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; 78(8). DOI:10.1002/jemt.22529 · 1.15 Impact Factor
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- "To overcome the problem of unintentional activation of cellular processes upon excitation with light in conventional fluorescence imaging, we constructed two bioluminescent indicators modelled after established luciferase-based indicators for Ca2+ 12 and cAMP13141516 (reviewed in ; Fig. 2a). These indicators contained a calmodulin-M13 Ca2+ sensor domain (cpGL-CaM) or a mouse protein kinase-A regulatory subunit cAMP-binding domain (cpGL-α-CT) fused to firefly luciferase. "
ABSTRACT: Molecular imaging is a powerful tool for investigating intracellular signalling, but it is difficult to acquire conventional fluorescence imaging from photoreceptive cells. Here we demonstrated that human opsin5 (OPN5) photoreceptor mediates light-induced Ca(2+) response in human embryonic kidney (HEK293) and mouse neuroblastoma (Neuro2a) cell lines using a luminescence imaging system with a fluorescent indicator and a newly synthesized bioluminescent indicator. Weak light fluorescence and bioluminescence imaging revealed rapid and transient light-stimulated Ca(2+) release from thapsigargin-sensitive Ca(2+) stores, whereas long-lasting Ca(2+) elevation was observed using a conventional fluorescence imaging system. Bioluminescence imaging also demonstrated that OPN5 activation in HEK293 cells induced a decrease in pertussis toxin-sensitive cAMP, confirming previous reports. In addition, ultraviolet radiation induced the phosphorylation of mitogen-activated protein kinases when OPN5 was stimulated in Neuro2a cells. These findings suggest that the combination of these imaging approaches may provide a new means to investigate the physiological characteristics of photoreceptors.Scientific Reports 06/2014; 4:5352. DOI:10.1038/srep05352 · 5.58 Impact Factor
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- "This makes them a widely used tool in a variety of in vitro and in vivo applications: in ATP-related assays from direct ATP measurements to estimation of bacterial contamination and pyrosequencing [4, 5], in in vivo molecular imaging and as a genetic reporter in molecular biology [6–8]. This enzyme was also shown to be a promising tool for molecular sensing of protein-protein interactions and different analytes [9–11], in analytical assays based on real time monitoring of polynucleotide amplification  and a label for immunoassays . "
ABSTRACT: Luciferase enzymes from fireflies and other beetles have many important applications in molecular biology, biotechnology, analytical chemistry and several other areas. Many novel beetle luciferases with promising properties have been reported in the recent years. However, actual and potential applications of wild-type beetle luciferases are often limited by insufficient stability or decrease in activity of the enzyme at the conditions of a particular assay. Various examples of genetic engineering of the enhanced beetle luciferases have been reported that successfully solve or alleviate many of these limitations. This mini-review summarizes the recent advances in development of mutant luciferases with improved stability and activity characteristics. It discusses the common limitations of wild-type luciferases in different applications and presents the efficient approaches that can be used to address these problems.Computational and Structural Biotechnology Journal 09/2012; 2(3):e201209004. DOI:10.5936/csbj.201209004