A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

Lima‐Fernandes, Evelyne; Misticone, Stanislas; Boularan, Cédric; Paradis, Justine S.; Enslen, Hervé; Roux, Philippe P.; Bouvier, Michel; Baillie, George S.; Marullo, Stéfano; Scott, Mark G. H.

doi:10.1038/ncomms5431

Cited by 21 publications

(18 citation statements)

References 55 publications

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“…Many uses of LumiFluors are feasible, for example as reporters in GEMM, as fusions with proteins to monitor real-time biological processes in cells, and as biosensor tags for antibodies or small molecule probes that home to select target cells, which could be used to visualize responses in vivo and to determine the margins of select tissues and/or tumors, to aid in surgical procedures (17,44,45). Finally, LumiFluors can also be used in traditional BRET assays to study protein:protein or ligand:protein interactions by developing a split LumiFluor reporter for complementation assays (46,47).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…As an example, a BRET (bioluminescence resonance energy transfer)-based biosensor of the PTEN (phosphatase and tensin homolog) phosphatase has been developed and is composed of the fulllength PTEN protein flanked by a donor Rluc and the acceptor YFP [38]. PTEN biosensor immunoprecipitated from cells displays the same phosphatase activity on PIP3 (phosphatidylinositol 3,4,5 trisphosphate) and AKT (or Protein kinase B) as the wild-type PTEN.…”

Section: Conformational Kinase-based Biosensormentioning

confidence: 99%

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Sizaire¹

2017

Protein Phosphorylation

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Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.

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“…However, the denatured enzymes after immobilization are likely to result in the failure of enzyme catalysis [18,19]. It is well known that suitable pores can act as cells to offer desired room for enzyme immobilization.…”

Section: Introductionmentioning

confidence: 99%

Preparing Co3O4 urchin-like hollow microspheres self-supporting architecture for improved glucose biosensing performance

Ding

Zhao

Fan

et al. 2016

Sensors and Actuators B: Chemical

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A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

Cited by 21 publications

References 55 publications

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

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

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Preparing Co3O4 urchin-like hollow microspheres self-supporting architecture for improved glucose biosensing performance

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