPs and transmissions occurring in the excitation (gray) and emission (light green) passbands.Relative excitation (Ex.) and emission detection (Em.) efficiencies are shown on the left and right sides of the spectra, respectively.Their products were used to calculate the correction factors (Supplementary

Supplementary Fig. 9 |
Chromophore maturation in bacteria.E. coli colonies expressing StayGold, mStayGold, and mStayGold2 on agar plates were directly imaged.a, Fluorescence development in bacterial cells after plating at 37 °C.Data points are shown as means ± s.e.m. (n = 12 different colonies from 3 independent experiments).A photomicrograph showing the three colonies exhibiting the same growth rate.Scale bar, 5 mm.b, Fluorescence development of anaerobically grown bacterial cells after exposure to air at 37 °C.Data points are shown as means ± s.e.m. (n = 18 different colonies from 3 independent experiments).See Supplementary Video 1. a, b, Fluorescence intensities were corrected for the respective spectral throughputs (see below).c, Spectral throughputs of StayGold, mStayGold, and mStayGold2 in the imaging system for observation of bacterial colonies.Normalized excitation (dotted line) and emission (solid line) spectra of individual FPs and transmissions occurring in the excitation (gray) and emission (light green) passbands.Relative excitation (Ex.) and emission detection (Em.) efficiencies are shown on the left and right sides of the spectra, respectively.Their products were used to calculate the correction factors.StayGold is abbreviated as SG.pH sensitivity of mStayGold and mStayGold2.The fluorescence intensity was plotted against pH and fitted using the Boltzmann sigmoid function.Data points are shown as means ± s.d.(n = 3 different experiments).Resistance of spectral properties of mStayGold and mStayGold2 to Cl -.Absorption (top) and fluorescence (bottom) spectra.KCl was added to the indicated concentration at pH 7.4 buffered with 50 mM HEPES/NaOH.

Supplementary Fig. 8 | Spectral throughputs of green-emitting FPs in the imaging system for observation of chromophore maturation.
Normalized excitation (dotted line) and emission (solid line) spectra of individual FPs and transmissions occurring in the excitation (gray) and emission (light green) passbands.Relative excitation (Ex.) and emission detection (Em.) efficiencies are shown on the left and right sides of the spectra, respectively.Their products were used to calculate the correction factors.StayGold is abbreviated as SG.See Fig. 3c.