Engineered luciferases for molecular sensing in living cells
ABSTRACT As a means for visualizing molecular physiology within living cells, new strategies are emerging for engineering luciferases into intracellular biosensors. These biosensors can be classified as bimolecular, relying on complementation of luciferase fragments, or intramolecular, relying on domain insertion within the luciferase structure. Multiple design strategies have recently surfaced for the development of intramolecular sensors, allowing dynamic detection of small molecules or post-translational modifications within cells. Building on successes achieved in cell culture, these sensors are now beginning to reveal molecular processes within living organisms.
- SourceAvailable from: Hirobumi Suzuki
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- "developmental biology (Akiyoshi et al., 2014), medical research (Horibe et al., 2014; Sramek et al., 2011), signal transduction analysis (Hall et al., 2012; Roger et al., 2008; Sugiyama et al., 2014), molecular interaction analysis (Binkowski et al., 2009; Cosby, 2009), and radiation biology (Pratx et al., 2012, 2013). "
ABSTRACT: Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.Microscopy Research and Technique 06/2015; DOI:10.1002/jemt.22529 · 1.17 Impact Factor
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- "Not only is luciferase activity readily measurable in a broad dynamic range with high sensitivity, luciferase activity can be measured continuously over time in response to various conditions making luciferase an excellent model enzyme to investigate deactivation. In addition to its broad use in gene reporter assays and in various bioanalytical assays (ATP detection down to attomole concentrations are possible), firefly luciferase was also used to study protein refolding as well as the effects of MHz electromagnetic radiation on biological systems   . Since luciferase is a fairly " typical " monomeric enzyme, detailed understanding of mechanisms of luciferase thermal stability will advance specific applications and can contribute to general understanding of mechanisms that affect stability of this and other enzymes under various conditions. "
ABSTRACT: The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.Biochimica et Biophysica Acta 10/2011; 1814(10):1318-24. DOI:10.1016/j.bbapap.2011.06.010 · 4.66 Impact Factor
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- "Genetic manipulation of firefly luciferase into a reversible biosensor of cAMP generation involved the insertion of the cAMP binding domain B from the regulatory subunit type IIb into a circularly permuted form of firefly luciferase (Fan et al., 2008; Binkowski et al., 2009). In vitro, the firefly luciferasebased cAMP biosensor has a pEC50 for cAMP of 6.3 and a large signal-to-noise window of approximately 70-fold (Fan et al., 2008). "
ABSTRACT: It is clear that the G protein-coupled receptor family play a key role in the pharmaceutical industry, with a significant proportion of approved drugs targeting this protein class. While our growing understanding of the complexity of G protein-coupled receptor pharmacology is playing a key role in the future success of these endeavours, with allosteric mechanisms now well integrated into the industrial community and G protein-independent signalling mechanisms establishing themselves as novel phenomenon to be exploited, it is still possible to underestimate the complexity of G protein signal transduction mechanisms and the impact that inappropriate study of these mechanisms can have on data interpretation. In this manuscript we review different approaches to measuring the cAMP signal transduction pathway, with particular emphasis on key parameters influencing the data quality and biological relevance.British Journal of Pharmacology 11/2010; 161(6):1266-75. DOI:10.1111/j.1476-5381.2010.00779.x · 4.99 Impact Factor