Three-Fragment Fluorescence Complementation Coupled with

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Three-Fragment Fluorescence Complementation Coupled with Photoactivated Localization Microscopy for Nanoscale Imaging of Ternary Complexes Minghai Chen,†,§,⊥ Sanying Liu,†,§,⊥ Wei Li,† Zhiping Zhang,† Xiaowei Zhang,† Xian-En Zhang,*,‡ and Zongqiang Cui*,† †

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China § University of Chinese Academy of Sciences, Beijing 100049, China ‡

S Supporting Information *

ABSTRACT: Many cellular processes are governed by molecular machineries that involve multiple protein interactions. However, visualizing and identifying multiprotein complexes such as ternary complexes inside cells is always challenging, particularly in the subdiffraction cellular space. Here, we developed a three-fragment fluorescence complementation system (TFFC) based on the splitting of a photoactivatable fluorescent protein, mIrisFP, for the imaging of ternary complexes inside living cells. Using a combination of TFFC and photoactivated localization microscopy (PALM), namely, the TFFC-PALM technique, we are able to identify the multi-interaction of a ternary complex with nanometer-level spatial resolution and single-molecule sensitivity. The TFFC-PALM system has been further applied to the analysis of the Gs ternary complex, which is composed of αs, β1, and γ2 subunits, providing further insights into the subcellular localization and function of G protein subunits at the single-molecule level. The TFFC-PALM represents a valuable method for the visualization and identification of ternary complexes inside cells at the nanometer scale. KEYWORDS: mIrisFP, TFFC, PALM, ternary complexes, G protein

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usually permit the analysis of interactions between two proteins. Recently, the three-chromophore-based FRET (3-FRET) system16 and the BiFC-based FRET (BiFC-FRET) assay17 were developed for the visualization of ternary complexes within cells. However, these assays have not been widely used because they require skilled personnel, sophisticated microscopes, and data analysis. In addition, the optical crosstalk and contamination that occur during these assays often complicate interpretation of the results. The development of a simple and convenient assay for analyzing ternary complexes inside living cells would therefore have a significant impact. Another major concern regarding the analysis of protein interactions is that the functions of protein complexes are

any cellular processes are governed by multiprotein machineries that rely on protein−protein interactions (PPIs). In particular, many signaling proteins such as G protein function as ternary complexes.1,2 However, visualizing and identifying multiprotein interactions such as those comprising ternary complexes remains a challenging task. At present, several fluorescence-based methods such as fluorescence resonance energy transfer (FRET),3,4 bioluminescence resonance energy transfer (BRET),5−7 singlet oxygen triplet energy transfer-based imaging technology (STET),8 fluorescence cross-correlation spectroscopy (FCCS),9 linear expression cassette coupled with bimolecular fluorescence complementation (LEC-BiFC),10 and bimolecular fluorescence complementation (BiFC)11−15 are available and used for studying PPIs inside cells. For example, the BiFC assay, which relies on the reconstruction of a reporter fluorescent protein from its two divided nonfluorescent fragments, is very popular for imaging intracellular PPIs. However, these methods © 2016 American Chemical Society

Received: May 29, 2016 Accepted: September 1, 2016 Published: September 1, 2016 8482

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Figure 1. Construction of mIrisFP-based BiFC systems. (a) The cleavage sites (arrows) of mIrisFP are indicated. (b) Green and red mIrisFP BiFC signals were detected in Vero cells for the cleavage sites between aa 150/151 and 165/166 for mIrisFP fragments based on the interactions between bJun and bFos or mbFos. The green form of mIrisFP was generated with 488 nm laser excitation, and the red form of mIrisFP was generated by 405 nm laser irradiation for 30 s prior to image acquisition with 561 nm laser excitation. (c) Quantitative analysis of the BiFC efficiency in (b). (d) Comparable expression of the fusion proteins in (b) determined by Western blotting with anti-HA and antiFlag antibodies. (e) No mIrisFP BiFC signal was detected when mIrisFP was split into fragments at the aa sites 134/135 and 157/158. The nucleus was stained with Hoechst 33342. Scale bars: 10 μm. All data are given as the mean ± SD (n = 60). The statistical significance was evaluated using a two-tailed Student’s t test. *** indicates p < 0.01.

imaging the ternary complexes inside living cells and for imaging the nanoscale subcellular distribution of individual ternary complexes with PALM. TFFC relies on the reconstruction of a reporter fluorescent protein from its three divided nonfluorescent fragments. mIrisFP, a recently developed monomeric photoactivatable and photoconvertible fluorescent protein,24 was first split at different sites to build the BiFC-PALM systems. Then, the TFFC-PALM system was constructed by splitting mIrisFP into three nonfluorescent fragments. This TFFC-PALM method was applied toward the study of the interactions of the αs, β1, and γ2 subunits of the heterotrimeric Gs protein, which plays important roles in several signal transduction pathways.25,26 The αsβ1γ2 heterotrimer was imaged inside cells at the single-molecule level, and the results revealed the different distribution patterns and biological functions of the αsβ1γ2 heterotrimer and the β1γ2 heterodimer at the nanometer scale.

closely related to their spatial distribution, and these complexes are often spatially regulated at the nanometer scale inside cells. However, the conventional fluorescence-based methods for PPI assays face limitations in terms of resolution. Due to the diffraction of light, the best spatial resolution achieved with such methods is ∼200 nm, which does not meet the demands of a nanometer-scale assay. In addition, these methods are incapable of imaging and analyzing individual PPIs. Several techniques, such as photoactivated localization microscopy (PALM)18 and stochastic optical reconstruction microscopy (STORM),19 have recently been developed to overcome the resolution limit of fluorescence microscopy. PALM is a method of sequentially localizing sparse subsets of photoactivatable fluorescent proteins to obtain a subdiffraction-limited fluorescent image. Based on stochastic switching and subdiffractive localization of individual fluorescent molecules, PALM has realized a ∼20 nm spatial resolution.20,21 More recently, BiFC systems based on split PAmCherry122 or mEos3.223 have been developed for the nanoscale imaging of PPIs with PALM. Unfortunately, however, a method for imaging ternary complexes with high resolution is still unavailable. In this work, we developed a three-fragment fluorescence complementation system (TFFC) based on split mIrisFP for

RESULTS AND DISCUSSION To develop the TFFC system, we first constructed an mIrisFPbased BiFC system. On the basis of the crystal structure of mIrisFP (PDB accession code 2VVH)27 and sequence alignments with other fluorescent proteins (mCherry and 8483

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ACS Nano YFP), three sites were selected among amino acids (aa) 134/ 135, 150/151, and 165/166, for splitting mIrisFP within the loops of its barrel-like secondary structures to develop the BiFC system (Figure 1a). The site aa 157/158 localized in the β-sheet of the mIrisFP’s secondary structures was selected as a control split site. The fragment pairs obtained were named mIN134/ mIC135, mIN150/mIC151, mIN157/mIC158, and mIN165/ mIC166. The coding sequences of these mIrisFP split reporter fragments were inserted into the pcDNA3.1 plasmid to construct the vectors pmIN134, pmIC135, pmIN150, pmIC151, pmIN157, pmIC158, pmIN165, and pmIC166. The well-known interaction of the bJun and bFos protein pair was then used to test the BiFC systems.11 Either bJun or bFos encoding sequences were inserted into pmIN134, pmIC135, pmIN150, pmIC151, pmIN157, pmIC158, pmIN165, and pmIC166, to express bJun-(mIN) N-terminal and bFos-(mIC) C-terminal fusion proteins. When the four sets of bJun-mIN and bFos-mIC proteins were coexpressed in Vero cells, the combination of bJunmIN150 and bFos-mIC151 yielded a strong green BiFC signal from the reconstitution of the mIrisFP molecules (Figure 1b). After the sample was irradiated with a 405 nm laser light for 30 s, the reconstituted green mIrisFP molecules were converted to the red form and a red BiFC signal could then be detected (Figure 1b). The combination of bJun-mIN165 and bFosmIC166 also yielded moderate green and red BiFC signals (Figure 1b). As a control, we also fused mbFos to mIC151 or mIC166 and coexpressed these proteins with bJun-mIN150 or bJun-mIN165, respectively. mbFos is a mutated bFos known to eliminate the interaction between bFos and bJun, but which has no effect on its interaction with other partners.11 As expected, in the presence of mbFos, only very weak fluorescence signals were visible (Figure 1b). Quantitative analyses confirmed the significant differences for the BiFC signals between these bJunbFos and bJun-mbFos BiFC systems. The bJun-bFos BiFC signals were ∼70-fold higher than bJun-mbFos BiFC signals (Figure 1c). Western blot analyses showed that these fusion proteins were, however, expressed at equivalent levels in the BiFC system (Figure 1d). For the other fragments obtained by splitting mIrisFP at the 134/135 or 157/158 sites, the combination of bJun-mIN134 with bFos-mIC135, and bJunmIN157 with bFos-mIC158, did not result in any observable BiFC signals (Figure 1e). This meant that the spatial configuration of the mIrisFP could not be reconstituted after the protein was split at the 134/135 or 157/158 sites. These results showed that the mIrisFP could be split at the 150/151 or 165/166 site to develop the BiFC system for imaging PPIs. Next, the mIrisFP-based BiFC fluorescent system was tested for the imaging of PPIs at the nanometer scale with the PALM technique. As shown in Figure 2, the red form of the reconstituted mIrisFP was imaged with a 561 nm excitation laser and a 488 nm light for on-switching after photoconversion with 405 nm laser irradiation for 30 s. The BiFC-reconstituted mIrisFP not only maintained the ability to reversibly photoswitch between the fluorescent and nonfluorescent states (Figure 2a) but also held similar photophysical properties to those of native mIrisFP (Figure S1, Video S1, and Figure S2). We then imaged the bJun/bFos complexes using the mIrisFPbased BiFC-PALM system. Single bJun/bFos complexes could be detected in single-molecule images of reconstituted mIrisFP, and each dot represents a single bJun/bFos complex (Figure 2b, Video S2). BiFC-PALM imaging provided a greater level detail and higher resolution (∼18 nm molecular organization

Figure 2. BiFC-PALM imaging of PPIs. (a) Reversible photoswitching of BiFC-reconstituted mIrisFP. (b) Example of singlemolecule images of reconstituted mIrisFP. (c) PALM (left) and TIRF (right) images of bJun/bFos complexes. Enlarged views of the regions marked by white boxes are shown below. mIrisFP was imaged with a 561 nm excitation laser and 488 nm light for onswitching after photoconversion with 405 nm laser irradiation for 30 s. (d, e) Distribution of σ (d) and photons (e) in cells expressing the bJun/bFos complexes. TIRF represents the conventional imaging in TIRF mode. Scale bars: 2 μm.

uncertainty, σ) compared with the traditional method of total internal reflection (TIRF) imaging (Figure 2c,d), acquiring an average of ∼900 photons per localization event (Figure 2e). The green form of reconstituted mIrisFP was also imaged using BiFC-PALM, but certain characteristics, such as the distribution of σ and the photon number, were inferior to those of the red form (Figure S3a−d). These results demonstrated that split mIrisFP could be used to successfully develop a BiFC-PALM system for the imaging of PPIs at the nanometer scale. After developing the BiFC-PALM fluorescent system based on mIrisFP, we attempted to split mIrisFP into three nonfluorescent fragments to construct a TFFC system for imaging multiprotein interactions. The schematic principle of the TFFC system is shown in Figure 3a. When the appropriate split sites are chosen, these three fragments can be brought together and reconstituted to emit fluorescence, as a result of the simultaneous and successful association of the three protein units fused to each mlrisFP fragment, thereby allowing the analysis of the ternary complex of interest. On the basis of the crystal structure of mIrisFP and the results obtained from the mIrisFP-BiFC systems, we designed four schemes for splitting mIrisFP into three fragments, and all split sites were localized within the unstructured flexible loops (Figure 3). The three fragments were denoted fragment 1, fragment 2, and fragment 3. The Fos-Jun-NFAT ternary complex was used as the test model for constructing the TFFC system. NFAT is a transcription factor required for T cell development and for many other cellular processes28 and is known to form a ternary complex with the Fos-Jun heterodimer to regulate many target genes.28,29 NFAT1 was fused to the fragment 1, while the basic 8484

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Figure 3. Schemes to split mIrisFP to construct the mIrisFP-based TFFC systems. (a) Schematic principle of TFFC. (b) Four schemes were used to split mIrisFP to construct the TFFC systems. The arrows indicate the cleavage sites. (c) The cartoon images show three mIrisFP splits (shown as red, green, and blue colors) in the barrel-like structure of mIrisFP corresponding to the four schemes in (b).

those of native mIrisFP (Figure S2). We first imaged the bJunbFos-NFAT1 heterotrimer using TFFC-PALM. Single bJunbFos-NFAT1 heterotrimers could be detected in singlemolecule images of reconstituted mIrisFP, and the spots with apparently higher fluorescence represent the bJun-bFosNFAT1 heterotrimers (Figure 5b, Video S3). The bJun-bFosNFAT1 heterotrimer exhibited a discontinuous dot distribution in the high-resolution PALM image (Figure 5c). TFFC-PALM imaging provided a greater level of detail with higher resolution (∼18 nm molecular organization uncertainty, σ) compared with TIRF imaging (Figure 5c,d), acquiring an average of ∼770 photons per localization event (Figure 5e). Similarly to the results obtained with BiFC-PALM imaging, the green form of mIrisFP-TFFC could also be used for TFFC-PALM, although this form was again inferior to the red form (Figure S5a−d). These results demonstrate that the mIrisFP TFFC fluorescent system is a powerful tool for imaging multiprotein interactions at the nanometer scale. With the mIrisFP-based TFFC-PALM fluorescent system, it is possible to identify and image ternary complexes with a subdiffraction-limit spatial resolution. To test-drive this TFFCPALM method, we next applied it toward the imaging of the Gs protein αβγ heterotrimers, as they play important roles in signal transduction. Heterotrimeric G proteins are composed of α and βγ subunits. The βγ complex dissociates only when denatured.30−32 Upon receptor activation, the GTP-bound α is disassociated from the βγ dimer and transmits signals to downstream molecules, and the GDP-bound α can then reassociate with the βγ dimer to reconstitute the heterotrimer. Although the subcellular localization, function, and regulation of the subunits of the G protein heterotrimers have been

region leucine zipper (bZIP) domain of Jun (bJun) and Fos (bFos) was fused to fragments 2 and 3, respectively. Our results showed that when mIrisFP was split at residues 150/151 and 165/166, the three mIrisFP fragments could be reconstituted to recover the fluorescence of native mIrisFP via the association of the bFos, bJun, and NFAT1 heterotrimer (Figure 4a−c). Split mIrisFP fragments obtained from the other three schemes did not result in any observable fluorescence (Figure 4d). When mNFAT1 (where three amino acids in NFAT1, R466A/I467A/T533G, were mutated to destroy its interaction with the bJun-bFos heterodimer)17 or mbFos was included in the system or when any one of the three mlrisFP fragments such as the short middle fragment was absent, no fluorescence signal could be detected (Figure 4a). In addition, coexpression of the three free mIrisFP fragments, mIN150, mIN(151−165), and mIC166, without fusion to the bFos-bJun-NFAT1 heterotrimer also did not produce a fluorescence signal (Figure S4). The statistical analysis of the complementary fluorescence intensities produced in these assays is shown as Figure 4b. Western blot analyses confirmed that all fusion proteins were expressed at comparable levels (Figure 4c). In summary, the mIrisFP protein was successfully split into three fragments in the region of residues 150/151 and 165/166 to generate a mIrisFP-TFFC system for identifying and imaging the ternary protein interactions. We next used the mIrisFP-based TFFC fluorescent system to image multiprotein interactions at the nanometer scale with the PALM technique. The red form of mIrisFP-TFFC PALM imaging is shown in Figure 5. The TFFC-reconstituted mIrisFP not only maintained a reversible photoswitching character (Figure 5a) but also held similar photophysical properties to 8485

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Figure 4. Construction of mIrisFP-based TFFC systems. (a) Green and red mIrisFP TFFC signals were detected in Vero cells due to the interaction of NFAT1-bJun-bFos, as determined by the cleavage sites at aa 150/151 and 165/166. (b) Quantitative analysis of the TFFC efficiency in (a) based on the reconstructed fluorescence intensities. (c) Comparable expression of fusion proteins in (a) was determined by Western blotting with anti-HA and anti-Flag antibodies. (d) No mIrisFP TFFC signal was detected when mIrisFP was split at the sites according to schemes 2 to 4 shown in Figure 3. The nucleus was stained with Hoechst 33342. Scale bars: 10 μm. All data are given as the mean ± SD (n = 60). The statistical significance was evaluated using a two-tailed Student’s t test. *** indicates p < 0.01.

extensively studied,33−35 the actual ternary αβγ complex has not yet been directly observed and investigated in cells because of the absence of the appropriate methodology. Here, by using our TFFC-PALM fluorescent system, we were able to visualize the αsβ1γ2 heterotrimer in cells at the single-molecule level. As shown in Figure 6a, the interactions among the αs, β1, and γ2 subunits resulted in the reconstitution of the three mIrisFP fragments, resulting in fluorescence emission in the TFFCPALM system. The αsβ1γ2 heterotrimer showed a discontinuous small dot distribution pattern on the cell membrane (Figure 6a and Figure S6a). Each dot in the high-resolution image represented a single αsβ1γ2 heterotrimer complex. As a control, when αs was mutated (denoted as αsm) to destroy its interaction with β1γ2,36 no fluorescence signal could be detected in the TFFC-PALM system (Figure S7). We also visualized the β1γ2 heterodimer using the mIrisFPbased BiFC-PALM system. As shown in Figure 6b, the β1γ2 heterodimer, which produced BiFC signals from the interaction between the β1 and γ2 units, was imaged at a single-molecule scale. The β1γ2 heterodimers were present in two forms, namely, clusters and small dots, and these were primarily located in the cytoplasmic zone (Figure 6b,c and Figure S6b), which differs from the cell membrane localization observed for the αsβ1γ2 heterotrimers. To further characterize the αsβ1γ2 heterotrimer, cells were treated with cholera toxin during the TFFC-PALM imaging assay. Cholera toxin can cause Gs protein to abnormally maintain activation, forcing Gαs to dissociate from the Gβγ

heterodimer.37 During the assay, Vero cells were transfected with a combination of pαs-mIN150, pβ1-mIN(151−165), and pγ2-mIC166 and then treated with 3 μg/mL of cholera toxin at 5 h after transfection. After 16−18 h of further incubation, the cells were imaged with PALM. The results showed that in cholera toxin-treated cells the quantity of αsβ1γ2 heterotrimers showing TFFC fluorescence decreased significantly compared to that in untreated cells, while the BiFC fluorescence signals from the β1γ2 heterodimers showed no apparent difference between the cholera toxin-treated and untreated cells (Figure 6d). These results suggested that cholera toxin inhibited the formation of the αsβ1γ2 heterotrimer, while it had no obvious influence on the formation of the β1γ2 heterodimer. In our experiments, we also attempted to use cholera toxin to reduce pre-existing TFFC signals arising from the αsβ1γ2 interaction. Vero cells with TFFC fluorescence signals arising from the αsβ1γ2 heterotrimers were treated with cholera toxin for various time periods (e.g., 20, 40, or 60 min) and then analyzed with PALM imaging. No obvious decrease in TFFC fluorescence signals was apparent in the cells treated with cholera toxin compared with untreated cells (Figure S8). As cholera toxin causes Gαs to dissociate from the Gβγ heterodimer, a reasonable explanation for this result may be that the mIrisFP TFFC system is irreversible, similar to most BiFC systems.12,38 The development of a convenient assay for the visualization and identification of ternary complexes at the nanometer scale will facilitate significant insights in protein interaction studies. In this study, we constructed the TFFC fluorescent system 8486

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inside cells with higher spatial resolution compared with traditional TIRF imaging. The cleavage sites in mIrisFP used to construct the TFFC system also provide a good reference point for the development of TFFC systems using other fluorescent proteins. The reconstituted form of mlrisFP comprising the three split mIrisFP fragments retains the photophysical properties of native mIrisFP, which validated its feasibility for use in the TFFC-based PALM imaging technique. We believe this breakthrough in the creation and application of TFFC systems will promote the study of multiprotein interactions inside cells. Of course, there are possible limitations for the TFFC system, such as inability to quantify interaction strength between protein partners in the complexes and the orientation influence of the fragments as fusion to the N- or C-terminus of the interaction proteins. During this study, several mIrisFPbased BiFC-PALM systems were also built for visualizing and identifying individual PPIs inside cells. These systems also add tools to the current suite of BiFC-PALM systems, which, until now, only comprised the recently developed PAmCherry1- and mEos3.2-based BiFC-PALM systems,22,23 thereby providing alternative tools for PPIs analyses at high resolution. The TFFC-PALM fluorescent system and BiFC-PALM were also used to study the heterotrimer subunits of the Gs protein, which play important roles in cell signaling pathways. Our results provided the previously unobserved localization patterns for the αsβ1γ2 heterotrimer and the β1γ2 heterodimer at the single-molecule level. The αsβ1γ2 heterotrimer showed a uniform small dot distribution pattern on the cell membrane, while the β1γ2 dimers were primarily localized in the cytoplasm in clusters or small dot forms. The different localization patterns observed for the αs β1γ2 heterotrimer and β1γ2 heterodimer correlate with their known biological functions and thus further indicate that the αs subunit plays key roles in the subcellular translocation of Gs protein during signal transduction. Cholera toxin treatment significantly decreased the formation of TFFC fluorescence dots comprising the αsβ1γ2 heterotrimer,

Figure 5. TFFC-PALM imaging of ternary complex interactions. (a) Reversible photoswitching of TFFC-reconstituted mIrisFP. (b) Example of single-molecule images of reconstituted mIrisFP. (c) TFFC-PALM (left) and TIRF (right) images of the bJun-bFosNFAT1 complexes. Enlarged views of the regions marked by white boxes are shown below. (d, e) Distribution of σ (d) and photons (e) in cells expressing the bJun-bFos-NFAT1 complexes. Scale bars: 1 μm.

based on mIrisFP for imaging ternary complexes inside living cells. By taking advantage of the photoactivatable properties of mIrisFP, we are able to visualize ternary complexes at the single-molecule level by combining the mIrisFP-based TFFC system with the PALM technique. Our TFFC-PALM fluorescent system therefore provides a powerful tool for the direct visualization and identification of ternary complexes

Figure 6. Super-resolution imaging of the Gαsβ1γ2 heterotrimer by TFFC-PALM. (a) TFFC-PALM imaging of the Gαsβ1γ2 heterotrimers. (b) BiFC-PALM imaging of the Gβ1γ2 heterodimers. (c) Statistical analysis of clusters for Gαsβ1γ2 and Gβ1γ2 (n = 10). (d) Imaging of Gαsβ1γ2 and Gβ1γ2 when treated with cholera toxin. Scale bars: 10 μm. 8487

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plasmids, αs (Sangon, Shanghai), β1 and γ2 were fused to the Nterminus of mIN150, mIN(151−165), and mIC166, respectively. The primers used in this study are listed in Table S1. All of the sequences were verified by DNA sequencing. Cell Culture and Transfection. Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C under 5% CO2 in a humidified incubator. The cells were seeded the day before transfection in 35 mm glass bottom coverslips at 70−80% confluency. Transfection of the Vero cells with the plasmids was conducted using 2 μg of each component with Lipofectamine 2000 (Invitrogen; USA) following the manufacturer’s instructions. For the experiment characterizing Gs subunits, 3 μg/mL cholera toxin was added to the medium 5 h after transfection. The transfected cells were incubated at 37 °C (5% CO2) for 8−12 h followed by 10−12 h at 27 °C (5% CO2) before imaging. For PALM imaging, cells were fixed in fresh 4.0% PFA for 30 min at room temperature and changed to PBS buffer after fixation. Confocal Fluorescent Microscopy, Image Acquisition, and Data Analysis. The cells were imaged using an UltraVIEW VOX Confocal system (PerkinElmer, Co.) with a 60×, 1.4 numerical aperture (NA), oil immersion objective lens. The green fluorescence from the mIrisFP green channel was excited at 488 nm. The red fluorescence from the mIrisFP red channel was excited at 561 nm after the sample was irradiated with a 405 nm laser light (10 mW cm−2) for 30 s. The nuclei were stained using Hoechst 33342, which was excited at 405 nm. The original fluorescence images were acquired using the same exposure time in the same channel. For fluorescent quantitative analysis, the green and red fluorescence intensities were acquired from their original images by subtracting their background fluorescence. The background values were the average fluorescence intensities obtained from a region (50 × 50 pixel2) without green and red signals. The average fluorescence intensities of approximately 60 fluorescent cells (from randomly selected 10−15 images) were used for the relative BiFC and TFFC efficiency calculations. Western Blot Analysis. Western blot analysis was performed to determine the expression levels of various fusion proteins. The cell lyses were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated with specific antibodies against the HA or Flag epitopes of the fusion proteins (Abcam), followed by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (Abcam). The signal was detected with a chemiluminescent detection system (BioRad). Super-resolution Imaging and Data Analysis. BiFC-PALM and TFFC-PALM imaging were conducted using a Nikon TiE inverted microscope equipped with a 100×, 1.49 NA, oil immersion objective lens (Nikon, A1MP STORM) and an Andor-897 EMCCD (Andor). Three lasers with emission wavelengths of 561 nm (Coherent, 50 mW), 488 nm (Coherent, 100 mW), and 405 nm (Coherent, 100 mW) were available for fluorescence excitation, photoswitching to the on state, and green-to-red photoconversion. The mIrisFP channel was collected with continuous 488 and 405 nm laser irradiation or continuous 561 and 488 nm laser irradiation after photoconversion by a 405 nm laser. The lasers were slowly adjusted for optimal photoswitching rates. Fluorescent spots above the threshold were acquired by subtracting background (the background was defined as the fluorescence intensity of the nonfluorescent region in the cell). Localization precision was determined as previously described.40,41 Super-resolution image reconstruction was performed using Insight 3 software, and then, the images were reanalyzed by a Gaussian fitting routine. TIRF illumination was used in all PALM imaging experiments.

while such treatment had no obvious influence on the formation of β1γ2 heterodimer. These observations further confirm that signal activation causes the αs to dissociate from the β1γ2 subunit, indicating the important role of αs in the recycling of the αsβ1γ2 heterotrimer and β1γ2 heterodimer. After the αsβ1γ2 subunits were observed to form a stable interaction in the mIrisFP TFFC system, the TFFC signals emitted as a result of the αsβ1γ2 interaction could not be reduced by cholera toxin treatment. One of the explanations for this result may be that the mIrisFP TFFC system is irreversible, as most BiFC systems.12,38,39 A reversible TFFC system allows studying the dynamic combination and dissociation of protein complexes. Further efforts should be dedicated to developing a reversible TFFC system, although the irreversible TFFC system offers significant advantages for the screening of weak or transient interacting partners.

CONCLUSION In conclusion, we developed the method TFFC-PALM for studying cellular multiprotein interactions with high spatial resolution. The TFFC-PALM fluorescent system represents a valuable tool for the imaging of ternary complexes at the singlemolecule level. Application of TFFC-PALM toward the study of the Gs protein αsβ1γ2 heterotrimer also provides insights into the subcellular localization and function of G protein subunits at the single-molecule level. METHODS Construction of Plasmids. The mIrisFP protein was split at four positions between aa 134/135, 150/151, 157/158, and 165/166 to construct the BiFC systems. The fragment pairs obtained were named mIN134/mIC135, mIN150/mIC151, mIN157/mIC158, and mIN165/mIC166, which correspond to the aa sequences of 1−134 and 135−226, 1−150 and 151−226, 1−157 and 158−226, and 1−165 and 166−226, respectively. For the construction of pbJun-mIN134, pbJun-mIN150, pbJun-mIN157, and pbJun-mIN165 plasmids, bJun was amplified by PCR from plasmid pbJun-iRN97.14 Kozak sequences and an HA tag were included in the forward primer and inserted into the corresponding sites of pcDNA3.1 (+) using NheI and KpnI restriction enzymes. mIN134, mIN150, mIN157, and mIN165 were amplified by PCR from the mIrisFP sequence (Sangon, Shanghai) and inserted into the corresponding sites of pcDNA3.1-bJun using BamHI and EcoRI restriction enzymes. The coding regions were connected by the linker sequence GGGGSGGGGS. To construct the plasmids of pbFos-mIC135, pbFos-mIC151, pbFos-mIC158, and pbFos-mIC166, bFos was amplified by PCR from plasmid piRC98-bFos.14 Kozak sequences and a flag tag were included in the forward primer and inserted into the corresponding sites of pcDNA3.1 (+) using NheI and KpnI restriction enzymes. mIC135, mIC151, mIC158, and mIC166 were amplified by PCR from the mIrisFP sequence and inserted into the corresponding sites of pcDNA3.1-bFos using BamHI and EcoRI restriction enzymes. The coding regions were also connected by the linker sequence GGGGSGGGGS. The plasmids of pmbFos-mIC151 and pmbFos-mIC166 were constructed by replacing bFos with mbFos at the same sites of pbFos-mIC151 and pbFos-mIC166. mbFos was amplified by PCR from plasmid piRC98-mbFos.14 We chose the Fos-Jun-NFAT1 ternary complex as the test model to construct the mIrisFP-TFFC systems because of their well-known strong interactions. We split mIrisFP into three nonfluorescent fragments. Fragment 1 was fused to NFAT1 (Sangon, Shanghai) or (R466A/I467A/T533G) NFAT1 (named mNFAT1), fragment 2 was fused to bJun, and fragment 3 was fused to bFos or mbFos. For the construction of plasmids pβ1-mIN150 and pmIC151-γ2, β1 and γ2 were PCR-amplified from random-primed HeLa cDNA and fused to the fragments mIN150 and mIC151, respectively. For the construction of pαs-mIN150, pβ1-mIN(151−165), and pγ2-mIC166

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b03543. Figures S1−S8 (PDF) Video S1 (AVI) 8488

DOI: 10.1021/acsnano.6b03543 ACS Nano 2016, 10, 8482−8490

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Video S2 (AVI) Video S3 (AVI)

AUTHOR INFORMATION Corresponding Authors

*E-mail (Z. Cui): [email protected]. *E-mail (X.-E. Zhang): [email protected]. Author Contributions ⊥

M. Chen and S. Liu contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Z.-Q.C. is supported by the National Nano Project (no. 2011CB933600), the National Natural Science Foundation of China (NSFC) (no. 31470269), and the Youth Innovation Promotion Association CAS. Z.-P.Z. is supported by NSFC (no. 31470837). X.-E.Z. is grateful for support from the Chinese Academy of Sciences (KJZD-EW-TZ-L04). We thank the Core Facility and Technical Support, Wuhan Institute of Virology for excellent technical support. REFERENCES (1) Khan, S. M.; Sleno, R.; Gora, S.; Zylbergold, P.; Laverdure, J. P.; Labbe, J. C.; Miller, G. J.; Hebert, T. E. The Expanding Roles of G Beta Gamma Subunits in G Protein-Coupled Receptor Signaling and Drug Action. Pharmacol. Rev. 2013, 65, 545−577. (2) Takida, S.; Wedegaertner, P. B. Heterotrimer Formation, Together with Isoprenylation, is Required for Plasma Membrane Targeting of G Beta Gamma. J. Biol. Chem. 2003, 278, 17284−17290. (3) Stryer, L. Fluorescence Energy-Transfer as a Spectroscopic Ruler. Annu. Rev. Biochem. 1978, 47, 819−846. (4) Jares-Erijman, E. A.; Jovin, T. M. Imaging Molecular Interactions in Living Cells by FRET Microscopy. Curr. Opin. Chem. Biol. 2006, 10, 409−416. (5) Xu, Y.; Piston, D. W.; Johnson, C. H. A Bioluminescence Resonance Energy Transfer (BRET) System: Application to Interacting Circadian Clock Proteins. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 151−156. (6) Xu, X. D.; Soutto, M.; Xie, Q.; Servick, S.; Subramanian, C.; von Arnim, A. G.; Johnson, C. H. Imaging Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET) in Plant and Mammalian Cells and Tissues. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 10264−10269. (7) Ogoh, K.; Takahashi, T.; Miyawaki, A.; Suzuki, H. Bioluminescence Resonance Energy Transfer (BRET) Image Analysis of Ras-Raf Interaction in Live Cells using Nanoluc Luciferase and Venus Yellow Fluorescent Protein and Bioluminescence Microscopy. Luminescence 2014, 29, 87−87. (8) To, T. L.; Fadul, M. J.; Shu, X. K. Singlet Oxygen Triplet Energy Transfer-Based Imaging Technology for Mapping Protein-Protein Proximity in Intact Cells. Nat. Commun. 2014, 5, 4072. (9) Oyama, R.; Takashima, H.; Yonezawa, M.; Doi, N.; MiyamotoSato, E.; Kinjo, M.; Yanagawa, H. Protein-Protein Interaction Analysis by C-Terminally Specific Fluorescence Labeling and Fluorescence Cross-Correlation Spectroscopy. Nucleic Acids Res. 2006, 34, e102. (10) Lin, J.; Wang, N.; Li, Y.; Liu, Z.; Tian, S.; Zhao, L.; Zheng, Y.; Liu, S.; Li, S.; Jin, C.; Xia, B. LEC-BiFC: A New Method for Rapid Assay of Protein Interaction. Biotech. Histochem. 2011, 86, 272−279. (11) Hu, C. D.; Chinenov, Y.; Kerppola, T. K. Visualization of Interactions among bZip and Rel Family Proteins in Living Cells using Bimolecular Fluorescence Complementation. Mol. Cell 2002, 9, 789− 798. (12) Filonov, G. S.; Verkhusha, V. V. A Near-Infrared BiFC Reporter for in vivo Imaging of Protein-Protein Interactions. Chem. Biol. 2013, 20, 1078−1086. 8489

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