Simultaneous visualization of multiple protein interactions in
living cells using multicolor fluorescence complementation
analysis
Chang-Deng Hu and Tom K. Kerppola*
Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan
Medical School, Ann Arbor, MI 48109-0650
Abstract
The specificity of biological regulatory mechanisms relies on selective interactions between different
proteins in different cell types and in response to different extracellular signals. We describe a
bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of
multiple protein interactions in the same cell. This approach is based on complementation between
fragments of fluorescent proteins with different spectral characteristics. We have identified twelve
new bimolecular fluorescent complexes that correspond to seven different spectral classes.
Bimolecular complex formation between fragments of different fluorescent proteins did not
differentially affect the dimerization efficiency or subcellular sites of interactions between the bZIP
domains of Fos and Jun. Multicolor BiFC enables visualization of interactions between different
proteins in the same cell and comparison of the efficiencies of complex formation among alternative
interaction partners.
Networks of protein interactions mediate cellular responses to environmental stimuli and direct
the execution of developmental programs. Each protein typically has a large number of
alternative interaction partners, and the selectivity of these interactions determines the
developmental potential of the cell and its responses to extracellular stimuli. We recently
described a new approach for the visualization of protein interactions in living cells designated
bimolecular fluorescence complementation (BiFC) analysis 1. The BiFC approach is based on
the formation of a fluorescent complex by fragments of the yellow fluorescent protein (YFP)
brought together by the association of two interaction partners fused to the fragments. This
approach enables visualization of the subcellular sites of protein interactions under conditions
that closely reflect the normal physiological environment.
Molecular engineering of the green fluorescent protein (GFP) has produced several variants
with altered spectral characteristics 2. These variants allow simultaneous visualization of the
distributions of multiple proteins in living cells. Moreover, fluorescence resonance energy
transfer (FRET) between different variants enables analysis of interactions between individual
pairs of proteins in living cells 3, 4. Thus far, it has not been possible to visualize multiple
interactions in the same cell.
Selected fragments of many proteins can associate to produce functional bimolecular
complexes. Such bimolecular complementation provides a convenient approach for detection
of protein interactions in cells if the protein fragments can associate only when they are brought
together by interaction partners fused to the fragments 1, 5-9. The unique characteristic of the
BiFC approach is that the bright intrinsic fluorescence of the bimolecular complex allows direct
*To whom correspondence should be addressed:Tel: (734) 764-3553, FAX: (734) 615-3397, e-mail: kerppola@umich.edu
NIH Public Access
Author Manuscript
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Published in final edited form as:
Nat Biotechnol. 2003 May ; 21(5): 539–545.
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visualization of the complex in living mammalian cells 1. Moreover, the large number of GFP
variants with distinct spectral characteristics provided the potential for parallel analysis of
multiple protein interactions in the same cell. In the present study, we have realized the promise
of multicolor BiFC analysis by characterizing twelve new bimolecular fluorescent complexes,
and we have used these complexes to compare the dimerization selectivity and subcellular sites
of interactions among basic region leucine zipper (bZIP) family proteins.
RESULTS
The spectral characteristics of bimolecular fluorescent complexes formed by fragments of YFP
were virtually identical to those of intact YFP 1. We reasoned that fragments of other GFP
variants might support bimolecular fluorescent complex formation, and that such complexes
might have distinct spectral characteristics. To identify such complexes, we investigated
fluorescence complementation using the corresponding fragments of the enhanced GFP and
cyan fluorescent protein (CFP) fused to the bZIP domains of Fos and Jun (bFos and bJun) (Fig.
1A). Each pair of fusion proteins was expressed in mammalian cells and the cells were
examined by fluorescence microscopy (Fig. 1B-D). No complementation was detected when
fragments of GFP (GN155 and GC155) fused to bFos and bJun were expressed in mammalian
cells. However, fragments of CFP (CN155 and CC155) exhibited fluorescence
complementation when fused to bFos and bJun. (Fig. 1D). All of the fusion proteins were
expressed at comparable levels as determined by Western analysis (Supplementary Fig. 1A
online).
To examine the selectivity of bimolecular complex formation, we tested fluorescence
complementation between all 9 combinations of fragments (Supplementary Fig. 2 online).
YN155 exhibited fluorescence complementation with YC155 and CC155, whereas CN155
exhibited fluorescence complementation only with CC155 when fused to bFos and bJun (Fig.
1B-D). Significantly, the fluorescence spectrum of cells expressing YN155 and CC155 fusions
was distinct from those of cells expressing either YN155 and YC155 or CN155 and CC155.
GN155 and GC155 did not exhibit detectable fluorescence complementation with any of the
other fragments. YC155 and CC155 differ from GC155 by single amino acid residues whereas
YN155 and CN155 differ from GN155 by four and three amino acid residues respectively (Fig.
1A). These amino acid substitutions determined the selectivity of bimolecular fluorescence
complementation among these fragments.
We used a genetic screen in E. coli 1 to identify a second pair of YFP fragments (YN173 and
YC173) that exhibit bimolecular fluorescence complementation when fused to bFos and bJun.
We examined fluorescence complementation between these fragments and the corresponding
fragments of GFP, CFP and the enhanced blue fluorescent protein (BFP) fused to bFos and
bJun. The sequences of the C-terminal fragments of GFP, CFP and BFP are identical (Fig. 1A),
and thus only YC173 and GC173 were tested. YC173 exhibited fluorescence complementation
with YN173, GN173 and CN173 when fused to bFos and bJun (Fig. 1E-G), whereas GC173
did not exhibit detectable fluorescence complementation with any of the fragments tested. The
two positions of fragmentation (155 and 173) enabled complementation between distinct
combinations of fluorescent protein fragments (Supplementary Fig. 2 online). Thus, a small
number of amino acid substitutions can influence the positions where proteins must be
fragmented to support bimolecular complementation.
GFP is a β barrel structure containing 11 strands surrounding a central α helix10.The two sites
of fragmentation in YFP that support BiFC are separated by one strand of the β barrel that
surrounds the fluorophore10. We examined fluorescence complementation between all 24
combinations of fragments truncated at these positions fused to bFos and bJun (Supplementary
Fig. 2 online). None of the N-terminal fragments truncated at residue 155 exhibited
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fluorescence complementation with C-terminal fragments truncated at residue 173. In contrast,
all of the N-terminal fragments truncated at residue 173 (YN173, GN173 and CN173) formed
bimolecular fluorescent complexes with CC155 and YC155 (Fig. 1H, I, J, data not shown).
The corresponding N-terminal fragment of BFP (BN173) also exhibited fluorescence
complementation with CC155 (Fig. 1K). Duplication of the segment separating the points of
truncation therefore facilitated bimolecular fluorescence complementation. This duplication
had no detectable effect on the spectra of the bimolecular complexes (Fig. 2).
To establish whether bimolecular fluorescence complementation between fragments of
different fluorescent proteins fused to bFos and bJun required the leucine zipper dimerization
interface, we examined complementation by bFos fusions in which the carboxy-terminal half
of the leucine zipper was deleted (bFosΔZIPYC155 and bFosΔZIPCC155). We compared the
complementation efficiencies of the wild type and mutant fusions with bJun fused to fragments
of different fluorescent proteins (Fig. 3, Supplementary Table 1 online). Mutation of the leucine
zipper resulted in a more than 10-fold reduction in the efficiencies of fluorescence
complementation between all fragments of fluorescent proteins tested. Cells expressing
fragments of different fluorescent proteins fused to wild type bFos and bJun produced different
intensities of fluorescence emissions at different times following transfection. Cells expressing
the same fragments, but with a deletion in the leucine zipper of Fos, produced either
undetectable or lower fluorescence emissions at all times following transfection
(Supplementary Table 1 online). Similar results were obtained when the fluorescence
emissions of the cell populations were measured using fluorescence spectroscopy (data not
shown). The mutant proteins were expressed at the same levels and were localized to the same
subnuclear sites as the wild type proteins. Thus, efficient fluorescence complementation
between all fragments of fluorescent proteins examined required a specific interaction
interface.
Multicolor BiFC allows comparison of the efficiencies of complex formation between
alternative interaction partners, providing that the fragments do not influence the selectivity of
protein interactions. To compare the effects of fragments of different fluorescent proteins on
dimerization efficiency, we examined the competition between proteins in which bJun was
fused to fragments of different fluorescent proteins for dimerization with bFosYC173 in
vitro (Fig. 4). The ratio of bimolecular fluorescent complexes formed by the alternative
interaction partners was calculated by fitting the spectrum of the mixture to the weighted sum
of the spectra of the two complexes. This ratio exhibited a perfect correspondence with the
ratio of bJun fusion proteins that was added to each reaction. Thus, the bZIP domains of Fos
and Jun have identical dimerization efficiencies when fused to fragments of different
fluorescent proteins, suggesting that the fusions do not have differential effects on interactions
between bFos and bJun in vitro.
The spectral differences between bimolecular complexes formed by fragments of different
fluorescent proteins enable comparison of the subcellular sites of interactions between different
proteins in the same cell providing that the fragments do not differentially affect complex
localization. To compare the subcellular locations of bFos-bJun heterodimers fused to
fragments of different fluorescent proteins, we co-expressed bJunCN173 and bJunYN173 with
bFosYC173 in COS-1 cells and imaged the cells using filters optimized for the detection of
CN173-YC173 and YN173-YC173 complexes respectively (Fig. 5A). Both complexes were
localized preferentially to the nucleoli, and exhibited perfect co-localization. Similar results
were obtained when bJunCN173 and bJunYN173 were co-expressed with bFosYC155 or with
bFosCC155. Although the efficiency of nucleolar localization of the complexes varied between
different cells in the population, the efficiencies of nucleolar localization of complexes
containing fragments of different fluorescent proteins were identical in individual cells. Thus,
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fragments of different fluorescent proteins did not have differential effects on the subcellular
sites of interactions between bFos and bJun.
To visualize the subcellular sites of interactions between different proteins in the same cell,
we co-expressed bJunCN173 and JunYN155 with bFosYC155. bJunCN173-bFosYC155
exhibited nucleolar localization, but JunYN155-bFosYC155 was localized to the nucleoplasm
(Fig. 5B). The two complexes exhibited distinct distributions in the same nucleus, indicating
that regions outside the bZIP domain of Jun affected the subcellular localization of the complex.
Similar results were obtained when bJunCN173 and JunYN155 were co-expressed with
bFosCC155. Thus, multicolor BiFC can be used to compare the subcellular sites of interactions
between different proteins in the same cell.
The bZIP domains of Fos, Jun and ATF2 can interact with each other in all pairwise
combinations in vitro, and activate different genes in cells 11-14. To investigate the competition
between alternative interaction partners in cells, we examined the relative efficiencies of bJun
interactions with bFos and bATF2. We expressed bJunCC155 with bFosCN173 and
bATF2YN155 separately as well as together, and compared the ratio between the fluorescence
emissions of the complexes (Fig. 6A, Supplementary Table 2 online). Cells expressing the two
pairs of proteins separately exhibited cyan and yellow fluorescence respectively. Coexpression
of a limiting amount of bJunCC155 with bFosCN173 and bATF2YN155 together
resulted in cyan fluorescence that was comparable to that observed in cells transfected with
bJunCC155 and bFosCN173, but yellow fluorescence that was 100-fold lower than that of cells
transfected with the same amounts of bJunCC155 and bATF2YN155 (Supplementary Table
2). The ratio between yellow and cyan fluorescence was therefore comparable for cells
expressing all three proteins and those expressing only bJunCC155 and bFosCN173 (Fig. 6A).
Similar results were obtained when bJunCC155 was expressed with bFosCN155 and
bATF2YN155 (data not shown). When excess bJunCC155 was expressed with bFosCN173
and bATF2YN155, both cyan and yellow fluorescence was observed in the cells. It is therefore
likely that the inhibition of bimolecular fluorescence complementation between bATF2YN155
and bJunCC155 in the presence of either bFosCN155 or bFosCN173 was caused by a higher
efficiency of complex formation by bJunCC155 with either bFosCN155 or bFosCN173 than
with bATF2YN155.
To confirm that the identity of the fragments fused to bJun, bFos and bATF2 did not affect the
preference for bimolecular fluorescent complex formation, we exchanged the fragments
between bFos and bATF2 (Fig. 6B). Cells expressing a limiting amount of bJunCC155 with
bFosYN155 and bATF2CN173 produced yellow fluorescence comparable to that of cells
transfected with bJunCC155 and bFosYN155, but cyan fluorescence that was 10-fold lower
than that of cells transfected with the same amounts of bJunCC155 and bATF2CN173
(Supplementary Table 2). Again, the ratio between yellow and cyan fluorescence for cells
expressing all three proteins was similar to that of cells expressing only bJunCC155 and
bFosYN173 (Fig. 6B). The fluorescent protein fragments were fused to the same positions in
bJun and bATF2 relative to the leucine zipper, and the same linker sequences were used for
all fusions. The competing proteins were expressed at comparable levels (Supplementary Fig.
1B online). Consequently, it is likely that the higher efficiency of complementation between
bJun and bFos fusions reflected preferential heterodimerization of bJun with bFos than with
bATF2 in cells.
To examine the relative efficiencies of bFos interactions with bJun and bATF2 in cells, we
expressed limiting bFosCC155 with bJunCN173 and bATF2YN155. The cells exhibited cyan
fluorescence comparable to cells transfected with bFosCC155 and bJunCN173, but markedly
lower yellow fluorescence than cells transfected with the same amounts of bFosCC155 and
bATF2YN155 (data not shown). A similar inhibition of bFos-bATF2 and bJun-bATF2
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heterodimer formation was observed in cells transfected using a limiting concentration of
bATF2YC155 with bFosYN155 and bJunCN173 (data not shown). The higher efficiency of
bFos-bJun heterodimer formation therefore did not require bimolecular complex formation
since bFosYN155-bJunCN173 heterodimers could not form a bimolecular fluorescent
complex. Thus, the bZIP domains of Fos and Jun favor dimerization with each other over
dimerization with the bZIP domain of ATF2 in cells.
To examine the relative efficiencies of Fos-Jun heterodimerization and Jun homodimerization
in cells, we expressed bJunCC155 with bFosYN155 and bJunCN173 (Fig. 6C). The cells
produced on average 60% of the cyan fluorescence and 35% of the yellow fluorescence
produced by the control cells. The fluorescence intensities of individual cells were normally
distributed and exhibited a moderate (r=0.83) correlation between cyan and yellow
fluorescence. Thus, Fos-Jun heterodimers and Jun homodimers can co-exist in cells, consistent
with the similar thermodynamic stabilities of these dimers in vitro 15.
DISCUSSION
The multicolor BiFC assay provides a unique approach for the simultaneous visualization of
multiple protein interactions in a living cell. Although we describe only cases where two
interactions were compared in parallel, the number of interactions that can be visualized
simultaneously is limited in principle only by the number of spectrally distinct bimolecular
fluorescent complexes. In practice, the number of complexes that can be visualized
simultaneously depends on the spectral resolution of the imaging system. In typical cases where
two different complexes need to be resolved, the broad range of spectral characteristics among
the bimolecular complexes identified here enables visualization of the complexes without
interference from spectral overlap using standard fluorescence microscopy.
Fragments of different fluorescent proteins did not differentially affect bZIP protein
dimerization or the localization of those dimers. These fragments therefore enable comparison
of the efficiencies and subcellular locations of complex formation with alternative interaction
partners in the same cell. It remains possible that these fragments have distinct effects on
interactions between other proteins or the localization of those complexes. It is therefore
necessary to compare the efficiencies of complex formation by proteins fused to these
fragments, and to establish the effects of the fragments on complex localization in order to use
multicolor BiFC for comparison of interactions among other proteins. In addition, since
formation of the bimolecular fluorescent complex is essentially irreversible, the efficiency of
fluorescence complementation represents the efficiency of the association between the
interaction partners at the time of complex formation, and does not reflect subsequent shifts in
the equilibrium among alternative interaction partners 1.
The systematic analysis of complementation between fragments of different fluorescent
proteins provides several insights into protein complementation. Complementation between
fragments of different fluorescent proteins was selective, and single amino acid substitutions
had greater than 100-fold effects on the efficiency of fluorescence complementation. These
same substitutions had little effect on the quantum yields of the intact proteins 2, and some
were located at a distance from the fluorophore. Substitutions that resulted in different
efficiencies of fluorescence complementation had only small effects on the kinetics of
fluorophore maturation (data not shown). It is therefore likely that these amino acid
substitutions influence the efficiency of bimolecular complex formation.
Two different positions were identified where fragmentation of fluorescent proteins enabled
bimolecular complementation. These positions are within loops at opposite ends of the β barrel
structure. The bFos-bJun interaction and the linker sequences used in these experiments
therefore provide sufficient flexibility for the fragments to associate with their N-termini
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separated by a distance of either 10 or 50 Å. We have used the multicolor BiFC assay to
investigate interactions among several structurally unrelated protein families. The results from
these experiments confirm that the interaction partners do not need to juxtapose the fragments
in a specific orientation, providing that the linkers that tether the fragments have sufficient
flexibility to allow the fragments to associate with each other. Multicolor BiFC therefore
provides a general approach for the analysis of complex formation among alternative
interaction partners in living cells.
The β barrel structure that surrounds the fluorophore in green fluorescent proteins is composed
of three segments of contiguous peptide sequence, each of which forms an anti-parallel β-sheet
that together form the β barrel10. Both of the truncations that allow fluorescence
complementation interrupt one of these β-sheet segments. Thus, all of the fragments that allow
fluorescence complementation contain an incomplete β-sheet. Fragments that were truncated
at the junctions between the three β-sheet segments did not exhibit fluorescence
complementation 1. Other structurally unrelated protein fragments that exhibit
complementation also contain incomplete domains that are unlikely to fold in the absence of
the complementary fragments 6, 7, 16-18. It is therefore likely that bimolecular
complementation by many protein fragments requires at least part of the fragments to be
unfolded, which may facilitate their association.
Comparison of the efficiencies of interactions between alternative interaction partners using
multicolor BiFC requires that the fusion proteins meet several criteria. First, fragments of
different fluorescent proteins must not differentially affect interactions between the proteins.
In the case of bFos-bJun heterodimers, identical efficiencies of complex formation were
observed between proteins fused to fragments of different fluorescent proteins. Second, the
efficiencies of complementation between the fragments must be equivalent once they have
been brought together by interactions between the alternative interaction partners. In the case
of bJun-bFos and bJun-bATF2 heterodimers fused to the same fragments, the fluorescence
emissions of bimolecular complexes formed separately were comparable, consistent with the
structural similarity between these complexes. Finally, the alternative interaction partners must
be expressed at similar levels and be localized to their normal subcellular compartments. The
bZIP domains of Fos, Jun and ATF2 were expressed at comparable levels and were localized
to the nucleoli. When these criteria are met, multicolor BiFC enables comparison of the
efficiencies and subcellular sites of complex formation between alternative interaction partners
in living cells.
EXPERIMENTAL PROTOCOL
Plasmid Construction.
The sequences encoding amino acid residues 1-154, 155-238, 1-172, and 173-238 of the
enhanced YFP, GFP, CFP and BFP (Clontech, Palo Alto, CA) were fused downstream of
sequences encoding Fos118-210 (bFos) and Jun257-318 (bJun) using linker sequences
encoding RPACKIPNDLKQKVMNH and RSIAT respectively. The chimeric coding regions
were cloned into pFLAG-CMV2 (Sigma, St. Louis, MO) and pHA-CMV (Clontech, Palo Alto,
CA) vectors for expression in mammalian cells and into pDS56 11 for expression in E. coli.
As a control, residues 179-193 in the leucine zipper of Fos were deleted to produce bFosΔZIP.
Plasmids encoding JunYN155, bATF2YN155, and bATF2YC155 were described previously
1.
Imaging and Spectral Analysis of Fluorescence in Cells.
COS-1 cells were transfected with plasmids encoding the fusion proteins indicated. Transfected
cells were incubated at 37°C for 24 h and then switched to 30°C for 0-24 h to promote
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fluorophore maturation. To image the fluorescence emissions of cells expressing different
combinations of fusion proteins, we used filter pairs centered at 436 nm excitation and 470 nm
emission (C) or 500 nm excitation and 535 nm emission (Y) together with dichroic mirrors
with transmission windows at 450-490 nm and 520-590 nm. The fluorescence intensities of
individual cells were quantified using automated feature recognition software (Compix). The
signal in an area of the field containing no cells was used as background and subtracted from
all values. There was less than 2% overlap between the signals from bimolecular fluorescent
complexes formed by YN173 and CN173 with either YC155 or CC155 using these filtes. Thus,
no correction for cross-talk was necessary. To measure the spectra of bimolecular fluorescent
complexes, the cells were washed and resuspended in PBS. All spectra were corrected for
background signal produced primarily by scatter by subtracting the spectrum of untransfected
cells. The level of expression of each fusion protein was quantified by Western blotting using
antibodies directed against the FLAG and HA epitopes (Sigma, St. Louis, MO).
Analysis of Fluorescence Complementation in vitro
Chimeric fusion proteins were purified from E. coli using nickel chelate affinity
chromatography in the presence of 6 M guanidine as described 1. The proteins indicated in
each experiment were heated to 95 °C for 5 min and diluted to a final concentration of 30 μg/
ml in buffer (50 mM NaPO4, pH 8.0, 150 mM NaCl, 5% glycerol (vol/vol), 0.1 mg/ml BSA
and 1mM DTT) at room temperature. Excitation and emission spectra were collected after no
further change in fluorescence was observed. The fluorescence intensities of the bimolecular
fluorescent complexes were comparable to those observed for the intact fluorescent proteins.
The spectra were corrected for scatter by subtracting the spectrum of the buffer alone.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGEMENTS
We thank members of the Kerppola laboratory for helpful discussions.
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Figure 1.
Visualization of bimolecular fluorescence complementation between fragments of different
fluorescent proteins fused to bFos and bJun. (A) Diagram of amino acid substitutions among
enhanced green fluorescent protein variants and the positions where they were fragmented (155
and 173). (B-K) Fluorescence images of COS-1 cells transfected with plasmids expressing the
protein fragments indicated in each panel fused to the bZIP domains of Fos and Jun. The Cterminal
fragments were fused to bFos and the N-terminal fragments were fused to bJun.
Structural models of the bimolecular fluorescent complexes shown to the right of each image
are based on the X-ray crystal structure of full-length GFP 10. The positions of fragmentation
are indicated by arrows in the structures. The position and structure of the duplicated β-strand
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shown in panels H-J is unknown. The excitation/emission maxima of each complex are shown
below each image. The bar represents 10 μm in all images.
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Figure 2.
Excitation and emission spectra of cells expressing fragments of different fluorescent proteins
fused to bFos and bJun. Solid lines correspond to bimolecular fluorescent complexes and
dashed lines to intact fluorescent proteins (A) The excitation spectra were collected by
measuring emissions at 535 nm through a 530 nm long pass filter, and were normalized by the
ratio between the emission maximum of each complex and the emission intensity at 535 nm.
(B) The emission spectra of YN173-YC173 and YFP were collected through a 500 nm longpass
filter using excitation at 480 nm and the other emission spectra were collected through a
450 nm long-pass filter using excitation at 430 nm, and were normalized by the ratio between
the excitation maximum of each complex and the excitation efficiencies at 480 nm and 430
nm respectively. The cells were transfected with 0.5μg of the plasmids encoding the fluorescent
protein fragments fused to bFos and bJun or with 0.05 μg of the plasmids encoding the full
length proteins.
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Figure 3.
Effects of a deletion in the leucine zipper on the efficiencies of complementation between
fragments of different fluorescent proteins fused to the bZIP domains of Fos and Jun. The
efficiencies of fluorescence complementation were determined in individual cells by
measuring the ratio between the fluorescence emissions of the bimolecular fluorescent complex
and that of a co-expressed intact fluorescent protein. Plasmids encoding the proteins indicated
in each panel were transfected into cells (0.25 μg of each fusion protein and 0.025 μg of the
ECFP or EYFP internal control) and the ratio of fluorescence emissions produced by the
bimolecular complex and the internal control (Y/C or C/Y) was measured. Note the logarithmic
scaling of the categories in the histograms.
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Figure 4.
Comparison of the efficiencies of bimolecular complex formation by fragments of different
fluorescent proteins fused to bFos and bJun. We examined the competition between different
ratios of bJun fused to fragments of different fluorescent proteins for a limiting concentration
of bFos fused to the complementary fragment. (A) The indicated ratios of GN173 and CN173
fused to bJun were mixed with a fixed concentration of YC173 fused to bFos. The excitation
spectra were collected after 12 hours by measuring emissions at 530 nm. (B) The indicated
ratios of YN173 and CN173 fused to bJun were mixed with a fixed concentration of YC173
fused to bFos. The emission spectra were collected after 12 hours during excitation at 450 nm.
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Figure 5.
Multicolor BiFC analysis of the subcellular sites of interactions between different proteins in
the same cell. (A) Co-localization of bJunCN173-bFosYC173 and bJunYN173-bFosYC173
bimolecular fluorescent complexes. (B) Differential localization of bJunCN173-bFosYC155
and JunYN155-bFosYC55 complexes. Plasmids encoding the proteins indicated below the
images were co-transfected into COS-1 cells. The images show the fluorescence emissions of
the same cell using 436 nm and 470 nm (C) or 500 nm and 530 nm (Y) filters. The bar represents
10 μm in all images.
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Figure 6.
Multicolor BiFC analysis of the competition between alternative interaction partners in cells.
Two combinations of interaction partners producing bimolecular complexes with different
spectra were transfected into different cell populations (cyan and yellow bars) or co-transfected
into the same cell population (green bars) as indicated above each graph. The ratio between
the fluorescence emissions corresponding to each complex (Y/(C+Y)) was determined in
individual cells in each population, and was plotted in the histograms. (A) Competition between
bFosCN173 and bATF2YN155 for dimerization with bJunCC155. (B) Competition between
bFosYN155 and bATF2CN173 for dimerization with bJunCC155. (C) Competition between
bFosYN155 and bJunCN173 for dimerization with bJunCC155. Plasmids encoding the
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proteins indicated in each panel were co-transfected into cells. The plasmid encoding the shared
interaction partner fused to CC155 was used at limiting concentration (0.1 μg), whereas the
other plasmids were used at a higher concentration (0.5 μg). The fluorescence intensities of
individual cells were quantified using 436 nm/470 nm (C) and 500 nm/535 nm (Y) filters and
the Y/(Y+C) ratios were plotted in the histograms.
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