Live Cell Visualization of Multiple ProteinâProtein Interactions with

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Live Cell Visualization of Multiple ProteinProtein Interactions with BiFC Rainbow Sheng Wang, Miao Ding, Boxin Xue, Yingping Hou, and Yujie Sun ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00931 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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ACS Chemical Biology

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow 1

1

Sheng Wang , Miao Ding , Boxin Xue, Yingping Hou and Yujie Sun* State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China *Corresponding Author, [email protected] 1

Contributed equally to this work

ABSTRACT As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during the recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made towards simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of 4 PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells. ACS Paragon Plus Environment

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INTRODUCTION Protein-protein interactions (PPIs), spatial and temporal-dependent, are basic modules for execution of most cellular functions.1 During the past decades, a number of methods have been developed to detect PPIs.2 Compared with the classical biochemical methods such as co-immunoprecipitation or GST-pull down which need to detect PPIs in lysed cells, fluorescence methods such as bimolecular fluorescence complementation (BiFC) can directly detect and visualize protein interactions in live cells with both spatial and temporal information.3 The BiFC method is based on the complementary reconstitution of a functional fluorescent protein from its split non-fluorescent fragments which are fused to two proteins of interest that are thought to interact. The interaction between the two proteins of interest facilitates complementation of the two non-fluorescent fragments. Because of its simplicity and sensitivity, BiFC assays have been expanded, optimized and applied to detect and identify PPIs in different cell types and organisms in the past decade. For example, BiFC with far-red and near-infrared fluorescent proteins have enabled visualization of PPIs directly in deep tissues and organs of whole mammals.4-6 BiFC with photochromic fluorescent proteins have enabled super-resolution imaging of PPIs and ternary protein complex formation.7-11 However, in spite of these advancements towards deeper and finer levels, little has been done for simultaneous imaging of multiple protein-protein interactions, which is important for understanding dynamic nature of signaling pathways. Even though multicolor BiFC has been demonstrated previously using different combinations of GFP variants, spectral crosstalk has constrained the number of BiFC colors.12 Recently, several LSS-FPs have been developed including two notably bright fluorescent proteins mT-Sapphire13 and CyOFP1.14 Imaging with LSS-FPs is advantageous because multicolor imaging can be easily performed with single excitation source.15 We noticed that BiFC with LSS-FPs may facilitate detection of multiple

PPIs

simultaneously

in

the

same

cell.

In

addition,

the

large

excitation/emission spectra separation of mT-Sapphire with CyOFP1 may also

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simplify intensity-based FRET detection in three-filter FRET microscopy.16 Here, we describe the first time to our knowledge the development, characterization and application of BiFC assays based on LSS-FPs mT-Sapphire and CyOFP1. We also further combine BiFC with FRET to visualize multiple protein interactions with a conventional wide-field three-filter FRET setup. Our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in live cells.

RESULTS AND DISCUSSION Development of LSS-FP-based BiFC assays in live cells. During the past decade, although many LSS-FPs have been generated, most of them showed relatively low brightness.17 Among the reported LSS-FPs, Green fluorescent protein mT-Sapphire (83% brightness of EGFP) and Orange fluorescent protein CyOFP1 (95% brightness of EGFP) showed dramatically improved brightness. We wondered whether the two fluorescent proteins could be used to develop BiFC assays and combined with previously reported mCerulean/mVenus-based BiFC assays18 for multiple PPI detection simultaneously in live cells. Since mT-Sapphire is directly derived from A. victoria GFP, and shows high sequence similarity with mVenus (Supporting Figure S1A) which previously has been demonstrated to support BiFC by splitting at amino acids 154/155, we therefore chose to split mT-Sapphire between amino acids Ala154 and Asp155, yielding two nonfluorescent fragments mT-Sapphire-N

and

mT-Sapphire-C,

respectively.

In

order

to

establish

mT-Sapphire-based BiFC assay, we used previously reported leucine zipper domains of β-Fos and β-Jun as a heterodimer interaction model to examine the fluorescence complementation of mT-Sapphire. As a negative control, β-Fos (∆Zip), a β-Fos mutant with deletion of 15 amino acids that prevents the heterodimerization, was used to quantify the background BiFC signal resulted from the spontaneous complementation. Specifically, β-Jun encoding sequence was tagged to the



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N-terminus of the mT-Sapphire-N sequence while β-Fos or β-Fos(∆Zip) encoding sequence was tagged to the N-terminus of the mT-Sapphire-C sequence respectively (Figure S1B). To quantify the efficiency of fragment complementation mediated by β-Fos/β-Jun heterodimerization, we cotransfected β-Fos/β-Jun or β-Fos(∆Zip)/β-Jun along with TagBFP (internal control) expression vectors into HeLa cells. As mT-Sapphire is a LSS-FP with violet light excitability, it is convenient to efficiently excite both mT-Sapphire and TagBFP simultaneously with a single excitation light while detect TagBFP and mT-Sapphire emission with little spectral crosstalk. The cells were incubated at 37℃ for 24 hours after plasmid transfection and bright green reconstituted fluorescence signal was detected in the nucleus with an enrichment in the nucleolus which indicated the specific subcellular localization of β-Fos/β-Jun heterodimerization (Figure 1A). Although the fluorescence from complemented mT-Sapphire fragments mediated by β-Fos/β-Jun heterodimerization is about 6-fold lower than its full length parental protein (Supporting Figure S2A), the complemented fluorescence signal is still over 8 folds brighter than cells coexpressing β-Jun-mT-Sapphire-N and β-Fos(∆Zip)-mT-Sapphire-C (Figure 1B, Supporting Figure S3A and S3C) indicating high specificity of mT-Sapphire-based BiFC. Similarly, since CyOFP1 is derived from mNeptune

19

which was also demonstrated

to support BiFC by splitting at amino acids 151/152, we split CyOFP1 between amino acid Asp151 and Gly152, yielding two nonfluorescent fragments CyOFP1-N and CyOFP1-C, respectively (Supporting Figure S1C). We fused β-Jun encoding sequence to the N-terminus of the CyOFP1-N sequence via a flexible linker RSIAT and β-Fos encoding sequence to the N-terminus of the CyOFP1-C sequence via a flexible linker RPACKIPNDLKQKVMNH,

respectively.

Contrary

to

the

results

in

the

mT-Sapphire-based BiFC experiments, HeLa cells coexpressing β-Jun-CyOFP1-N and β-Fos-CyOFP1-C showed no detectable reconstituted orange fluorescence signal (data not shown). Since previous studies suggested that the conformations of fusion proteins or compositions of the linker sequence could affect the complementation efficiency of fragments, we tagged β-Jun/β-Fos to either N-terminus or C-terminus of


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CyOFP1-N or CyOFP1-C with a uniform 2×GGGGS flexible linker (Supporting Figure S1D and S4A). We cotransfected cells with various combinations of CyOFP1-N/CyOFP1-C tagged β-Fos/β-Jun along with EGFP (internal control) which could be imaged together with CyOFP1 using a single excitation light (Figure 1A). Of all the tested 8 combinations, 5 combinations showed bright orange fluorescence signal (Supporting Figure S4B) while cells coexpressing CyOFP1-N tagged β-Fos(∆Zip) and CyOFP1-C tagged β-Jun showed very low fluorescence, indicating the specificity of CyOFP1-based BiFC assay in detecting PPIs in live cells. Qualitatively, although the fluorescence from complemented CyOFP1 fragments mediated by β-Fos/β-Jun heterodimerization is about 9-fold lower than its full length parental protein (Supporting Figure S2B), the complemented fluorescence is still over 12-fold

brighter

than

cells

coexpressing

β-Jun-CyOFP1-C

and

β-Fos(∆Zip)-CyOFP1-N (Figure 1 B, Supporting Figure S3B and S3D) indicating high specificity of CyOFP1-based BiFC. The spectra of complemented mT-Sapphire and CyOFP1 also reserved as their original parental fluorescent protein (Figure S5A and S5B). Additionally, the high specificity of both established BiFC assays were also confirmed by rapamycin-inducible FRB/FKBP interaction system (Supporting Figure S6).20 Simultaneously visualize two PPI pairs in live cells with a single excitation light. In order to test whether the newly developed BiFC assays could be coupled with previously established mCerulean/mVenus-based BiFC assays to detect two pairs of PPI directly in live cells with a single excitation light, we visualized the subcellular sites of interactions between different proteins in the same cell using two PPI pairs (Jun/β-Fos and β-Jun/β-Fos). We tagged mT-Sapphire-N to the C-terminus of β-Jun and

co-expressed

β-Jun-mT-Sapphire-N/β-Fos-mT-Sapphire-C

along

with

Jun-mCerulean-N173 (Jun-CrN173)/β-Fos-CFP-C155 (β-Fos-CC155). As shown in Figure 1C, Jun-CrN173/β-Fos-CC155 exhibited nucleoplasm localization, but β-Jun-mT-Sapphire-N/β-Fos-mT-Sapphire-C was mainly localized to the nucleolus. The distribution of the two distinct PPI pairs could be well discerned with cyan and



green emission via a single violet light excitation in live cells. Similarly, we tagged CyOFP1-C and CyOFP1-N to the C-terminus of β-Jun and β-Fos respectively and co-expressed β-Jun-CyOFP1-C/β-Fos-CyOFP1-N along with Jun-mVenus-N173 (Jun-VN173)/ β-Fos-CC155 in live cells. The distribution of the two distinct PPI pairs could also be well discerned with green and red emission via a single cyan light excitation in live cells. These results clearly demonstrated that the newly developed BiFC assays could be easily combined with mCerulean/mVenus-based BiFC assays to detect two PPI pairs simultaneously with a single excitation light in live cells. Multicolor BiFC in live cells by cross-complementation between fragments of mT-Sapphire, mCerulean and mVenus. Since there is only one amino acid difference of fragment VC155 or two amino acids difference of fragment CC155 with that of fragment mT-Sapphire-C (Supporting Figure S1A), we wondered whether VN173 or CrN173 could complement with mT-Sapphire-C to reconstitute fluorescence upon PPIs. In order to test the cross-complementation between fragments of mT-Sapphire and those of mCerulean or mVenus, we tagged Jun to CrN173 or β-Jun to VN173 and β-Fos to mT-Sapphire-C. HeLa cells coexpressing Jun-CrN173 and β-Fos-mT-Sapphire-C showed bright fluorescence signal in the nucleus, which could be efficiently excited and detected in the mCerulean channel (Supporting Figure S7A). This result indicates that the N-terminus of mCerulean (CrN173) could efficiently complement with the C-terminus of mT-Sapphire (mT-Sapphire-C) by PPIs and the emitted fluorescence reserves the characteristics of mCerulean (Supporting Figure S5C). Similarly, cells coexpressing Jun-VN173 and β-Fos-mT-Sapphire-C also showed bright fluorescence signal in the nucleolar but the fluorescence signal can be efficiently excited and detected in the GFP channel rather than the mVenus channel (Supporting Figure S7A). This result indicates that the N-terminus of mVenus (VN173) could also efficiently complement with the C-terminus of mT-Sapphire (mT-Sapphire-C) by PPIs and the emitted fluorescence was much like EGFP and blue-shifted (Supporting Figure S5D) compared with mVenus. This result is also consistent with the previous report that Y203 is important in the red-shifting of YFP fluorescence spectrum.21 Since either ACS Paragon Plus Environment

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CrN173 or mT-Sapphire-N could efficiently complements with mT-Sapphire-C, it is convenient to visualize and differentiate Jun/β-Fos and β-Jun/β-Fos interactions in subcellular localizations by different emission in the same cell (Figure 2). Similarly, multicolor BiFC can also be performed in the same cell with VN173 and mT-Sapphire-N complemented with the same mT-Sapphire-C fragment and the subcellular localizations of Jun/β-Fos and β-Jun/β-Fos interactions could also be differentiated by different excitations rather than emission (Figure 2). We also tested the complementation efficiency of mT-Sapphire-N with either CC155 or VC155 mediated by β-Jun/β-Fos heterodimerization and the complemented fluorescence was very low, which precluded further analysis and applications in detecting PPIs in live cells (date not shown). Thus, our newly developed multicolor BiFC assays based on cross-complementation between fragments of mT-Sapphire and those of mCerulean and mVenus enables visualization of the subcellular distributions of several protein complexes in the same cell and allows analysis of the competition between mutually exclusive interaction partners for binding to a common partner. We also tested the cross-complementation of fragments from CyOFP1 with those from mT-Sapphire, mCerulean and mVenus. The results clearly showed that fragments from Anthozoan-derived

CyOFP1

could

not

complement

with

fragments

from

Hydrozoan-derived mT-Sapphire, mCerulean and mVenus (Supporting Figure S7B). Simultaneously visualize and track four PPI pairs in the same cell with BiFC rainbow. We next examined whether the combination of mT-Sapphire or CyOFP1 with mCerulean and mVenus could be used to simultaneously visualize four interactions in the same cell. For this purpose, in addition to β-Jun/β-Fos and Jun/β-Fos, Bcl-xL/Bak22 and Lifeact targeting to polymerized G-actin23 were selected for their well-known interactions. We expressed β-Jun, β-Fos, Jun, Bcl-xL, Bak and Lifeact as fusion proteins with BiFC fragments derived from mT-Sapphire, mCerulean, mVenus and CyOFP1 in HeLa cells. As shown in Figure 3, cyan, green and red fluorescence could be visualized in the same HeLa cell after 24 h of plasmids transfection, which represented four different interactions: β-Jun/β-Fos (red, nucleoli), Jun/β-Fos (green, ACS Paragon Plus Environment


nucleus), Bak/Bcl-xL (light green, pseudo colored in magenta, mitochondria) and Lifeact/Lifeact (cyan, F-actin). The subcellular localizations of these different PPIs are also consistent with previous reports. In addition, long-term live cell imaging and tracking of multiple PPIs simultaneously in the same cell (Figure 3) could be easily performed with alternate violet and cyan light excitations which are commonly available in most fluorescence microscope. These results may also provide the exciting potential of live cell imaging and tracking of multiple PPIs in the complicated signal transduction interactome networks. Visualize ternary protein complex formation by BiFC-FRET in live cells. In order to test whether the newly developed mT-Sapphire/CyOFP1-based BiFC assays could be combined with FRET to detect ternary protein complex formation, we visualized the ternary β-Jun-β-Fos-NFAT1 interactions in live HeLa cells.24 As the excitation or emission spectrum of mT-Sapphire has large separation with that of CyOFP1 and the emission spectrum of mT-Sapphire also completely overlaps with the excitation spectrum of CyOFP1, mT-Sapphire and CyOFP1 should be an ideal FRET pair, which could facilitate intensity-based FRET measurements on a typical three-filter FRET setup. As shown in Figure 4A, HeLa cells expressing mT-Sapphire showed bright fluorescence signal in the mT-Sapphire channel with no signal in the CyOFP1 channel and very low fluorescence signal in FRET channel, indicating dramatically reduced donor leakage compared with previously reported CFP/YFP FRET pair.25 Similarly, HeLa cells expressing CyOFP1 showed bright fluorescence signal in the CyOFP1 channel with no signal detected in the mT-Sapphire channel and also very low fluorescence signal in the FRET channel, indicating dramatically reduced acceptor excitation by the donor excitation light. Moreover, as shown quantitatively in Figure 4B, cells coexpressing mT-Sapphire and CyOFP1 (mT-Sapphire + CyOFP1) exhibited no FRET with a FRET ratio (FR) = 1.02+0.06 (n=14). In contrast, cells coexpressing the mT-Sapphire-CyOFP1 concatemer, a positive control for FRET, showed significantly increased FRET signal with a FR = 5.18+0.15 (n=27). Corrected FRET (FC) images in Figure 4A showed strong FRET signal in both cytoplasm and nucleus of HeLa cells expressing mT-Sapphire-CyOFP1 ACS Paragon Plus Environment

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fusion protein, while cells coexpressing mT-Sapphire and CyOFP1 showed no FRET signal. These results clearly demonstrate that mT-Sapphire and CyOFP1 could be used in a typical three-filter FRET setup. We then tried to combine the newly developed LSS-FP-based BiFC assays with three-filter FRET to measure and visualize ternary β-Jun-β-Fos-NFAT1 interactions in live cells. To this end, we tagged NFAT1 to the C-terminus of CyOFP1 and cotransfected it with β-Jun-mT-Sapphire-N/β-Fos-mT-Sapphire-C in HeLa cells. As shown in Figure 4A, HeLa cells coexpressing β-Jun-mT-Sapphire-N/β-Fos-mT-Sapphire-C and CyOFP1-NFAT1 showed bright complemented green fluorescence and red fluorescence in both mT-Sapphire and CyOFP1 channels, respectively. Strong FRET signal could also be detected in the FRET channel with a FR = 3.87+0.10 (n=24), indicating formation of the ternary protein complexes. In contrast, tagging mNFAT1, an interaction surface mutated NFAT1, to the C-terminus of CyOFP1 and cotransfected

it

with

β-Jun-mT-Sapphire-N/β-Fos-mT-Sapphire-C

resulted

in

dramatically decreased FRET efficiency with FR = 1.12+0.10 (n=20) (Figure 4B), despite that mNFAT1 still co-localized with complemented β-Jun-β-Fos heterodimers in the cell nucleus. The corrected FRET images in Figure 4A also clearly showed the heterotrimerization

of β-Jun-β-Fos-NFAT1

in

the

cell

nucleus

while

no

heterotrimerization of β-Jun-β-Fos with mNFAT1 could be detected. These results prove that the newly developed LSS-FP-based BiFC assays could be easily combined with FRET to visualize ternary protein complex formation in live cells. Live cell imaging and tracking of multiple PPIs simultaneously in the same cell is not only important to elucidate the entire map of cell signaling networks but also the hierarchy of individual interacting protein components in the signal transduction cascades.26 During the past decade, BiFC assays have been widely used for its simplicity and sensitivity in detecting PPIs in live cells with both spatial and temporal information. Although the recent years have witnessed the development of BiFC assay towards either deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins, there is still limited reports of BiFC assays which can detect multiple PPIs simultaneously in live ACS Paragon Plus Environment


cells. Thus, simultaneously visualizing PPIs at a multiplexing high-throughput level represents a direction for further development of BiFC assays.27 In this study, we developed BiFC assays based on two bright LSS-FPs which facilitate the detection and visualization of multiple PPIs in live cells. As the derivate variant of GFP, mT-Sapphire-based BiFC assay demonstrates excellent compatibility with previously established mCerulean/mVenus-based BiFC assays and multicolor BiFC can be easily applied to compare the subcellular distributions of several protein complexes in the same cell and analyze the competition between mutually exclusive interaction partners for binding to a common partner. For example, the competition of three interested proteins tagged with mT-Sapphire-N, CrN173 and VN173 fragments binding to a common partner tagged with mT-Sapphire-C fragment could be simultaneously visualized and analyzed, which are not available before. Coupled with mCerulean/mVenus-based BiFC assays, two PPI pairs can be simultaneously visualized using a single excitation, which not only simplifies the required excitation sources, but also minimizes the light exposure times leading to toxicity during long-term live cell imaging. It is also to be noted that beyond the capability of visualizing four PPI pairs simultaneously in the same cell as demonstrated in this work, there is still space to add one or two pairs of PPI detection using previously established far-red or near-infrared fluorescent protein-based BiFC assay, turning the BiFC rainbow as a powerful tool for multiplexing PPI detection and visualization in live cells. However, the main drawback of BiFC assays based on Hydrozoan or Anthozoan-derived fluorescence proteins are irreversible which precludes the detection and monitoring of dynamic PPIs. Contrary to BiFC, Fluorescence resonance energy transfer (FRET) can detect dynamic PPIs in live cells based on its reversibility.28 Nevertheless, intensity-based intermolecular FRET measurement is notoriously complicated by spectral cross-talk and spectral bleed-through between fluorescent donor and acceptor. In our experiment, intensity-based intermolecular FRET measurement greatly benefitted from mT-Sapphire as donor and CyOFP1 as acceptor due to the large excitation and emission spectral separation which largely ACS Paragon Plus Environment

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minimize the excitation cross-talk and emission bleed-through and the complete spectra overlap of donor emission with acceptor excitation which increases the FRET efficiency for detecting weak and monitoring transient PPIs. Moreover, compared with previous FRET pairs such as CFP and YFP which are from the same species, mT-Sapphire and CyOFP1 are derived from different species which share distinct protein interfaces, basal FRET signal caused by fluorescent protein self-association could be minimized. Combining BiFC with FRET also enables detection and monitoring of ternary protein complex formation directly in live cells. Similarly, it could be expected that large-Stokes-shift fluorescent protein-based BiFC or BiFC-FRET

further

combined

with

flow

cytometry29

may

also

benefit

high-throughput PPI detection or even drug screening30 based on large-Stokes-shift fluorescent protein-based biosensors.31 Although, mT-Sapphire and CyOFP1-based BiFC assays have been developed in this work, there is still space to improve the performance of both LSS-FP-based BiFC assays. For example, much effort is still needed to improve the imaging contrast of mT-Sapphire-based BiFC while complementation and maturation efficiency at physiological conditions (e.g.37℃) in mammalian cells should also be optimized for CyOFP1-based BiFC. In summary, we have developed new BiFC assays based on LSS-FPs, i.e. mT-Sapphire and CyOFP1. We demonstrate that these assays can be easily combined with previously established BiFC assays based on mCerulean/mVenus to enable multicolor BiFC or BiFC rainbow for multiplexing detection and visualization of multiple PPIs in the same cell. We also demonstrate intensity-based intermolecular FRET measurement with less spectral cross-talk and bleed-through using mT-sapphire as donor and CyOFP1 as acceptor. In addition, we also combine newly developed BiFC with FRET to detect ternary protein complex formation in live cells. The newly developed BiFC assays and intensity-based FRET measurement using mT-Sapphire and CyOFP1 not only facilitate multiple PPI detection and visualization but also offer the potentials for simultaneous detection and imaging of multiple cellular events based on fluorescent protein-based biosensors.33 ACS Paragon Plus Environment

32


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METHODS Plasmid construction To construct pBiFC-mT-Sapphire-N or pBiFC-CyOFP1-N plasmid, the cDNA sequence of mT-Sapphire (residue 1-154aa) or CyOFP1 (residue 1-151aa) was amplified by PCR and insert into the pBiFC-VN173 plasmid (provided by Dr. Chang-Deng Hu, Purdue University) with XbaI and BamHI sites to replace VN173 sequence. To construct pBiFC-mT-Sapphire-C or pBiFC-CyOFP1-C plasmid, the cDNA sequence of mT-Sapphire (residue 155-238aa) or CyOFP1 (residue 152-230aa) was amplified by PCR and inserted into the pBiFC-VC155 plasmid (provided by Dr. Chang-Deng Hu, Purdue University) with XhoI and NotI sites to replace VC155 sequence. To construct pBiFC-β-Jun-mT-Sapphire-N or pBiFC-β-Jun-CyOFP1-N plasmid, the β-Jun (c-Jun (residues 257-334aa)) cDNA sequence was amplified by PCR and inserted into pBiFC-mT-Sapphire-N or pBiFC-CyOFP1-N plasmid by EcoRI

and

XbaI

sites.

To

construct

pBiFC-β-Fos-mT-Sapphire-C

or

pBiFC-β-Fos-CyOFP1-C plasmid, the β-Fos [c-Fos (residues 118-211aa)] cDNA sequence was amplified by PCR and inserted into pBiFC-mT-Sapphire-C or pBiFC-CyOFP1-C plasmid by EcoRI and KpnI sites. Accordingly, the pBiFC-β-Fos (∆Zip)-mT-Sapphire-C or pBiFC-β-Fos (∆Zip)-CyOFP1-C plasmid was constructed by inserting the interaction domain (179–193aa) truncated β-Fos into the pBiFC-mT-Sapphire-C or pBiFC-CyOFP1-C plasmid by EcoRI and KpnI sites. To construct plasmids expressing β-Fos, β-Fos (∆Zip),β-Jun fusion to either N or C-terminal of CyOFP1-N or CyOFP1-C fragment, the cDNA of β-Fos, β-Fos (∆Zip),β-Jun were fused with cDNA of CyOFP1-N or CyOFP1-C with flexible linker GGGGSGGGGS by PCR and cloned into pcDNA3.1(+) vector with NheI and BamHI sites. To construct mT-Sapphire-C-Bcl-xL or mT-Sapphire-N-Bak plasmid, the cDNA of Bcl-xL or The BH3 peptide of Bak fused with GGGGSGGGGS and mT-Sapphire-C or mT-Sapphire-N fragment was amplified by PCR and inserted into pcDNA3.1(+) vector with NheI and BamHI sites. To construct Lifeact-CrN173 or Lifeact-CC155 plasmid, the cDNA of Lifeact was tagged to the N-terminal of CrN173 or CC155


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fragment by PCR and inserted into pcDNA3.1 (+) with BamHI and EcoRI sites. To construct pmT-Sapphire-C1 or pCyOFP-C1 plasmid, the cDNA of mT-sapphire or CyOFP1 was amplified by PCR and inserted into pEGFP-C1 (Clontech) plasmid to replace EGFP gene with NheI and HindIII sites. The vector pmT-Sapphire-CyOFP1 coding for the mT-Sapphire-CyOFP1 fusion protein was generated by inserting CyOFP1

cDNA

into

pmT-Sapphire-C1

vector

using

a

spacer

peptide

GASTVPRARDPPVAT between mT-Sapphire and CyOFP1. To construct plasmid encoding mT-sappire-NFAT1 or mT-sapphire-mNFAT1 protein, the cDNA of NFAT1 or mNFAT1 was amplified by PCR and inserted into pCyOFP1-C1 vector using EcoRI and BamHI sites. To construct pBiFC-Jun-CrN173 and pBiFC-Jun-VN173 plasmid, the cDNA of Jun was amplified by PCR and inserted into pBiFC-CrN173 or pBiFC-VN173 with EcoRI and XbaI sites. To construct pBiFC-β-Fos-CC155 plasmid, the cDNA of β-Fos was amplified by PCR and inserted into pBiFC-CC155 vector with EcoRI and KpnI sites. To construct pBiFC-FKBP-mT-Sapphire-N plasmid, the cDNA sequence of FKBP was amplified by PCR and inserted in to pBiFC-mT-Sapphire-N plasmid. To construct pBiFC-FRB-mT-Sapphire-C plasmid, the cDNA sequence of FRB was amplified by PCR and inserted into pBiFC-mT-Sapphire-C plasmid. To construct pcDNA3.1 (+)-FKBP-CyOFP1-C or pcDNA3.1 (+)-FRB-CyOFP1-N plasmid, 2×GGGGS linker was used. All the constructs were sequenced to ensure correct reading frame, orientation and sequences. Cell culture and transfection HeLa cell line was grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and antibiotics in a 5% CO2 incubator. Exponentially growing cells were dispersed with trypsin, seeded at 2×105 cells/35mm glass bottom dish in 1.5 mL of culture medium. The transfection of fusion protein constructs were carried out using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. After transfection, the cells were grown in DMEM complete medium for 24-72 h. For ratio analysis, plasmids encoding β-Jun and β-Fos [β-Fos (∆Zip)] fusion protein along with TagBFP or EGFP were cotransfected in a ratio of 2.5:2.5:1(0.75µg:0.75µg:0.3µg). Here, TagBFP or EGFP serves as an internal control to measure the BiFC efficiency. ACS Paragon Plus Environment


For two-color BiFC study, 0.4µg of each BiFC plasmid was used for co-transfection; for four-color BiFC study, 0.2µg of each BiFC plasmid was used for co-transfection. After transfection, the cells were incubated at 37℃ for 24-48 h and then 25℃ for 5-8 h (specifically for CyOFP1-based BiFC) before imaging. Fluorescence microscopy and image processing Olympus IX81 fluorescence microscope equipped with a 100×,NA = 1.45 oil immersion objective lens and cooled-coupled device controlled by MetaMorph software was used to acquire images. Excitation light was delivered by an X-cite light source. In most experiments, the excitation intensity was attenuated down to 25% of the maximum power of the light source. Images were acquired using 1×1 binning mode and 1s integration times. The excitation and emission were controlled and selected by Lambda 10-3 Shutter wheel changers (AutoMate Scientific). The filter sets used for each fluorescence channel were mT-Sapphire channel (Excitation filter: 387/11nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 529/39nm; Semrock), mCerulean channel (Excitation filter: 427/10nm; Dichroic mirror: 440/521/607/700nm; Emission filter: 472/30nm; Semrock), GFP channel (Excitation filter: 485/20nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 529/39nm; Semrock), mVenus channel (Excitation filter: 504/12nm; Dichroic mirror: 440/521/607/700nm; Emission filter: 529/39nm; Semrock), CyOFP1 channel (Excitation filter: 485/20nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 607/36nm; Semrock), TSC channel (Excitation filter: 387/11nm; Dichroic mirror: 444/521/608nm; Emission filter: 472/30nm; Semrock). FRET channel (Excitation filter: 387/11nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 607/36nm; Semrock).All fluorescence images were corrected by subtracting the background and analyzed using ImageJ (http://rsbweb.nih.gov/ij/) and Adobe Photoshop 7.0. The ratio value representative of fluorescence complementation efficiency was calculated as IBiFC-mT-Sapphire/ITagBFP or IBiFC-CyOFP1/IEGFP, wherein I represent background subtracted fluorescence intensity. Intensity-based FRET measurement with three-filter microscopy HeLa cells were plated onto 0.17 mm thick bottom glass dishes and were transiently ACS Paragon Plus Environment

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transfected with Lipofectamine 2000 (Invitrogen) 24 h later. The cells were washed twice with phosphate buffer saline (pH 7.4) and covered with 1 mL fresh medium. Then, images were taken with an Olympus IX81 inverted microscope equipped with a 100×,NA = 1.45 oil immersion objective lens and cooled-coupled device. Excitation light was delivered by an X-cite light source. For images acquisition, the MetaMorph software was used. In most experiments, the excitation intensity was attenuated down to 25% of the maximum power of the light source. Images were acquired using 1×1 binning mode and 400 ms integration times. For quantitative FRET measurements, the method of sensitized FRET was previously described in detail.34 Images were acquired sequentially through CyOFP1, FRET and mT-Sapphire filter channels. Here, the filter sets used were mT-Sapphire channel (Excitation filter: 387/11nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 529/39nm; Semrock), CyOFP1 channel (Excitation filter: 485/20nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 607/36nm; Semrock), FRET channel (Excitation filter: 387/11nm; Dichroic mirror: 410/504/582/669nm; Emission filter: 607/36nm; Semrock). The background images were subtracted from the raw images before carrying out FRET calculation. Corrected FRET (FC) was calculated on a pixel-by-pixel basis for the entire image using the following equation: FC = FRET – (a×CyOFP1) – (b×mT-Sapphire), where FRET, mT-Sapphire and CyOFP1 correspond to background subtracted images of cells coexpressing mT-Sapphire and CyOFP1 acquired through the FRET, mT-Sapphire and CyOFP1 channels respectively. The “a” and “b” are the fractions of bleed-through of CyOFP1 and mT-Sapphire fluorescence through the FRET filter channel, respectively. In our system, a = 0.04 ± 0.002(n = 30), b = 0.06 ± 0.003(n = 30). To quantify FRET efficiency, we used FR = [FRET-(b×mT-Sapphire)]/(a×CyOFP1), a relative value that varies with changes in energy transfer to quantify the FRET signal. The FRET ratio (FR) represents the fractional increase in CyOFP1 emission due to FRET. Thus, in the absence of energy transfer, FR has a predicted value of 1. Statistics All results are expressed as means ± SD values. Data were analyzed using two-tailed Student’s t test for comparison of independent samples. Differences were ACS Paragon Plus Environment


considered significant at p < 0.05.

AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

NOTES The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful to C. D. Hu for providing the plasmids pBiFC-β-Jun-VN173, pBiFC-β-Fos-VC155, pBiFC-β-Fos (∆Zip)-VC155, pBiFC-VN173, pBiFC-CrN173, pBiFC-VC155 and pBiFC-CC155. This work is supported by grants from the National Science Foundation of China 21573013, 21390412, 31271423 and 31327901, 863 Program SS2015AA020406 and CAS Interdisciplinary Innovation Team for Y. J. Sun.

ASSOCIATED CONTENT Supporting Information includes supplementary figures and references. The Supporting Information is available free of charge via the internet at http://pubs.acs.org


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FIGURES AND LEGENDS



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Figure 1. Identification of large-Stokes-shift fluorescent proteins for BiFC analysis. (A) HeLa cells were cotransfected with plasmids encoding β-Jun-mT-Sapphire-N, β-Fos-mT-Sapphire-C

and

internal

control

TagBFP

or

β-Fos-CyOFP1-N,

β-Jun-CyOFP1-C and internal control EGFP. Images were acquired 24 h after transfection. Scale bar, 40 µm in mT-Sapphire channel, 20 µm in CyOFP1 channel, 10 µm in merged images. (B) Quantitative analysis of mT-Sapphire and CyOFP1-based BiFC efficiency. All data are given as mean + S.D. (n >50). *p

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