ИСТИНА

Optical reporters are widely used in cell biology to tag and monitor molecular processes non-invasively in living cells or in whole intact animals. While less bright than fluorescent probes, bioluminescent systems have several distinct advantages since they do not require external illumination and have extremely low background signals. The applications of luciferase reporters fall into two main groups. Firstly, bioluminescence can be used to monitor temporal changes in gene expression and protein-protein interactions. Secondly, specific types of cells can be marked with constitutively high levels of luciferase and then imaged in intact animals to follow tumor growth or spread of pathogens. One can monitor either a total light output from an object with a photomultiplier tube or perform a continuous 2D-imaging with a highly sensitive camera. The particular benefit of the beetle luciferin/luciferase system is the ability to perform continuous long-term real-time monitoring both in live cell cultures and in animals up to several weeks and with up to single cell resolution. We report novel mutants of Photinus pyralis firefly luciferase that outperform the widely used beetle luciferases (Eluc, luc2) in brightness in live mammalian cells. Furthermore, some mutants provide faster response to transcriptional changes (surpassing the efficiency of the CL1-PEST degradation tag in reducing luciferase half-live). Interestingly, the shorter half-life of the latter mutants is mediated by a real-time inhibition of luciferase activity in the course of the recording and not due the protein degradation. Thus, their fast performance is expected to be less dependent on the state of proteasomal machinery. Of note, beetle luciferases from different species show widely different protein and mRNA half-lives (associated with their protein and coding DNA sequences). For example, the mutant LmGTS of Luciola mingrelica luciferase provides a faster response as inducible reporter due to its short-lived mRNA and relatively short protein half-life compared to short-lived variants of P. pyralis luciferase. Our findings promote the design of superior beetle luciferase reporters for bioluminescence imaging and monitoring in living systems.