Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo

Abstract

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

Keywords: bacterial phytochrome; fluorescence imaging; fluorescent proteins; rational design.

Figure 1

Rational design of a far‐red fluorescent protein iBlueberry. (a) Modeled structure of mIFP based on IFP2.0 (PDB: 4CQH), with the single mutation I251C. The chromophore BV is shown in purple. (b) Structure of CphP1 (PDB: 2VEA). The chromophore PCB is shown in cyan. (c) Purified iBlueberry. (d) Coomassie blue staining and zinc‐induced fluorescence assay (bottom) of mIFP, iBlueberry (i.e., mIFP/I251C) and other mutants. The molecular mass standards are shown on the right. (e) Excitation spectra of mIFP and its I251C mutant (i.e., iBlueberry). (f) Emission spectra of mIFP and its I251C mutant. A structure model of mIFP with introduced mutations. The chromophore BV is shown in purple. (g) Photobleaching of mIFP and iBlueberry (i.e., mIFP/I251C). Normalized fluorescence intensity over time under illumination of 100% 640 nm laser. The laser power is 13.4 mW and illumination light intensity is 1.83 W/cm². (h) Fluorescence of purified iBlueberry against pH.

Figure 2

Centrosome dynamics in radial glia (RG) neural progenitors revealed by time‐lapse in vivo imaging in the developing zebrafish brain. (a) A schematic shows the experimental design. (b) Representative images show that the centrosomes of RG progenitors, labeled by iBuleberry‐Centrin, predominantly locate at the ventricular zone (VZ) surface. V: ventricle. Scale bar: 50 μm. (c) Representative montage of selected images from time‐lapse in vivo imaging, tracking a single fluorescently labeled RG progenitor across space and time. Time “0” is defined as the time point of metaphase when the condensed chromosome is distinguishable. Minus time indicates the time before metaphase, while plus time means the time after metaphase. The mother RG progenitor undergoing division is indicated by white arrowheads, while the two daughter cells are indicated by yellow arrowheads. Arrows point to the two centrosomes in the dividing RG progenitor. Scale bar: 10 μm.

Figure 3

Two‐color protein and tumor labeling using iBlueberry and mIFP. (a) Two‐color protein labeling in living HEK293 cells: actin filaments marked by Lifeact‐iBlueberry (left), nucleus labeled by histone 2B (H2B)‐mIFP (middle). The merged image is shown on the right. Scale bar, 10 μm. (b, c) Two‐color tumor labeling in mice. The tumor on the right part of the mouse body expresses iBlueberry IRES GFP, whereas the tumor on the left part expresses mIFP IRES GFP. The fluorescence from GFP (b), iBlueberry (c, top), and mIFP (c, middle) was detected using appropriate filters. See text for details.