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Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can associate to form a fluorescent complex and that the association of the fragments can be facilitated by fusing them to two proteins that interact with each other. The non-covalent association of the fragments produces a bimolecular fluorescent complex. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other since they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at concentrations comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.
Studies of protein interactions have provided fundamental insights into the regulation of cellular functions. Many methods have been developed to investigate protein interactions. Most methods that enable direct detection of protein interactions, such as co-purification and affinity precipitation assays, require removal of the proteins from their native environment. In contrast, methods that enable detection of protein interactions in cells, such as genetic suppressor analysis, generally rely on indirect consequences of the protein interactions.
The visualization of protein interactions in living cells provides the potential for direct detection of protein interactions with minimal perturbation of their normal environment. Many strategies for the visualization of protein interactions in cells have been developed. The most commonly employed are fluorescence resonance energy transfer (FRET) 1 and bimolecular fluorescence complementation (BiFC) 2. Others include fluorescence correlation spectroscopies 3, 4, image correlation spectroscopy 5 and complementation approaches using fragments of other proteins 6-8
FRET and BiFC analysis are fundamentally different approaches and have complementary advantages and disadvantages. FRET is based on the transfer of excitation energy between two fluorophores that are in close spatial proximity and have permissive relative orientations 1. This energy transfer results in a change in the fluorescence intensities and lifetimes of the two fluorophores. FRET analysis of a protein interaction requires quantitation of the change in the fluorescence intensity or lifetime of the donor and acceptor fluorophores in the presence versus the absence of energy transfer between the fluorophores.
BiFC is based on the association between fragments of a fluorescent protein when they are tethered in the same macromolecular complex (Figure 1) 2. Thus, the association produces a fluorescent complex from non-fluorescent constituents. BiFC analysis requires determination of the difference in fluorescence intensities produced by the association of the fluorescent protein fragments in the presence versus the absence of an interaction between the proteins fused to the fragments. BiFC analysis has been used for the visualization of interactions between many different proteins in many cell types and organisms (see Table 1).
FRET enables, in principle, instantaneous monitoring of protein interactions whereas BiFC produces a signal after a delay required for the chemical reactions that generate the fluorophore. BiFC theoretically allows detection of interactions at lower protein concentrations and is predicted to be affected less by changes in cellular conditions that can alter the fluorescence intensities and lifetimes of fluorescent proteins. FRET requires close spatial proximity between the fluorophores whereas BiFC requires that the fluorescent protein fragments have the dynamic flexibility to associate in the protein complex. FRET requires that a large fraction of the fluorescent proteins associate with each other whereas BiFC can detect an interaction involving only a small subset of each fusion protein. Each approach is therefore applicable for different purposes. The fundamental principles and recent applications of the BiFC assay have been reviewed 9.
The BiFC assay is applicable for visualization of the steady-state distributions of complexes formed between virtually any combination of proteins in a wide variety of cell types and organisms. Although the approach is applicable for studies of protein interactions in a wide variety of organisms (see Table 1), the chemistry of fluorophore formation requires molecular oxygen, making this approach unsuitable for use in organisms that are obligate anaerobes. The proteins must be able to accept fusions to fluorescent protein fragments without disruption of their functions. The complex must tolerate stabilization of the interaction by the association between the fluorescent protein fragments without changes in function or deleterious effects for the cell. The BiFC assay is particularly valuable for determining the subcellular locations of protein interactions, which can provide insight into the biological functions of newly discovered protein complexes. Fragments of several proteins that can associate when they are brought together by an interaction between proteins fused to the fragments have been identified. These include fragments of ubiquitin, β-galactosidase and dihydrofolate reductase 6-8. The advantage of BiFC analysis is that association between fragments of fluorescent proteins produces a complex with intrinsic fluorescence, eliminating the need for exogenous stains and enabling direct detection of the protein complex. We have developed several extensions of the BiFC assay that enable visualization of multiple protein interactions in the same cell 10, 11 as well as covalent protein modifications 12. Recent studies have also suggested that BiFC analysis can be used to determine the topology of membrane proteins 13 and for high throughput screening for the effects of small molecules on protein complexes 14. Future efforts will undoubtedly identify many new applications for fluorescence complementation.
The BiFC assay has several characteristics that limit its applicability and should temper interpretations based on results from this assay. One limitation of the BiFC approach is the time required for fluorophore maturation, which reflects the chemical reactions required for formation of the cyclic fluorophore. This prevents real-time detection of rapid changes in interactions using the BiFC assay. It is possible that some of the chemical reactions required for fluorophore formation occur in the isolated fragments, accelerating fluorescence complementation under some conditions 15.
Bimolecular fluorescent complex formation is also likely to affect the dynamics of complex dissociation and partner exchange. Under many in vitro conditions, formation of the bimolecular fluorescent complex is essentially irreversible 2, 16, 17. However, in some experiments, rapid changes in the fluorescence signal have been observed 18, 19. It is possible that bimolecular fluorescent complex formation is reversible under these conditions, but it is difficult to exclude the possibility that other processes, such as protein degradation, affect the signal in living cells. The signal from fluorescence complementation has also been shown to decrease under conditions predicted to reduce complex formation under some in vitro conditions 15, 18. However, dissociation of the fusion proteins has not been directly demonstrated.
Finally, fluorescent protein fragments have a finite ability to associate with each other independent of an interaction between proteins fused to the fragments. This major source of background signal in the BiFC assay varies depending on the identities of the fusion proteins and their levels of expression. Generally, this problem can be alleviated by expression of the fusion proteins at concentrations approximating their endogenous counterparts. In cases where this may not be possible, alternative fusions can be tested for the specificity of fluorescence complementation.
There are many characteristics of the BiFC assay that make it useful for the study of protein interactions. First, it enables direct visualization of protein interactions and does not rely on their secondary effects. Second, the interactions can be visualized in living cells, eliminating potential artifacts caused by cell lysis or fixation. Third, the proteins are expressed in a relevant biological context, ideally at levels comparable to those of their endogenous counterparts. This increases the likelihood that the results reflect the properties of native proteins, including potential effects of post-translational modifications. Fourth, the BiFC assay does not require stoichiometric complex formation but can be used to detect interactions between subpopulations of each protein. Finally, BiFC does not require specialized equipment, apart from an inverted fluorescence microscope equipped with objectives that allow imaging of fluorescence in cells. The direct detection of bimolecular complex formation requires no post-acquisition image processing for interpretation of the data.
This protocol focuses on the visualization of protein interactions in cultured mammalian cells using the BiFC assay 2, but the general principles described are applicable to many other experimental systems. Although this protocol describes the use of transiently transfected plasmid vectors, it is likely that other expression strategies (retroviruses, lentiviruses etc.) can be used with only slight modifications to the protocol.
Inverted fluorescence microscope equipped with:
Several combinations of fluorescent protein fragments support bimolecular fluorescence complementation 11; those recommended for BiFC analysis are listed in Table 2. For most purposes, fragments of YFP truncated at residue 155 (designated YN155 and YC155) are recommended, as they exhibit a relatively high complementation efficiency when fused to many interaction partners, yet produce low fluorescence when fused to proteins that do not interact with each other 2. Fragments of YFP truncated at residue 173 (designated YN173 and YC173) can also be used 11, and may exhibit a different efficiency of complementation due to differences in the steric constraints imposed by tethering of the fragments to the protein complex. Fragments of Venus (a mutated GFP with high fluorescence intensity) 22 truncated at either residue 155 or 173 (designated VN155 and VC155 or VN173 and VC173 respectively) produce a significantly brighter fluorescent signal when fused to specific interaction partners 20. However, these fragments also produce a brighter signal when fused to proteins that do not selectively interact with each other 20. These fragments have the great advantage that the bimolecular fluorescent complex is readily detectable at 37°C, which avoids the incubation at 30°C that is generally necessary to detect complementation using YFP fragments. Other combinations of fluorescent protein fragments can also be used, especially when using BiFC analysis for the visualization of multiple protein complexes in the same cell 11.
Determine the positions of the fusions empirically to fulfill three criteria.
The linkers must provide flexibility for independent motion of the fluorescent protein fragments and the interaction partners, allowing the fragments to associate when the proteins interact. We have used the RSIAT and RPACKIPNDLKQKVMNH linker sequences in many fusion constructs used for BiFC analysis 2, 11. These linkers have been used for the visualization of interactions between many structurally unrelated proteins. The sequence AAANSSIDLISVPVDSR encoded by the multiple cloning sites of the pCMV-FLAG vector (Sigma) has also been successfully used as a linker in many BiFC experiments. A peptide sequence designed to be flexible such as (GGGS)n can also be used, although it can potentially affect the degradation of the fusion protein. Although these linker sequences have worked well for the proteins examined previously, it is possible that linkers of a different length or sequence are optimal for BiFC analysis of interactions between other proteins.
Choose a cell culture system that represents the biological context to be investigated, and allows efficient introduction of DNA into a large fraction of the cells. Cells that grow as an adherent monolayer are generally easier to image. The BiFC assay has been used for the analysis of protein interactions in many mammalian cell lines including COS-1, HEK293, HeLa, Hep3B, αTN4, and NIH3T3 cells as well as in intact organisms 2, 10, 12, 18, 19, 23-67.
Choose either transient expression (A) or stable expression (B) strategies, based on the purpose of the experiment.
This approach may be adequate to determine if a pair of proteins can interact in cells and to determine the subcellular location of the complex. To minimize protein mislocalization and formation of non-native complexes due to overexpression, express the fusion proteins at levels comparable to the endogenous proteins. This can be achieved by using plasmids with weak promoters, by transfecting small amounts of plasmid DNA and by observing the cells as soon as signal is detectable.
More reproducible levels of expression can be obtained by using inducible expression vectors integrated into the genomes of stable cell lines. This allows for the control of protein expression at relatively uniform levels in the entire cell population and replication of experiments at constant expression levels, independent of transfection efficiency and other factors that are difficult to control in transient assays.
As fluorescent protein fragments are able to form fluorescent complexes with a low efficiency in the absence of a specific interaction, it is essential to include negative controls in each experiment. Spontaneous complementation is generally reduced when the fragments are fused to proteins that do not interact with each other; appropriate negative controls are fusion proteins in which the interaction interface has been mutated and fused to the fluorescent protein fragments in a manner identical to the wildtype fusion proteins (Figure 3) 2, 10. Compare the level of expression and localization of the mutated and wild-type fusion proteins by immunoblot and indirect immunofluorescence analyses (using any standard or commercially available method). Quantify and compare the efficiencies of fluorescence complementation between the wild-type and mutated proteins. If there is no prior knowledge of the location or the structural nature of the interaction interface, it is possible to screen for mutations that alter the efficiency of bimolecular fluorescence complementation, and thereby determine if the complementation reflects a specific interaction. The BiFC assay can therefore be used to determine whether two proteins interact in cells without prior knowledge of the location or the structural nature of the interaction interface.
Use some of the numerous fusion proteins whose interactions have been visualized using the BiFC assay as positive controls (see Table 1). However, the failure to detect fluorescence complementation between the proteins under investigation does not demonstrate the absence of an interaction (see ANTICIPATED RESULTS).
Construct plasmid expression vectors, using the appropriate vectors, by fusing the sequences encoding the selected fluorescent protein fragments (see Table 2) to the sequences encoding the proteins of interest. Any standard cloning techniques can be used. Whenever possible, test fusions to both the N- and C-terminal ends of the proteins to be investigated (Figure 2). Construct negative control plasmids that encode mutated non-interacting variants of the proteins (see step 6) using the same strategy. Positive controls should be included to ensure that a known interaction can be detected (see step 6).
Seed cells the day before transfection at an appropriate density. This density should allow for cell proliferation over the course of the experiment while taking into consideration the effects of cell growth and density on the interaction under investigation. Cluster plates are convenient for processing multiple transfections in parallel. If short-working-distance objectives will be used to visualize the interaction, grow the cells in slide chambers or on glass coverslips.
Transfect cells (using the optimal procedure for the cells) with appropriate amounts (e.g. 0.25 μg) of the BiFC plasmids encoding the fusion proteins when an appropriate confluency (e.g. ~50%) is reached. In parallel, transfect cells with the negative and positive control plasmids. For quantitation of the efficiency of fluorescence complementation, all plasmids should be co-transfected with the same amount of an internal control plasmid (e.g. a plasmid that expresses CFP, see Fig. 3)
Grow cells under conditions appropriate for the cell-type until fluorescence is detected (12 to 36 hr). If necessary, incubate the cells at 30°C with 5% CO2 to promote maturation of the fluorophore and to increase the signal. Results obtained under low temperature conditions should be interpreted with care, as incubation at a lower temperature could alter protein interactions.
Wash the cells once with an amount of PBS sufficient to remove dead cells and cell debris, then add fresh medium.
Image the cells using an inverted fluorescence microscope. When using cells grown on plastic, a long-working-distance objective is convenient, but produces lower signal (due to the lower numerical aperture) than a short-working distance objective. A 20× objective is useful for observing large numbers of cells and can provide general subcellular localization information, whereas 60× or 100× objectives can be necessary for detailed localization within subcellular compartments. For detection of complementation between YFP or Venus fragments an excitation filter with 500 ± 10 nm transmission and an emission filter with 535 ± 15 nm transmission are appropriate. Confirm that fluorescent cells are alive by comparing their morphology to that of non-transfected cells. Cells grown on coverslips can be fixed and individual proteins can be visualized by indirect immunofluorescence analysis. Use protocols for fixation and immunofluorescence that have been established for the cell line and antibody to be used.
Compare the levels of fusion protein expression with those of the endogenous proteins by immunoblot analysis. The cultures used for imaging can be subsequently processed for immunoblotting or separate cultures can be prepared in parallel. Use protocols for immunoblotting that have been established to work for the cell line and antibody to be used. Use the ratio between the intensities of the bands corresponding to the transfected and endogenous proteins together with the transfection efficiency to estimate the relative levels of transfected and endogenous proteins. Ideally, the amount of transfected protein should not exceed that of the endogenous proteine in the cells.
Compare the intensities and numbers of fluorescent cells observed when the cells are transfected with the wildtype interaction partners with those observed when the cells are transfected with the negative control constructs. For quantitative analysis of the efficiencies of fluorescence complementation, divide the fluorescence intensities produced by fluorescence complementation by the fluorescence intensities produced by intact fluorescent protein in individual cells (see Fig. 3). Higher fluorescence intensity and an increased number of fluorescent cells for the wild type proteins is consistent with a specific interaction.
The construction of plasmid vectors for the expression of fusion proteins can be accomplished in a few days once the design for the vectors has been completed. Preparation of cells that express the fusion proteins can vary a great deal from days in transient transfection experiments to months or years for the production of stable cell lines or transgenic organisms. In transient expression experiments, fluorescence from specific interactions can generally be detected between 12 and 30 hours after transfection. In the case of complementation between YFP or CFP fragments, this generally requires a short incubation at 30°C to facilitate fluorophore maturation. In the case of complementation between Venus fragments, this step is generally not necessary. Longer incubation should be avoided since this may result in higher expression of fusion proteins and complementation due to nonspecific interactions. Images can be recorded and analyzed in less than an hour for each combination of fusion proteins.
The fluorescence intensity produced by bimolecular fluorescence complementation in living cells is generally less than 10% of that produced by intact fluorescent proteins. It is likely that only a subset of the fragments associate with each other since the fluorescence intensity of BiFC complexes produced in vitro is comparable to that of intact fluorescent proteins 2. Several variants of the BiFC assay have been developed that enable visualization of multiple protein interactions in the same cell as well as covalent protein modifications 9, 11, 67
I thank Dr. Changdeng Hu for his participation in the design and implementation of the BiFC assay in mammalian cells and all members of the Kerppola laboratory for their contributions to the improvement and adaptation of the BiFC approach.