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 heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.

Phytochromes: An Atomic Perspective on Photoactivation and Signaling

“…Their advantages over other commonly used reporters, such as the suite of green fluorescent protein derivatives, are their smaller monomeric sizes (220 amino acids), the ability to regulate chromophore availability and spectrally tune light absorption, and, importantly, the fact that the excitation and emission spectra of some are in the red/far-red regions of the visible spectrum, which extends their utility to otherwise opaque tissues or even whole organisms (Shu et al, 2009). Improvements have been made recently in fluorescence brightness and extension to near-infrared wavelengths through directed evolution, thus adding Phy mutantbased tags to the arsenal of fluorescence imaging tools (Filonov et al, 2011;Lecoq and Schnitzer, 2011;Auldridge et al, 2012;Yu et al, 2014).…”

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc

“…We tracked BAC-encoded Hh:GFP and Ptc:mCherry in wing discs that expressed membrane-tethered infrared fluorescent protein (Yu et al, 2014) in the ptc expression domain ( ptc-Gal4 UAS-IFP2.0). Cytonemes extending from A compartment cells were detected by their infrared fluorescence (Fig.…”

“…Stocks were obtained from Bloomington Drosophila Stock Center (BDSC): UAS-disp RNAi (#27247), UAS-dia RNAi (#28541), tub-Gal80 ts (#7018), UAS-CD8:GFP (#5137), yw, ubi-RFP.nls FRT40A/CyO (#34500) and UAS-Nrg RNAi (#37496). UAS-CD8: mCherry (described in Roy et al, 2011); UAS-Hh:CD2 (described in Strigini and Cohen, 1997); and UAS-CD4:IFP2.0-T2A-HO1 (described in Yu et al, 2014). Stocks were also obtained from Vienna Drosophila Research Center (VDRC) Stock Center: UAS-SCAR RNAi (#21908) and UAS-Syb RNAi (102922KK).…”

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

“…BV is a catabolic metabolite of heme by heme oxygenase and is nonfluorescent by itself. BV binds to the GAF domain noncovalently and forms a thioether bond with a conserved cysteine near N terminus of IFPs (3,6).…”

“…This might suggest an intrinsic limitation of the protein topology of GFP, which was introduced into molecular imaging two decades ago. Recently engineered infrared fluorescent proteins (IFPs) from bacterial phytochromes (BphPs) provide a new scaffold for designing such reporters (3,4).…”