Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.

No abstract

Venus ͉ Cerulean ͉ YFP ͉ cross-talk ͉ NFAT P rotein-protein interactions play a pivotal role in mediating signal-transduction pathways and executing cellular functions. Defining how each protein interacts with all possible partners in cells provides insight into cellular roles of individual proteins. This task has become more demanding given that many interacting networks have been generated in the postgenome era (1, 2). Although a number of methods are available for protein interaction study, several fluorescent protein-based methods, such as FRET and bimolecular fluorescence complementation (BiFC), are most widely used because of direct visualization and easy operation (3-5).FRET is an assay that can measure the proximity or distance between a donor and an acceptor. If two fluorophores are fused to a pair of interacting proteins, a fraction of an excited donor fluorophore nonradiatively transfers energy to an acceptor molecule. The efficiency of the energy transfer is defined as the fraction of donor excitation events that results in energy transfer to an acceptor. Hence, FRET efficiency can be used as an indicator of protein-protein interactions (6). Although the most commonly used fluorescent proteins for FRET analysis are CFP and YFP (4-6), development of new fluorescent proteins with improved properties has significantly expanded our choice of selecting appropriate fluorescent proteins for live-cell imaging (4). For example, the YFP mutant Venus and the CFP mutant Cerulean have been developed, and these mutants have shown better properties for live-cell imaging (7-11).We previously used CFP and YFP to develop a BiFC assay and a multicolor BiFC assay for visualization of protein interactions in living cells (12,13). Over the past few years, these assays have been widely used for visualization of protein-protein interactions in living cells and in different model systems (3). Because the YFP-and CFP-based BiFC assays required a preincubation of cells at lower temperature before visualization of BiFC signals because of their sensitivity to higher temperatures, we recently improved the system and demonstrated that several newly developed fluorescent proteins, such as Venus and Cerulean, can be used for BiFC and multicolor BiFC analysis (14). The use of Venus and Cerulean not only allows for BiFC analysis under physiological conditions but also increases the signal output and the specificity (14).Although determination of individual interacting proteins by FRET and BiFC assays provides useful information for function of a pair of interacting proteins, proteins often form multiple protein complexes such as ternary complexes. In particular, many signaling proteins function as ternary complexes. These complexes include membrane-bound receptors, cytoplasmic signaling molecules, and transcriptional regulatory complexes in the nucleus. For example, the formation of ternary complexes of transcriptional regulatory proteins is critical for gene transcription. These complexes include cross-talk between different families...

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.

ATF2 and c‐Jun are key components of activating protein‐1 and function as homodimers or heterodimers. c‐Jun–ATF2 heterodimers activate the expression of many target genes, including c‐jun, in response to a variety of cellular and environmental signals. Although it has been believed that c‐Jun and ATF2 are constitutively localized in the nucleus, where they are phosphorylated and activated by mitogen‐activated protein kinases, the molecular mechanisms underlying the regulation of their transcriptional activities remain to be defined. Here we show that ATF2 possesses a nuclear export signal in its leucine zipper region and two nuclear localization signals in its basic region, resulting in continuous shuttling between the cytoplasm and the nucleus. Dimerization with c‐Jun in the nucleus prevents the export of ATF2 and is essential for the transcriptional activation of the c‐jun promoter. Importantly, c‐Jun‐dependent nuclear localization of ATF2 occurs during retinoic acid‐induced differentiation and UV‐induced cell death in F9 cells. Together, these findings demonstrate that ATF2 and c‐Jun mutually regulate each other by altering the dynamics of subcellular localization and by positively impacting transcriptional activity.