Trends in Biotechnology
ReviewFluorescence complementation: an emerging tool for biological research
Introduction
Molecular interactions in cells execute a huge range of cellular events, ranging from transmitting genetic information to controlling cellular functions. Direct visualization of molecular interactions can provide important insight into their underlying mechanisms. During the past decade, engineering and applications of fluorescent proteins with distinct spectral properties have increased our understanding of a wide variety of molecular events 1, 2, 3, 4. Among the many applications of fluorescent proteins, fluorescence complementation (FC) has emerged as a new tool for visualizing molecular events, particularly those involving molecular interactions in living cells and organisms. The basic strategy of FC is to split a fluorescent protein into two non-fluorescent fragments that are fused to a pair of interacting proteins (Figure 1a). The interaction between the two proteins brings the two fragments into close proximity, allowing reconstitution of an intact fluorescent protein. Thus, the reconstituted fluorescence is an indication for the interaction of two proteins. The applications of this basic design for visualization of a variety of protein–protein interactions, for screens of interacting proteins and for drug discovery are reviewed in detail elsewhere 5, 6, 7, 8, 9. This review will focus specifically on FC-based technologies that have been developed over the past five years for visualization of many other molecular events that go beyond those involving a single pair of interacting proteins. We will use the term ‘FC’ to refer to any method that involves the use of fragments that are derived from one or more fluorescent proteins and the reconstitution (hence ‘complementation’) of intact fluorescent protein molecules induced by molecular events. Other protein-fragment complementation assay (PCA)-based methods that utilize ubiquitin [10] or enzymes such as dihydrofolate reductase (DHFR) [11], β-glactosidase [12], luciferase [13] and β-lactamase 14, 15 and a protein-splicing-based ligation of a split green fluorescent protein (GFP) [16] are not covered here.
Section snippets
FC-competent proteins
The first demonstration of complementation based on fluorescent protein fragments was the antiparallel leucine zipper-mediated reassembly of GFP [17] (Figure 1a). By mixing two α-helix-tagged fragments of GFP, each by itself non-fluorescent, either in vitro or by coexpression in Escherichia coli, fluorescence could be restored [17]. It was also shown in HeLa cells that the interaction between calmodulin and the M13 peptide that had been fused to fragments of enhanced yellow fluorescent protein
Visualization of multiple protein interactions and multiple protein complexes
Many proteins selectively interact with specific partners in response to specific stimuli. Demonstration of a selective interaction among numerous possible interacting proteins in response to a specific signaling event can therefore be used to provide indication of the underlying signaling pathways. Furthermore, signal transduction often requires an integration of multiple upstream signals through the formation of multiple protein complexes. Such interactions are converging points of signaling
Visualization of post-translational modifications
Several post-translational modifications have important roles in regulating protein functions and can lead to changes in subcellular location and interactions with signaling molecules. The FC technology has been used to visualize one of these modifications, the ubiquitylation of proteins. By fusing non-fluorescent protein fragments of EYFP to ubiquitin and to its putative substrate, an ubiquitin-mediated fluorescence complementation (UbFC) assay was developed that made it possible to visualize
Visualization of protein folding and aggregation
Protein folding has long been intensively investigated by researchers working in the field of protein science. However, most protein-folding studies are performed in vitro because only limited methods for their in vivo study are available. Recently, a so-called superfolder GFP (sfGFP) was identified [37] that exhibits an increased global stability of its β-can structure arising from the introduction of six additional mutations into the previously reported folding reporter GFP [38].
Visualization of conformational changes and topology
Traditionally, conformational changes in proteins have been studied using spectroscopic methods. However, the use of GFP variants, which can serve as FRET pairs, has stimulated the development of numerous FRET-based biosensors, such as protein kinase and small GTPase biosensors 4, 42, 43, 44, 45. As an alternative to FRET-based biosensors, a split-GFP-based biosensor has been developed to measure maltose concentration [46]. This maltose sensor constitutes the maltose binding protein (MBP), to
Visualization of molecular events involving other biomolecules
In addition to the widespread use of FC for visualizing protein interactions, FC-based technologies have also been developed for the detection of molecular events involving RNA and DNA. The Brown group developed a trimolecular fluorescence complementation (TriFC) assay for visualizing RNA–protein interactions [25] (Figure 3a). TriFC is based on the use of a reporter mRNA that contains the well-characterized coat protein-RNA operator from the bacteriophage MS2, as well as the mRNA sequence of
FC-based applications in C. elegans
C. elegans is an excellent model organism for biological research, and its transparent body makes it particularly useful for the applications of fluorescent proteins [53]. Using the leucine zipper-based FC of split GFP (i.e. NZGFP and CZGFP) first reported by the Regan group [17], Zhang et al. demonstrated several elegant applications in living C. elegans that used two different promoters to drive the expression of NZGFP and CZGFP [54] (Figure 3e). The coexpression of NZGFP and CZGFP in any
Challenges and perspectives
Although the FC-based technologies have already found applications in direct visualization of various molecular events, the underlying technologies are still in their infancy. To maximize their biological applications, further improvements and developments in several areas are needed.
Conclusion
FC-based technologies have already revolutionized many aspects of current biological research. One unique feature of these approaches is the direct visualization of protein–protein interactions in living cells and organisms. FC-based assays have been used for the identification and analysis of events such as post-translational modifications, protein folding and aggregation, conformational changes and protein topology. Other molecular events, such as RNA localization, RNA–protein interactions
Acknowledgements
We are grateful to Elizabeth J. Taparowsky and Joseph L. Borowitz for their critical reading of this manuscript. We thank the members of the Hu laboratory and many other laboratories for their contributions to the development of FC-based technologies. C.D.H. was supported by the Purdue Cancer Center and by grants from the National Science Foundation and the American Heart Association. Owing to space constraints, we have emphasized the development of novel FC-based technologies in this review,
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