Near-infrared fluorescent proteins engineered from bacterial phytochromes

Abstract

Near-infrared fluorescent proteins (NIR FPs), photoactivatable NIR FPs and NIR reporters of protein-protein interactions developed from bacterial phytochrome photoreceptors (BphPs) have advanced non-invasive deep-tissue imaging. Here we provide a brief guide to the BphP-derived NIR probes with an emphasis on their in vivo applications. We describe phenotypes of NIR FPs and their photochemical and intracellular properties. We discuss NIR FP applications for imaging of various cell types, tissues and animal models in basic and translational research. In this discussion, we focus on NIR FPs that efficiently incorporate endogenous biliverdin chromophore and therefore can be used as straightforward as GFP-like proteins. We also overview a usage of NIR FPs in different imaging platforms, from planar epifluorescence to tomographic and photoacoustic technologies.

Figure 1. Phenotypes of near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochrome photoreceptors (BphPs) and their chromophore photochemistry

(a) In contrast to GFP-like FPs, BphP-based NIR FPs have both excitation and emission peaks in the NIR window (650-900 nm) where mammalian tissue is most transparent to light, and scattering and autofluorescence are low. Therefore, NIR FPs are the optimal fluorescent probes for in vivo imaging, while bright GFP-like FPs are the good choice for microscopy. (b) The domain architecture of BphPs. The photosensory module of BphPs consists of the PAS, GAF and PHY domains. Light irradiation activates the photosensory module, which then transmits the signal to the effector domain to initiate a molecular signaling pathway. As a chromophore, BphPs use biliverdin (BV), which is located in a pocket of the GAF domain and is covalently bound to the Cys in the PAS domain. (c) In eukaryotic cells BV is produced as a product of heme degradation by heme oxygenase. (d) Multicolor permanently fluorescent FPs differ in the way they bind the BV chromophore. The red-shifted NIR FPs (top) bind it the same way as natural BphPs, i.e. by the Cys in the PAS domain via C3² carbon atom of the pyrrole ring A. The engineered blue-shifted NIR FPs (bottom) bind BV by the Cys in the GAF domain via either C3² or C3¹ carbon atoms of side chain of the ring A. In the resulting chromophore adducts, one double bond in the ring A is removed from the conjugation with the rest of the π-electron system resulting in the spectral blue shift. (e) Photoactivatable NIR FPs consist of a whole photosensory module and, similarly to natural BphPs, the bound BV undergoes the cis-trans isomerization of the C15/C16 double bond. In contrast to natural BphPs, the light-induced transition from the fluorescent Pr to non-fluorescent Pfr state is blocked and slowly occurs in darkness only. (f) The bimolecular fluorescence complementation (split) reporters are based on the reconstitution of a fluorescent NIR FP molecule in response to physical interaction of a pair of the fused protein partners. In the process of NIR FP reconstitution, BV chromophore enters the pocket of the GAF domain and may remain non-covalently bound or may form a covalent bond with the Cys in the PAS domain.

Figure 2. Applications of NIR FPs as probes for fluorescence microscopy and whole-body imaging

(a) Whole-body imaging of a dorsal tumor obtained as a metastasis lesion after tail vein injection of iRFP713-labelled IGR-37 melanoma cells (left), and a brain tumor derived by intracranial injection of iRFP713-labelled U87 MG glioblastoma cells (right). Adapted from [32]. Copyright 2014 Society of Photo Optical Instrumentation Engineers. (b) Whole-body imaging of cardiac progenitor cells labelled with either EGFP (left mouse) or iRFP713 (right mouse) transplanted to the heart after myocardial infarction. The fluorescence images are overlaid with the X-ray images Adapted from [37]. Copyright 2014 (available under a Creative Commons Attribution license). (c) Advanced NIR FPs are non-cytotoxic and well expressed in all tissues. The image shows newborn iRFP713-transgenic mice and wild-type non-fluorescent littermates. Adapted from [38]. Copyright 2014 by the Japanese Association for Laboratory Animal Science. (d) Microscopy image showing that iRFP713 is ubiquitously expressed in the primary culture of mouse hippocampal neurons infected with iRFP713-encoded lentivirus. (e) Two-color image of a mammary gland tumor derived from iRFP670-labelled MTLn3 cells and a liver expressing iRFP713 after infection with iRFP713-encoding adenovirus (left mouse). Two-color image of two MTLn3 tumors, one expressing iRFP670 and another iRFP720 (right mouse). Two-color epifluorescence microscopy of HeLa cells transiently co-expressing iRFP670 and iRFP720 targeted to nucleus and mitochondria. The fluorescence signals in all images are presented in pseudocolors. Adapted from [13]. (f) Multicolor in vivo imaging of five types of MTLn3-derived tumors expressing different iRFPs in mice (left). The 19 images in different spectral channels were obtained and used for linear spectral unmixing. Multicolor imaging of MTLn3 cells expressing iRFP670, iRFP682, iRFP702 and iRFP720 obtained using confocal microscopy with spectral detection and subsequent linear unmixing (right). The fluorescence signals in all images are presented in pseudocolors. Adapted from [13]. (g) Photoactivatable NIR FPs allow to increase imaging sensitivity. The contrast enhancement is achieved by acquiring two images: before (left) and after (middle) photoactivation. By subtracting the former image from the latter one the contribution from autofluorescence is largely decreased (right). The tumors were derived from PAiRFP1-expressing MTLn3 cells. Adapted from [19]. (h) Schematics of the rapamycin induced interaction of FKBP and FRB proteins studied with iSplit reporter (left). Without rapamycin, non-interacting fusion proteins do not cause complementation of a NIR FP. After addition of rapamycin, FKBP and FRB interact with each other and bring the fused PAS and GAF domains in a close proximity required for reconstitution of NIR fluorescence. GAFm denotes a mutated version of the GAF domain from iRFP713 optimized for the complementation in the iSplit reporter. In vivo imaging of rapamycin-induced protein-protein interaction in MTln3-based tumor co- expressing PAS-FRB and FKBP-GAFm fusion proteins (right). Adapted from [20]. (i) Schematics of cAMP-induced dissociation of two protein kinase A (PKA) subunits studied with IFP PCA reporter (left). Initially reconstituted IFP PCA is fluorescent, because the PAS and GAF domains are fused to interacting catalytic subunit α and regulatory subunit IIβ. Signaling from β₂-adrenergic receptor induces an increase in cAMP concentration and dissociation of two subunits of PKA, which can be detected by a decrease in NIR fluorescence. Microscopy images of U2OS cells co-expressing two constructs are shown before and after activation of β₂-adrenergic receptor causing cAMP increase (right). Adapted from [21], with permission from Nature Publishing Group.

Figure 3. Detection of NIR FPs using different in vivo imaging techniques

(a) Schematics of planar fluorescence imaging setup that involves wide-field excitation and detection of emission light from a specimen. (b) Visualization of metastasis in mice as an example of planar fluorescence imaging. The metastasis in lymphatic channels (indicated by arrows) originate from a mammary gland tumor (covered) formed after implantation of iRFP713-labeled MDA-MB-231 cells. (Courtesy of EM. Sevick-Muraca, University of Texas, TX). (c) Schematics of fluorescence diffuse tomography setup. The scanning light source in transillumination mode allows to obtain a series of 2D images used for reconstruction. (d) 3D two-color visualization of a tumor derived from iRFP670-labelled MTLn3 cells and a liver expressing iRFP713. Adapted from [13]. (e) Schematics of a fluorescence tomography combined with X-ray computed tomography (XCT). Acquisition of multiple 2D projections is possible by using either multiple source-detector pairs, a scanning excitation source or a rotary gantry-based system. Co-registration of X-ray computed tomography (XCT) provides anatomical information and is used for more precise tomographic reconstruction of the fluorescence signal. (f) Hybrid fluorescence/Positron Emission Tomography (PET)/XCT imaging of a prostate tumor derived from iRFP713-expressing PC3 prostate cancer cells. XCT signal is in blue (bones), PET signal is in yellow (liver, prostate and bladder) and iRFP713 signal (tumor) is in red. Adapted from [31]. Copyright 2014 Society of Photo Optical Instrumentation Engineers. (g) Hybrid fluorescence molecular tomography (FMT)/XCT imaging of a mouse brain tumor derived from iRFP713-expressing glioblastoma U87 cells. Adapted from [45]. Copyright 2014 Springer group. (h) Schematics of a fluorescence tomography with a lifetime contrast combined with XCT. For detection of the fluorescence decay curves (right), fluorescence lifetime imaging with the time-domain (TD) detection uses an ultrafast scanning laser (left). This technique allows to separate autofluorescence from NIR FP signal on the basis of their characteristic fluorescence lifetimes. (i) Tomography with fluorescence lifetime contrast of a brain tumor derived from iRFP720-expressing MTLn3 cells. (left). Planar image showing an overlay of the unmixed signals from autofluorescence (green) and iRFP720 (red). The tomographic reconstruction of a tumor co-registered with XCT (middle and right). Adapted from [46]. (j) Schematics of a photoacoustic (also called optoacoustic) tomography. An animal is illuminated with a pulsed laser that induces the thermoelastic expansion of the localized absorbing objects. This expansion generates ultrasonic waves, which are detected. In addition to NIR FPs, the photoacoustic tomography allows to visualize natural absorbers, such as hemoglobin in blood. (k) Photoacoustic imaging of a mammary gland tumor derived from iRFP713-expressing MTLn3 cells (blue) surrounded by blood vessels (red). The image shows an overlay of the spectrally unmixed signals of iRFP713 and total hemoglobin in blood. Adapted from [48]. (l) Photoacoustic imaging of a brain glioblastoma tumor (green) derived from iRFP713-labelled glioblastoma U87 MG cells. The image shows an overlay of a spectrally unmixed signal of iRFP713 and an anatomical image of the head obtained by detection of total hemoglobin in blood. Adapted from [45]. Copyright 2014 Springer group.