Abstract
Kinetic simulation is a useful approach for the elucidation of complex cell-signaling systems. To perform the numerical simulations required for kinetic modeling, kinetic parameters such as the protein concentration and dissociation constant (Kd) are essential. However, only a limited number of kinetic parameters have been determined experimentally in living cells. Here, we describe a method for quantifying the concentration and dissociation constant (Kd) of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in (KI) and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked-in at the end of the MAPK1 gene, which encoded ERK2, through homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ). Next, the HaloTag gene was further knocked-in at the end of the RSK2 gene. Protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag were quantified in living cells by fluorescence correlation spectroscopy (FCS), revealing substantial cellular heterogeneities. Interestingly, the levels of ERK2-mEGFP and RSK2-HaloTag were strongly positively correlated, suggesting a global mechanism underlying their expressions. In addition, FCCS measurement revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to EGF stimulation. Our method provides a new approach for quantification of the endogenous protein concentration and dissociation constant in living cells.
Introduction
In response to extracellular signals, mammalian cells process information through an intracellular signaling network comprised of chemical reactions, eventually leading to cell fate decision making. Extensive studies of cell signaling have identified numerous proteins, pathways, and feedback or feedforward regulations, and have expanded our understanding beyond the simple view of the linear signaling cascade (1). Computer-assisted systems biology approaches may provide a promising strategy for the comprehensive understanding of such complicated cell signaling networks (2). Indeed, various simulation models of signal transduction pathways have been developed in a series of studies over the past 10 years (3–5). Nevertheless, the numerical simulations in most of these systems were conducted with kinetic parameters that have not been measured experimentally, and thus these parameters tend to differ among models, even when the reactions themselves are identical. For this reason, experimentally determined kinetic parameters are essential for the development of quantitative and reliable simulation models.
Kinetic parameters of cell signaling are roughly classified into four categories: protein concentration, dissociation constant (Kd), diffusion coefficient or transport rate, and enzymatic reaction rate parameters. Classically, these kinetic parameters have been measured by in vitro biochemical analyses, which require a large number of cells or molecules (6, 7). However, these parameters, e.g., protein concentration, are known to show non-genetic cell-to-cell variability (8, 9). Moreover, some kinetic parameters might differ significantly between in vitro and in vivo. For instance, we have shown that ERK MAP kinase represents entirely different phosphorylation patterns, namely processive and distributive phosphorylation, under an intracellular and an in vitro environment, respectively (7, 10). In addition, the Kd values measured in vivo were substantially higher than the in vitro Kd values by an order of 1 or 2 (11). Therefore, it is of critical importance to measure kinetic parameters in living cells.
Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are techniques that employ fluctuations of fluorescent molecules in the confocal volume (~ 1 fL) (12–14). FCS examines the auto-correlation function of temporal fluorescence fluctuation, enabling us to determine the number of fluorescent molecules in the confocal volume and diffusion coefficient. In FCCS, the cross-correlation function is calculated between fluctuations of two different florescent species in order to quantify the extent to which these two species form a complex. Thus, FCS and FCCS are capable of measuring Kd values in living cells—i.e., in vivo Kd. We have previously applied FCS and FCCS to mammalian cells exogenously expressing target proteins used with EGFP and HaloTag for the measurement of in vivo Kd (11). It has been reported that the endogenous protein concentration and in vivo Kd value were successfully measured in budding yeast with FCS and FCCS (15, 16). However, there have been no reports of the in vivo Kd values based on the measurement of endogenous proteins in mammalian cells, mainly because of the technical difficulties of knock-in (KI) of a fluorescent protein gene to label the protein of interest.
Recent advances in genome-editing tools have paved the way for tagging endogenous proteins with fluorescent proteins. These genome-editing tools, such as the CRISPR/Cas9 system, enable KI of a gene of interest through DNA double-strand break (DSB) repair mechanisms (17, 18). Homology-directed repair (HDR) is a mechanism by which a homologous template is used as a source of DNA repair. On the other hand, microhomology-mediated end joining (MMEJ) is a mechanism of alternative non-homologous end joining (NHEJ), which also seals DSB. In contrast to classical NHEJ, MMEJ repairs DNA DSB using a 5-25 base pair (bp) microhomologous sequence (19). HDR uses a relatively longer homologous sequence (0.1-10 kbp) to seal the DSB, thereby ensuring an error-free repair. Nonetheless, even though MMEJ is an error-prone process of end joining, a recent study demonstrated that CRISPR/Cas9-mediated KI was more efficient than HDR (20).
In this study, we demonstrate a new approach to quantifying the concentration and Kd of endogenous proteins by combining FCS and FCCS with CRISPR/Cas9-mediated KI of fluorescent proteins in mammalian cells.
Results
Design of donor vectors and KI strategy
First, we attempted to knock-in the mEGFP gene at the 3’ site of the human MAPK1 gene encoding ERK2 in HeLa cells. To do this, we constructed a selection cassette for the donor vector, which contained the mEGFP gene, a loxP sequence, a porcine teschovirus-1-derived self-cleaving 2A peptide (P2A) sequence, a bi-functional fusion protein between a truncated version of herpes simplex virus type 1 thymidine kinase (dTK) and the bacterial neomycin phosphotransferase (neo) genes (dTKneo), a poly(A) addition sequence, and a loxP sequence (Fig. 1A and Fig. S1). To develop a donor vector for KI at the hMAPK1 gene, this cassette was further sandwiched between the longer homology arms (L. arm and R. arm) or the shorter homology arms (40 bp for each) for HDR- or MMEJ-mediated DSB repair (Fig. 1A, top and bottom). The dTKneo was generated based on the previous reports (21, 22) as a positive/negative selection marker, and connected to mEGFP via cDNA of the P2A sequence, which is a self-cleaving peptide so that genes sandwiching the P2A peptide are separately expressed (23). Therefore, the expression of dTKneo requires both in-frame integration and endogenous promoter activity of the gene prior to the KI cassette, resulting in the suppression of false-positive cells harboring random insertions of the cassette. dTKneo expression renders cells resistant to G418 and sensitive to Ganciclovir. Of note, it has been reported that neo is preferable for targeting moderate or low expression genes with the promoter-less targeting vector (24). After KI of the donor vector, Cre-mediated recombination removes the P2A, dTKneo and poly(A) addition sequence. Ganciclovir treatment selects the cells whose selection cassette has been removed successfully (Fig. 1A).
In our experimental schedule, we transfected 1.0 μg pX459 CRISPR/Cas9 vector and 50 ng donor DNA into HeLa cells, then selected the transfected cells by 1.0 μg/mL puromycin treatment. Three days after transfection, we began to select KI cells with 0.5 mg/ml G418. Thirteen days after selection, the KI cells were sorted by flow cytometry based on mEGFP fluorescence, followed by single cell cloning for an additional 14 days. The cells were then infected with adeno-associated virus (AAV), which transiently induced the expression of Cre recombinase, to remove the P2A-dTKneo cassette. One week after AAV infection, the cells were further subjected to negative selection with Ganciclovir.
Establishment of KI HeLa cells expressing ERK2-mEGFP
As a proof-of-concept, we targeted the MAPK1 gene to generate cells expressing ERK2-mEGFP from the endogenous locus. HeLa cells were transfected with pX459-hMAPK1 and a donor vector for HDR- or MMEJ-mediated KI (Fig. 1A). After G418 selection, the genomic DNA were extracted from parental HeLa cells or bulk HeLa cells transfected with pX459-hMAPK1 and HDR- or MMEJ-donor vector, and subjected to PCR with either the forward (F) primer or reverse (R) primer or with both primers (FR). As expected, PCR products were only observed in the sample from cells introduced with the HDR donor or MMEJ donor when we used both forward and reverse (FR) primers (Fig. 2A), indicating KI of donor vectors into the 3’ site of the MAPK1 gene.
Next, we sorted GFP-positive cells in transfected HeLa cells by flow cytometry, and isolated two populations based on the intensity of low GFP fluorescence (Low) or high GFP fluorescence (High) (Fig. 2B). The Low and High cells accounted for 17.2% and 2.0% of the parent population in HeLa cells transfected with HDR-donor vector, respectively (Fig. 2B). Meanwhile, 5.6% and 0.1% of the parent population in cells transfected with the MMEJ-donor vector were categorized as Low and High cells, respectively. The immunoblotting data with an anti-GFP antibody and an anti-ERK1/2 antibody demonstrated ERK2-mEGFP expression, which corresponded to approximately 69 kDa (arrowhead), in both Low and High cells transfected with HDR- or MMEJ-vector (Fig. 2C). Intriguingly, in both HDR- and MMEJ-mediated KI, High cells showed stronger non-specific integration mEGFP bands and weaker specific ERK2-mEGFP bands than Low cells (Fig. 2C).
We subsequently isolated several single clones of Low cells by the limiting dilution method. Immunoblotting with the anti-ERK1/2 antibody demonstrated that isolated cell lines expressed comparable levels of ERK2-mEGFP (Fig. S2A). Western blot analysis revealed that, in comparison to parental HeLa cells, some of the isolated cells showed only a positive signal of ERK2-mEGFP around 69 kDa (#13, #16, and #20 with the HDR-donor vector and #1, #3, #5, #10, and #12 with the MMEJ-donor vector). The other cells exhibited not only the positive ERK2-mEGFP signal but also apparently negative GFP signals, indicating higher or lower molecular weights than we expected (Fig. S2A). Thus, we picked up the former cell clones, and confirmed that the positive ERK2-mEGFP signals were also detected with anti-ERK2 antibody (Fig. S2B). The KI efficiencies were roughly 15% (3 out of 20) and 40% (5 out of 12) with the HDR- and MMEJ-donor vectors, respectively (Fig. S2).
For further analysis, HDR-mediated KI cell #13 (HDR #13) and MMEJ-mediated KI cell #10 (MMEJ #10) were infected with AAV expressing Cre recombinase, followed by the analysis of expression levels of ERK2-mEGFP (Fig. 2D). Removal of the P2A-dTKneo-poly(A) cassette increased the ERK2-mEGFP levels in both cell lines (Fig. 2D, left). Of note, the expression levels of endogenous ERK2 in the KI cell lines were almost identical with that of the parental cells (Fig. 2D, right), suggesting that mEGFP genes were knocked-in into one or two of the multiple MAPK1 gene loci in HeLa cells, which are known to demonstrate aneuploidy (25). It is well-known that mitogen stimulation induces nuclear accumulation of ERK, thereby causing phosphorylation of several transcription factors and subsequent gene expression (26, 27). As expected, treatment with fetal bovine serum (FBS) provoked nuclear translocation of ERK2-EGFP from the cytoplasm (Fig. 2E).
Establishment of RSK2-HaloTag KI HeLa cells
Because RSK2 is known to interact with ERK2 (28), we next targeted the RSK2 gene for labeling with HaloTag, which covalently linked to HaloTag ligands such as the tetramethylrhodamine (TMR)-ligand (29). For HaloTag gene integration, we tested the aforementioned MMEJ-donor and precise integration into the target chromosome (PITCh)- like KI vector (20)(Fig. 3A). The PITCh-vector includes the insertion cassette, which is further sandwiched by recognition sites of the Cas9/gRNA complex that target the RSK2 gene locus (Fig. 3A, lower). Therefore, the Cas9/gRNA complex digests not only the RSK2 gene locus but also the PITCh-like donor vector, generating linear double strand donors within the cells.
To obtain HeLa cells expressing ERK2-mEGFP and RSK2-HaloTag from this endogenous locus, we introduced Cas9 and the sgRNA expression vector (pX459-hRSK2), which targeted a site close to the stop codon of the RSK2 gene, and linearized the MMEJ-donor vector (Fig. 3A, upper) or the PITCh-like donor plasmid (Fig. 3A, lower) into HeLa/ERK2-mEGFP (MMEJ#10) cells (Fig. 3B). After G418 selection and single cell cloning, several clones were analyzed by PCR using two primers that recognized the 3’ end of RSK2 or the 5’ end of HaloTag (Fig. 3C). Almost half of the clones used in either donor vector showed the expected PCR product (Fig. 3C). However, subsequent DNA sequencing of these PCR products revealed the repeated sequence of the homology arm in PITCh #1, 2, 6, and 7 cells, resulting in a frame shift. Meanwhile, MMEJ #1, 4, 6, and 7 cells had no insertion or deletion. In agreement with these data, the expressions of RSK2-HaloTag were observed only in MMEJ #1, 4, 6, 7 and PTICh #3 by immunoblotting with anti-RSK2 antibody (Fig. 3D). As with the case of ERK2-mEGFP, the removal of the selection marker also enhanced the expression of the RSK2-HaloTag (Fig. 3E). We picked up a cell line PITCh #3 (HeLa/ERK2-mEGFP/RSK2-HaloTag). To visualize the RSK2-HaloTag, we stained the HeLa/ERK2-mEGFP/RSK2-HaloTag cells with 100 nM TMR-Ligand, which was used in all subsequent experiments, and treated them with 10% FBS. As reported previously (30), ERK-mEGFP entered into the nucleus after administration of FBS, followed by the nuclear translocation of RSK2-HaloTag-TMR (Fig. 3F).
Quantification of the endogenous protein concentration and in vivo Kd value by FCS and FCCS
Finally, we measured the protein concentration and the in vivo Kd values of ERK2-mEGFP and RSK2-HaloTag in HeLa/ERK2-mEGFP/RSK2-HaloTag cells by FCS and FCCS, respectively. The auto-correlation functions of ERK2-mEGFP and RSK2-HaloTag-TMR and cross-correlation functions between ERK2-mEGFP and RSK2-HaloTag-TMR were calculated in a single HeLa cell (Fig. 4A). We performed repeated measurements with FCS and FCCS, and obtained the distribution of the endogenous protein concentration of ERK2-mEGFP and RSK2-HaloTag-TMR from 198 cells (Fig. 4B). The average concentrations of ERK2-mEGFP and RSK2-HaloTag-TMR were 0.078 μM and 0.097 μM, respectively (Fig. 4B). These histograms of ERK2-mEGFP and RSK2-HaloTag-TMR concentration exhibited non-Gaussian distribution, but a long-tail was seen in both the ERK2-mEGFP and RSK2-HaloTag-TMR distributions (Fig. 4B). Interestingly, the ERK2-mEGFP concentration was positively correlated with the RSK2-HaloTag-TMR concentration, showing a correlation coefficient of 0.64 (Fig. 4B). The average in vivo Kd value between ERK2-mEGFP and RSK2-HaloTag-TMR was approximately 0.63 (Fig. 4C). The in vivo Kd values also showed a long-tail distribution (Fig. 4C), but demonstrated little or no positive correlation with the protein concentrations of ERK2-mEGFP and RSK2-HaloTag-TMR (Fig. 4D).
Finally, in order to take full advantage of this strategy, we quantified the temporal changes in in vivo Kd values at the subcellular level. HeLa/ERK2-mEGFP/RSK2-HaloTag cells were stained with the TMR-HaloTag ligand, then treated with epidermal growth factor (EGF), followed by measurement of the protein concentration and in vivo Kd values in the cytoplasm and nucleus. The in vivo Kd values in the cytosol were higher than those in the nucleus over the course of EGF stimulation (Fig. 4E). In addition, the Kd value in the cytosol, but not that in the nucleus, was transiently increased 10 min after EGF stimulation (Fig. 4E). These results indicated that the ERK2-RSK2 complex was more stable in the nucleus than in the cytosol under both the unstimulated and stimulated conditions, and EGF stimulation transiently reduced the apparent affinity of the ERK2-RSK2 complex only in the cytoplasm. Consistently, the values of the ratio of complex to ERK2-mEGFP and RSK2-Halotag-TMR were significantly reduced 10 min after EGF treatment in the cytoplasm (Fig. 4F), and the ratio of the complex to RSK2 was gradually increased in the nucleus (Fig. 4F).
Discussion
Here, we established a method for quantifying the endogenous protein concentration and dissociation constant in living cells with a CRISPR/Cas9 genome editing technique and FCS/FCCS. These techniques allow us to quantify these kinetic parameters at the single cell level without any antibodies. While several research groups have reported methods for endogenous tagging in cultured mammalian cells (20, 31, 32), gene KI is still challenging in cultured cells even when using the CRISPR/Cas9 technique. The KI efficiency is much lower than the random integration efficiency in HeLa cells. Therefore, it is important not only to increase the KI efficiency (33–35), but also to decrease the emergence of negative clones. Further, we found that the selection cassette had a considerable effect on the expression of the tagged protein; the remaining selection cassette reduced the expression level of the protein fused with fluorescent protein (Figs. 2 and 3). For this reason, removal of the selection cassette by Cre recombination is essential for the quantitative assessment of protein concentration.
We quantified the endogenous protein concentrations of ERK2-mEGFP and RSK2-HaloTag-TMR by FCS, and found that there was substantial heterogeneity in the concentrations of these proteins among the individual cells (Fig. 4). The average concentration of ERK2-mEGFP measured in this study, 0.078 μM, was considerably lower than the ERK2 concentration reported previously, 0.68 μM for ERK2 in HeLa cells (11). This may have been due to the multiplication of MAPK1 genes, because it is evident that the reduction of unlabeled EKR2 by KI of the mEGFP gene was quite limited (Fig. 2). Therefore, the gene amplification, which is frequently observed in cancer cells, is a limitation of this technique. On the other hand, the RSK2-HaloTag-TMR concentration, 0.097 μM, was highly similar to the previously reported RSK2 concentration, 0.15 μm (11) (Fig.3). Indeed, KI of the HaloTag gene reduced the endogenous RSK2 protein level by half or to zero (Fig. 3D), implying monoallelic- and biallelic KI, respectively. It is possible to estimate the total protein concentration by comparing the tagged protein with unlabeled protein by western blotting (Fig. 2C and Fig. 3D). The distributions of ERK2-EGFP and RSK2-HaloTag-TMR appeared to be non-Gaussian, but rather resembled a long-tailed distribution such as a gamma distribution (Fig. 4B). This was in good agreement with the analytical distributions of stochastic gene expression (36, 37). Further, the positive correlation between the protein abundance of ERK2-mEGFR and RSK2-HaloTag-TMR (Fig. 4B) could be fully explained by the idea of global noise; the level of global factors that are involved in all protein expressions, such as the expressions of mRNA polymerase and ribosomes, dictates the global protein abundance (38).
We also quantified in vivo Kd values for the binding of ERK2-mEGFP and RSk2-HaloTag-TMR by FCCS (Fig. 4). The average in vivo Kd value measured in endogenously-tagged ERK2 and RSK2 in this study, 0.63 μM (Fig. 4C), was lower than the in vivo Kd value in overexpressed ERK2-mEGFP and RSK2-HaloTag, 1.3 μM (11). PEA-15 is known to function as a scaffold to enhance RSK2 activation by ERK (39). Thus, the overexpression of ERK2-mEGFP and RSK2-HaloTag might exceed the amount of endogenous PEA-15, resulting in an overestimation of the in vivo Kd values in the previous study. Further, the in vivo Kd values of ERK2-mEGFP and RSK2-HaloTag-TMR binding were hardly correlated with the expression levels of those proteins (Fig. 4D), suggesting that the binding between ERK2 and RSK2 is fluctuating in a manner dependent on unknown mechanisms, such as competitive binding, molecular crowding, posttranslational modification and/or scaffold proteins. In the future, quantitative analysis of the hetero-trimer complex could be a fascinating way to verify the scaffolding function of PEA-15 for ERK2-RSK2 binding (40).
Following EGF stimulation, ERK2-mEGFP was transiently dissociated from RSK2-HaloTag-TMR (Fig. 4E). It has been reported that ERK binding is differently regulated among RSK isoforms; RSK1 dissociated completely from ERK1/2 upon EGF stimulation, RSK2 only partially dissociated from ERK1/2, and RSK3 remained bound to ERK1/2 (28). These previous findings are consistent with our finding that half of the complex of ERK2-mEGFP and RSK2-HaloTag-TMR was dissociated 10 min after EGF stimulation, while the other half of the complex remained (Fig. 4E). The finding that the activation of RSK2 upon EGF stimulation is comparable to that of RSK1 suggested that the phosphorylation of RSK2 by ERK1/2, PDK1 and/or autophosphorylation (28, 41) reduced the affinity to ERK2 to some extent.
In summary, we established a method for quantifying the endogenous protein concentration and the Kd value in a living cell. Further studies will be needed to improve the KI efficiency, brightness of fluorescent proteins, and quantification of proteins localized at specific cellular compartments such as the plasma membrane.
Experimental procedures
Design of gRNAs of CRISPR/Cas9 for gene KI
pX459 (pSpCas9(BB)-2A-Puro) was a gift from Feng Zhang (Addgene plasmid #62988). All guides are designed to overlap the stop codon of MAPK1 or RSK2, so that these guide RNAs do not recognize the target sequences after KI. Guide sequences (without PAM sequence) were as follows: for MAPK1, TCTTAAATTTGTCAGGTACC; for RSK2, CTCACTGAGGTCACTTCACA.
Construction of KI donor vectors
Truncated-thymidine kinase (dTK) originated from pPB-CAG.OSKM-puDtk (a gift from Kosuke Yusa and Allan Bradley (42)) and the neomycin-resistance gene (neo) were conjugated by PCR (Fig. S1). KOD FX neo (TOYOBO, Osaka, Japan) was used for all PCR reactions. Homology arms of the HDR vector were amplified from genomic DNA of HeLa cells extracted with QuickExtract DNA Extraction Solution (Epicentre, Madison, WI) and attached to an insertion cassette by PCR. 40 bp homology arms for the MMEJ donor were attached by PCR.
Cell culture
HeLa cells were purchased from the Human Science Research Resources Bank (Sennanshi, Japan). HEK-293T cells were obtained from Invitrogen as Lenti-X 293 cells (Invitrogen, Carlsbad, CA). HeLa cells and HEK-293T cells were maintained in DMEM (Wako, Osaka, Japan) supplemented with 10% FBS. For imaging, HeLa cells were plated on 35-mm glass-base dishes (Asahi Techno Glass, Tokyo). At least three hours before observation, HeLa cells were maintained with FluoroBrite DMEM (LifeTechnologies, Carlsbad, CA) supplemented with 1% GlutaMAX (LifeTechnologies).
For HaloTag imaging, the cells were incubated with 100 nM HaloTag-tetramethylrhodamine (TMR) for at least 18 hours. After that, the cells were washed two times with PBS, followed by incubation with FluoroBrite DMEM supplemented with 1% GlutaMAX for 30 min. The medium was again replaced with the same medium.
Establishment of the KI cell line
HeLa cells were plated on a 24-well plate, and transfected with 1 μg pX459 vectors and 50 ng KI donor DNA by using Polyethyleneimine “Max” MW 40,000 (Polyscience Inc., Warrington, PA), followed by puromycin selection. Three days after transfection, transfected cells were seeded onto 35-mm dishes and treated with 0.5 mg/ml G-418 (Invivogen, San Diego, CA) for more than 10 days. The genomic DNAs of these cells were extracted with QuickExtract DNA Extraction Solution (Epicentre, Madison, WI), and analyzed by PCR.
To isolate MAPK1-EGFP KI cells, the selected cells were analyzed and sorted by FACSaria IIu (BD Biosciences, San Jose, CA). mEGFP fluorescence was detected using 488 nm laser light and a 518-548 nm BP emission filter, followed by sorting of mEGFP-positive cells and limiting dilution for single cell cloning. For single cell isolation of RSK2-HaloTag KI cells, G418-selected cells were subjected to the limiting dilution method.
Adeno-associated viruses expressing Cre and removal of selection marker
The cDNA of Cre was inserted into pAAV-MCS (Cell Biolabs Inc., San Diego, CA), generating pAAV-Cre. HEK-293T cells were co-transfected with pAAV-Cre, pAAV-DJ, and pHelper to produce recombinant AAV expressing Cre recombinase. Three days after transfection, HEK-293T cells were collected and resuspended in 1 mL DMEM, followed by four freeze-thaw cycles. After the final thaw and centrifugation, 10 μL supernatant was added to each well of a 24-well plate to remove the selection marker. At least 6 days after AAV-Cre infection, the cells were selected with 50 nM Ganciclovir (WAKO, Osaka, Japan).
Immunoblotting
Cells were lysed in 2x SDS sample buffer. After sonication, the samples were separated by 5-20% or 7.5% SDS-polyacrylamide gel electrophoresis (Nacali Tesque Inc., Kyoto, Japan), and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with Odyssey Blocking Buffer (TBS) (LI-COR Biosciences Inc., Lincoln, NE) for 1 hour, the membranes were incubated with primary antibodies diluted in blocking buffer, followed by secondary antibodies diluted in blocking buffer. Fluorescence levels were detected by Odyssey infrared scanner (LI-COR Biosciences Inc.).
The following antibodies were used in this study: anti-green fluorescent protein (GFP) antibody (632375; Clontech, Palo Alto, CA), anti-p44/p42 MAP kinase (ERK1/2) antibody (4695; Cell Signaling Technology, Danvers, MA), anti-RSK2 (sc-9986; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HaloTag antibody (G9281; Promega, Madison, WI), IRDye680LT goat anti-rabbit IgG antibody (no. 925–68021; LI-COR), and IRDye800CW donkey anti-mouse IgG antibody (no. 925–32212; LI-COR).
Imaging by confocal microscope
Cells were imaged with a laser scanning confocal microscope (FV1200; Olympus, Tokyo) equipped with gallium arsenide phosphide (GaAsP) detectors. The excitation lines were set at 488 nm and 559 nm. The excitation beam was reflected by a DM 405/488/559 dichroic mirror and focused by an oil immersion objective lens (Uplsapo 60XO, NA 1.35; Olympus). The emitted light was detected through a bandpath filter with wavelengths of 495 to 540 nm for mEGFP and 575 to 630 nm for HaloTag-TMR. FCS and FCCS data were analyzed as described previously (11).
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article.
AUTHOR CONTRIBUTIONS
K.A. and A.T.K. designed and performed the experiments. K.A., A.T.K. and M.M. wrote the manuscript.
ACKNOWLEDGMENTS
We thank the members of the Matsuda Laboratory and Aoki Laboratory for their helpful input. This work was supported by the Spectrography and Bioimaging Facility and Functional Genomics Facility, NIBB Core Research Facilities. A. Kawagishi, K. Hirano, N. Nishimoto, and K. Onoda are also to be thanked for their technical assistance. K.A. and M.M. were supported by the Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, and Science, Japan, and CREST, JST. K.A. was supported by JSPS KAKENHI Grants No. 16H01425, 16H01447, and 16KT0069, the Hori Sciences and Arts Foundation, and the Nakajima Foundation. A.T.K. was supported by a JSPS Grant-in-Aid for a JSPS Research Fellow.