High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of splitRenillaluciferase

Abstract

Nucleocytoplasmic trafficking of proteins in response to extraor intracellular stimuli is an essential step for regulating the magnitude and specificity of gene expressions. The trafficking is regulated by posttranslational modifications of proteins, which include ligand-receptor binding, protein phosphorylation, and proteolysis. The nuclear localization of those proteins is altered in the cells that are exposed to specific exogenous chemicals, of which potential effects on living animals are the major concern. Also, because various nuclear proteins are mislocalized in cancer cells, there is an intense interest in identifying small molecules that redirect the proteins to the correct compartments. Hence, development of a rapid screening system to detect the nucleocytoplasmic trafficking is essential for the discovery of novel compounds that have anticancer activity or for testing the toxicity of chemicals, from which new insights into the mechanism of nucleocytoplasmic trafficking could be provided (1-4).A technique for monitoring the dynamics of the protein movement inside single living cells relies on the use of immunocytochemistry or optical imaging with genetically tagged GFP (5). These analyses are effective for imaging the spatial and temporal dynamics of proteins of interest within single living cells. For high-throughput analysis of the protein movement inside the cells, automated fluorescence microscopy has been developed (2). Although such technological progress is important, image acquisition can be slow and tedious. In particular, algorithms to automatically determine nuclear vs. cytoplasmic localization in an acquired image still remain imprecise and slow. The obtained results are qualitative rather than quantitative because of the limited number of analyzed cells. In addition, analyses of the protein localization in living animals require complex assay procedures such as extraction of an organ and dividing it into sliced sections, which hampers temporal and quantitative analyses.In our previous studies, a system of split firefly luciferase reconstitution by protein splicing has been developed for detecting protein-protein interactions in cell lines and in living mice, in which the novel use of the new luminescent reporter protein was demonstrated (6, 7). We and others have also reported a split Rluc complementation system for imaging tyrosine phosphorylation and to monitor protein-protein interactions in living cells, where bioluminescence originated from Rluc complementation was sensitively detected (8, 9). A method for identifying mitochondrial proteins by using split GFP reconstitution by protein splicing has recently been developed by our group (10). These studies suggest a great potentiality of Rluc for rapid sensing of intracellular protein movements in living cells and animals.Taking advantage of the bioluminescence of Rluc with background-fluorescence-free and high-sensitive detection, we developed an indicator with general applicability for detecting transport into the nucleus of a particul...

Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

“…38,39 Even though the functions of different proteins are determined by posttranslational modification, the only way by which proteins trafficking within and between cells can be currently studied is by immunocytochemistry or by tagging with fluorescent proteins. 39,40 However, the subcellular localization of proteins by these microscopic techniques is not suitable for quantitative analysis and for the highthroughput screening of a larger number of samples. Hence, the strategy developed in this study compensates for both of these drawbacks and also provides an additional advantage for the use of this system in living animals via noninvasive imaging.…”

Establishment of a newly improved detection system for NF-κB activity

“…There are several candidates for suppressing background signals in the absence of stimulations. The first candidate is a split luciferase system, which has the advantage of being able to detect protein-protein interaction in vivo with a low background level [21].…”

Methods for Imaging and Analyses of Intracellular Organelles Using Fluorescent and Luminescent Proteins

“…2C). 16,17 The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (Rluc) by protein splicing. A target cytosolic protein fused to the C-terminal fragment of Rluc is expressed in mammalian cells.…”

“…The indicator enabled noninvasive in vivo imaging of the translocation of androgen receptor into the nucleus in the brains of living mice with a charge-coupled device imaging system. 16 This basic principle was also used to detect phosphorylation-or proteolysis-induced nuclear transports of a target protein. 17 Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation-and proteolysis-induced nuclear transport, respectively.…”