An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging

Abstract

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.

Figure 1

Directed evolution of a bright and monomeric infrared fluorescent protein (IFP2.0) and engineering of its cofactor biosynthesis. (a) Schematic diagram showing directed evolution of IFP2.0. (b) Crystal structure of IFP2.0 at atomic-resolution (1.13 Å). The mutations relative to IFP1.4 are labeled. 7 (in green) of 11 mutations are newly introduced, the remaining 4 (in cyan) are reverse mutations from IFP1.4 to IFP1.2. 3 of these 4 are located at the c-terminus (barely seen behind the chromophore, Fig. S3). BV is shown in purple. (c) Phytochrome-based IFPs and iRFP incorporate BV as the chromophore, which is an initial product of heme by heme oxygenase (HO1) and becomes fluorescent only when bound to IFPs. (d) Comparison of the cellular brightness of IFP1.4, IFP2.0, iRFP, and IFP2.0 + HO1 in three cell types: primary hippocampal neuron, glioma cell LN229 and cervical cancer cell HeLa. Fluorescence is normalized by co-expressed GFP. The error bar represents standard deviation (n = 10). (e) Representative fluorescence images of neurons showing significant increase of cellular brightness by co-expression of HO1. Scale bar, 40 μm.

Figure 2

IFP2.0 with engineered biosynthesis of the cofactor improves neuronal imaging in Drosophila larvae Cellular membrane of dendritic arborization (da) neurons labeled by (a, d, e) IFP2.0 fused to CD4 with expression of HO1 that produces the cofactor (CD4-IFP2.0 + HO1); (f) CD4-IFP2.0; (g) CD4-iRFP. (b) Fluorescence intensity profile along the line in (a); the green and red arrow point to the dendrites in (a). (c) Normalized fluorescence intensity of the cell body pointed by the yellow arrow in (a) and (f). (e) Confocal image of the area (blue box) in (d), with arrows pointing to dendritic spikes. Scale bar: (a, d, f, g), 100 μm; (e), 20 μm.

Figure 3

IFP2.0 with engineered biosynthesis of the cofactor improve imaging of tumors in mice. (a) Bright field (left), fluorescence (middle), and overlay (right) images of the brain tumor (glioblastoma) in intact mice expressing IFP2.0 + HO1 (top), iRFP (bottom). The fluorescence intensity in the overlay image of iRFP is 3-fold (3x) brightened compared to that of IFP2.0 + HO1. The scale bar indicates fluorescence radiant efficiency. (b) Average fluorescence intensity of the brain tumor in intact mice with standard deviation. (c) Bright field (top) and fluorescence overlay (bottom) images of the extracted brain. (d) Confocal images of the brain slices. The right panels show 3x brightened images. Size scale bar: (a), 1 cm; (c), 3 mm; (d), 40 μm.