Amit P. Jathoul

Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promegas codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λmax=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy

Genetically expressed fluorescent proteins have been shown to provide photoacoustic contrast. However, they can be limited by low photoacoustic generation efficiency and low optical absorption at red and near infrared wavelengths, thus limiting their usefulness in mammalian small animal models. In addition, many fluorescent proteins exhibit low photostability due to photobleaching and transient absorption effects. In this study, we explore these issues by synthesizing and characterizing a range of commonly used fluorescent proteins (dsRed, mCherry, mNeptune, mRaspberry, AQ143, E2 Crimson) and novel non-fluorescent chromoproteins (aeCP597 and cjBlue and a non-fluorescent mutant of E2 Crimson). The photoacoustic spectra, photoacoustic generation efficiency and photostability of each fluorescent protein and chromoprotein were measured. Compared to the fluorescent proteins, the chromoproteins were found to exhibit higher photoacoustic generation efficiency due to the absence of radiative relaxation and ground state depopulation, and significantly higher photostability. The feasibility of converting an existing fluorescent protein into a non-fluorescent chromoprotein via mutagenesis was also demonstrated. The chromoprotein mutant exhibited greater photoacoustic signal generation efficiency and better agreement between the photoacoustic and the specific extinction coefficient spectra than the original fluorescent protein. Lastly, the genetic expression of a chromoprotein in mammalian cells was demonstrated. This study suggests that chromoproteins may have potential for providing genetically encoded photoacoustic contrast.

An MRAS, SHOC2, and SCRIB Complex Coordinates ERK Pathway Activation with Polarity and Tumorigenic Growth

SHOC2 is mutated in Noonan syndrome and plays a key role in the activation of the ERK-MAPK pathway, which is upregulated in the majority of human cancers. SHOC2 functions as a PP1-regulatory protein and as an effector of MRAS. Here we show that SHOC2 and MRAS form a complex with SCRIB, a polarity protein with tumor suppressor properties. SCRIB functions as a PP1-regulatory protein and antagonizes SHOC2-mediated RAF dephosphorylation through a mechanism involving competition for PP1 molecules within the same macromolecular complex. SHOC2 function is selectively required for the malignant properties of tumor cells with mutant RAS, and both MRAS and SHOC2 play a key role in polarized migration. We propose that MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway spatiotemporal dynamics with polarity and that this complex plays a key role during tumorigenic growth.

In vivo photoacoustic imaging of tyrosinase expressing tumours in mice

Two human tumour cell lines (K562, 293T) were stably transfected to achieve the genetic expression of tyrosinase, which is involved in the production of the pigment eumelanin. The cells were injected subcutaneously into nude mice to form tumour xenografts, which were imaged over a period of up to 26 days using an all-optical photoacoustic imaging system. 3D photoacoustic images of the tumours and the surrounding vasculature were acquired at excitation wavelengths ranging from 600nm to 770nm. The images showed tumour growth and continued tyrosinase expression over the full 26 day duration of the study. These findings were confirmed by histological analysis of excised tumour samples.

Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging

BackgroundIn vivo imaging using genetic reporters is a central supporting tool in the development of cell and gene therapies affording us the ability to selectively track the therapeutic indefinitely. Previous studies have demonstrated the utility of the human norepinephrine transporter (hNET) as a positron emission tomography/single photon emission computed tomography (PET/SPECT) genetic reporter for in vivo cellular imaging. Here, our aim was to extend on this work and construct a tricistronic vector with dual optical (firefly luciferase) and nuclear (hNET) in vivo imaging and ex vivo histochemical capabilities. Guiding this development, we describe how a fluorescent substrate for hNET, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), can be used to optimise vector design and serve as an in vitro functional screen.MethodsVectors were designed to co-express a bright red-shifted firefly luciferase (FLuc), hNET and a small marker gene RQR8. Genes were co-expressed using 2A peptide linkage, and vectors were transduced into a T cell line, SupT1. Two vectors were constructed with different gene orientations; FLuc.2A.RQR8.2A.hNET and hNET.2A.FLuc.2A.RQR8. hNET function was assessed using ASP+-guided flow cytometry. In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.ResultsSupT1/FLuc.2A.RQR8.2A.hNET cells resulted in >4-fold higher ASP+ uptake compared to SupT1/hNET.2A.FLuc.2A.RQR8, suggesting that 2A orientation effected hNET function. SupT1/FLuc.2A.RQR8.2A.hNET cells were readily visualised with both BLI and SPECT, demonstrating high signal to noise at 24 h post 123I-meta-iodobenzylguanidine (MIBG) administration.ConclusionsIn this study, a pre-clinical tricistronic vector with flow cytometry, BLI, SPECT and histochemical capabilities was constructed, which can be widely applied in cell tracking studies supporting the development of cell therapies. The study further demonstrates that hNET function in engineered cells can be assessed using ASP+-guided flow cytometry in place of costly radiosubstrate methodologies. This fluorogenic approach is unique to the hNET PET/SPECT reporter and may prove valuable when screening large numbers of cell lines or vector/mutant constructs.

Development of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging

Firefly luciferase (Fluc) catalyzes a bioluminescent reaction using the substrates ATP and beetle luciferin in the presence of molecular oxygen (Fig. 1A). Because of its use of ATP and the simplicity of the single-enzyme system, firefly luciferase is widely used in numerous applications, notably those involving detection of living organisms, gene expression or amplification in both in vivo and in vitro systems.

Evaluation of genetically expressed absorbing proteins using photoacoustic spectroscopy

Genetically expressed contrast agents are of great interest in the life sciences as they allow the study of structure and function of living cells and organisms. However, many commonly used fluorescent proteins present disadvantages when used in mammalian organisms, such as low near-infrared absorption and photostability. In this study, a variety of genetically expressed fluorescent proteins and novel chromoproteins were evaluated using photoacoustic spectroscopy. The results showed that chromoproteins provide stronger photoacoustic signals, better spectral stability, and exhibit less photobleaching than fluorescent proteins.

I23 Tracking transplanted human foetal cells using bioluminescence imaging in a huntington’s disease model

Background Efforts in cell replacement therapy for Huntington’s disease (HD) are hindered by uncertainty of graft fate after transplantation. Current cell tracking methods fall short to either: 1) Detect labelled transplanted cells, or 2) Differentiate between live and dead cells, or 3) Provide long term tracking of growing grafts. Bioluminescence imaging (BLI) addresses these problems and could provide a cheaper, more reliable and less inconvenient alternative to currently used intracerebral cell tracking techniques. Aim We aimed to describe methods for long term tracking of transplanted Human Foetal Cells (HFC) using BLI in a HD rat model. Methods In-vitro: We have developed protocols to transduce Human Embryonic Kidney cells, Medium Spiny Neuron Progenitors and eventually HFCs with a lentivirus carrying a firefly luciferase gene. In-vivo: Using quinolinic acid lesioned adult female Sprague-Dawley rats as a HD model, 4 animals underwent transduced-cells transplantation followed immediately by BLI using intracerebral (IC) luciferin injection. These animals had pre and post-operative MRI for comparison. Five animals underwent BLI with IC luciferin at 12 weeks. Eight animals underwent acute BLI after transplantation using intraperitoneal (IP) Luciferin alternatives, namely: AkaLumin, D-Luciferin, D-Luciferin packaged in a nano vector and InfraLuciferin. Results In-vitro: All tested cell types were reliably transduced with a viral vector to express engineered luciferase. HFC viability inversely correlated with duration of incubation with the virus. In-vivo: HFC grafts were detected using BLI acutely and at 12 weeks after transplantation. D-Luciferin packaged in a nano vector showed promising results after IP injection, however signal is considerably weaker than IC injection. Conclusion BLI reliably and conveniently tracks transplanted HFC in small animals. This technique has great potentials in studying graft fate in terms of localization, viability, growth and integration.

ΔFlucs: brighter photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi‐rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an “omega‐loop” motif (T352‐G360) significantly enhanced activity, altered kinetics, reduced Km for D‐luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL‐infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω‐loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.