Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging

De, Abhijit; Jasani, Akshi; Arora, Rohit; Gambhir, Sanjiv S.

doi:10.3389/fendo.2013.00131

Cited by 44 publications

(36 citation statements)

References 41 publications

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“…BRET is similar to FRET except that luciferase is used as the energy donor (Lohse et al ., *; De et al ., ). One advantage over FRET is the absence of potential direct excitation of the energy acceptor.…”

Section: Tools and Strategies To Investigate Physical Proximity Betwementioning

confidence: 97%

In vivo opioid receptor heteromerization: where do we stand?

Massotte

2014

British J Pharmacology

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to correct some mis-citations in text and tables, these are marked by an asterisk.Opioid receptors are highly homologous GPCRs that modulate brain function at all levels of neural integration, including autonomous, sensory, emotional and cognitive processing. Opioid receptors functionally interact in vivo, but the underlying mechanisms involving direct receptor-receptor interactions, affecting signalling pathways or engaging different neuronal circuits, remain unsolved. Heteromer formation through direct physical interaction between two opioid receptors or between an opioid receptor and a non-opioid one has been postulated and can be characterized by specific ligand binding, receptor signalling and trafficking properties. However, despite numerous studies in heterologous systems, evidence for physical proximity in vivo is only available for a limited number of opioid heteromers, and their physiopathological implication remains largely unknown mostly due to the lack of appropriate tools. Nonetheless, data collected so far using endogenous receptors point to a crucial role for opioid heteromers as a molecular entity that could underlie human pathologies such as alcoholism, acute or chronic pain as well as psychiatric disorders. Opioid heteromers therefore stand as new therapeutic targets for the drug discovery field. ]enkephalin; DRG, dorsal root ganglion; GNTI, guanidinonaltrindole; GRP, gastrin-releasing peptide; HIV, human immunodeficiency virus; IHC, immunohistochemistry; ISH, in situ hybridization; i.t., intrathecal; KDOP, κ opioid receptor δ opioid receptor agonist antagonist; KOP, κ opioid receptor; MDAN, μ opioid receptor δ opioid receptor agonist antagonist; MOP, μ opioid receptor; NNTA, N-naphthoyl-β-naltrexamine; norBNI, nor-binaltorphimine; pbFRET, photobleaching FRET; TAT, transactivating transcriptional activator LINKED ARTICLESThis The opioid systemThe opioid system is composed of three families of endogenous peptides, the enkephalins, dynorphins and β-endorphin, and three homologous GPCRs, the μ opioid receptor (MOP), δ opioid receptor (DOP) and κ opioid receptor (KOP) (Filizola and Devi, 2013;Cox et al., 2015; receptor nomenclature follows Alexander et al., 2013a). Opioid receptors and endogenous opioid peptides are expressed throughout the nervous system (Le Merrer et al., 2009). The opioid system plays a key role in reward and motivation, and regulates emotional responses and cognition. The system also modulates nociception, neuroendocrine physiology and autonomic functions (see Walwyn et al., 2010;Feng et al., 2012). The involvement of the three opioid receptors in pain control, drug abuse and mood disorders has been extensively studied and has been the focus of recent reviews (Bruchas et al., 2010;Gaveriaux-Ruff and Kieffer, 2011;Pradhan et al., 2011;Raehal et al., 2011;Lutz and Kieffer, 2012;Nadal et al., 2013;Charbogne et al., 2014).Several decades of opioid pharmacology have uncovered the complexity of the opioid system physiology. In particular, the analysis of the effects of opioid drugs ...

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Section: Tools and Strategies To Investigate Physical Proximity Betwementioning

confidence: 97%

In vivo opioid receptor heteromerization: where do we stand?

Massotte

2014

British J Pharmacology

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“…Conversely, bioluminescent enzymes are limited by wide variations in signal intensity and duration. To resolve these problems, bioluminescence resonance energy transfer-based (BRET) reporters employing direct fusion of a donor luciferase moiety and a fluorescent acceptor moiety have emerged as promising tools for monitoring complex biological processes, including tumor development and progression (19,20). The majority of BRET reporters are designed with Renilla luciferase ( RLuc ) and variants thereof, which serve as the donor molecule to a yellow fluorescent acceptor molecule, although firefly luciferase ( FLuc ) BRET fusions have also been made.…”

Section: Introductionmentioning

confidence: 99%

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

et al. 2015

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Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

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“…In this instantiation, both the intensity and spectral properties of bioluminescent proteins are altered when they are associated with fluorescent proteins in a process termed bioluminescence resonance energy transfer. [31][32][33] Luciferase proteins have also [34][35][36][37][38] Given the expanding toolbox of bioluminescent proteins and their wide variety of applications (see reviews by Badr and Tannous 39 and Saito and Nagai 40 ), it is timely to look at methods of detection of bioluminescence in laboratories not necessarily set up for bioluminescence imaging per se or unwilling to purchase commercially available bioluminescence imagers (e.g., Olympus LV200 microscope, IVIS Spectrum animal imager). In this paper, we discuss advantages and disadvantages of the various methods we have utilized for detection and quantification of bioluminescent signals from live cells and animals.…”

Section: Introductionmentioning

confidence: 99%

Bioluminescence imaging in live cells and animals

et al. 2016

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Abstract. The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging

Cited by 44 publications

References 41 publications

In vivo opioid receptor heteromerization: where do we stand?

In vivo opioid receptor heteromerization: where do we stand?

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

Bioluminescence imaging in live cells and animals

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