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.
"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). "
"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. "
[Show abstract][Hide abstract] 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.
"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 . "
[Show abstract][Hide abstract] 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
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.