Endogenous Membrane Receptor Labeling by Reactive Cytokines and Growth Factors to Chase Their Dynamics in Live Cells

Abstract

•Membrane receptors can be selectively labeled with reactive cytokines and growth factors•Supramolecular approach for reactive cytokines and growth factors is simple and flexible•Real-time imaging of endogenous membrane receptor dynamics is achieved Cytokine and growth factor receptors play crucial roles in signaling cascades that modulate cell survival, adhesion, proliferation, and migration. Because the dynamic behaviors of these receptors are highly related to their functions and to changes in ligand-dependent signals, reliable techniques for visualization of these receptors are strongly desired. Here, we have developed a method of covalently and tracelessly labeling membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with an organocatalyst that facilitates a selective acyl transfer reaction on the target receptors. Construction of the reactive cytokines and growth factors relies on non-covalent coordination chemistry in an in situ manner, and this supramolecular approach is relatively simple and flexible. It can be applied to not only the labeling of various receptors but also real-time imaging of ligand-induced dynamics of endogenously expressed receptors in live cells. Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells. Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells. Cytokine and growth factor receptors, such as receptor tyrosine kinases (RTKs), Toll-like receptors, and G-protein-coupled receptors (GPCRs), play crucial roles in signaling cascades that modulate cell survival, adhesion, proliferation, and migration. Because these receptors are also deeply involved in the development and growth of various disease-related cells,1Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (3100) Google Scholar, 2Casaletto J.B. McClatchey A.I. Spatial regulation of receptor tyrosine kinases in development and cancer.Nat. Rev. Cancer. 2012; 12: 387-400Crossref PubMed Scopus (237) Google Scholar, 3Haugh J.M. Localization of receptor-mediated signal transduction pathways: the inside story.Mol. Interv. 2002; 2: 292-307Crossref PubMed Scopus (41) Google Scholar, 4Friedl P. Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer.Nat. Rev. Mol. 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Cancer. 2004; 4: 540-550Crossref PubMed Scopus (1911) Google Scholar Cytokine and growth factor receptors also show dynamic behaviors in live cells, such as lateral movement on the cell surface, internalization, and recycling, which are highly related to not only their functions but also alteration in cognate ligand-dependent signals.2Casaletto J.B. McClatchey A.I. Spatial regulation of receptor tyrosine kinases in development and cancer.Nat. Rev. Cancer. 2012; 12: 387-400Crossref PubMed Scopus (237) Google Scholar Therefore, reliable techniques are strongly desired for live-cell imaging of these receptors. To date, cytokines and growth factors conjugated with synthetic dyes have been applied to fluorescence imaging of receptors in live cells.9Sako Y. Minoguchi S. Yanagida T. Single molecule imaging of EGFR signal transduction on the living cell surface.Nat. Cell. Biol. 2000; 2: 168-172Crossref PubMed Scopus (748) Google Scholar, 10Lidke D.S. Nagy P. Heintzmann R. Arndt-Jovin D.J. 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Some methods that covalently label receptors by using genetically encoded protein tags, such as the SNAP tag12Kobayashi T. Komatsu T. Kamiya M. Campos C. González-Gaitán M. Terai T. Hanaoka K. Nagano T. Urano Y. Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins.J. Am. Chem. Soc. 2012; 134: 11153-11160Crossref PubMed Scopus (97) Google Scholar, 13Sun X. Zhang A. Baker B. Sun L. Howard A. Buswell J. Maurel D. Masharina A. Johnsson K. Noren C.J. et al.Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.ChemBioChem. 2011; 12: 2217-2226Crossref PubMed Scopus (193) Google Scholar and BL tag,14Mizukami S. Watanabe S. Hori Y. Kikuchi K. Covalent protein labeling based on Noncatalytic β-lactamase and a designed FRET substrate.J. Am. Chem. Soc. 2009; 131: 5016-5017Crossref PubMed Scopus (124) Google Scholar have been reported recently. Although these methods are indeed powerful, overexpression of the genetically modified target proteins is still needed. Because the functions of cytokine and growth factor receptors are strictly regulated by interactions with other receptors and the associated proteins,15Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3520) Google Scholar it is preferable to analyze receptor functions in an intrinsic state without artificial overexpression of the target proteins. We have recently reported that 4-dimethylaminopyridine (DMAP) covalently coupled to antibodies is a powerful tool for tag-free and selective labeling of endogenous proteins, termed affinity-guided DMAP (AGD) chemistry.16Hayashi T. Yasueda Y. Tamura T. Takaoka Y. Hamachi I. Analysis of cell-surface receptor dynamics through covalent labeling by catalyst-tethered antibody.J. Am. Chem. Soc. 2015; 137: 5372-5380Crossref PubMed Scopus (49) Google Scholar In this method, the DMAP catalyst selectively binds to the target protein through an antibody-epitope interaction, which facilitates the DMAP-catalyzed acyl transfer reaction from an acyl donor to a nucleophilic amino acid located near the epitope of the target protein. However, tedious multiple steps are needed to prepare a DMAP-conjugated antibody, including cysteine point mutation, cysteine-maleimide conjugation, and purification under mild conditions to retain the recognition capability of the antibody. Because most cytokines and growth factors are relatively small and labile, and contain indispensable disulfide bonds to maintain their three-dimensional structure, it is difficult to obtain covalently DMAP-conjugated cytokines and growth factors by our previous method. Here, we describe a supramolecular approach to simply and rapidly constructing DMAP-conjugated cytokines and growth factors that are valid for covalently labeling their corresponding receptors on a live-cell surface. These DMAP-conjugated cytokines and growth factors were successfully prepared through non-covalent coordination chemistry between an oligo-histidine tag (His tag) and Ni(II)-nitrilotriacetate (Ni(II)-NTA)17Lata S. Reichel A. Brock R. Tampe R. Piehler J. High-affinity adaptors for switchable recognition of histidine-tagged proteins.J. Am. Chem. Soc. 2005; 127: 10205-10215Crossref PubMed Scopus (325) Google Scholar in an in situ manner without the need for any purification processes (Figure 1A). We demonstrated that epidermal growth factor receptor (EGFR), C-X-C chemokine receptor type 4 (CXCR4), and Neuropilin 1 transiently expressed in live HEK293T cells were selectively and covalently labeled with DMAP-conjugated EGF, stromal cell-derived 1α (SDF1α), and vascular endothelial growth factor 165 (VEGF165), respectively. Moreover, this strategy allowed us to fluorescently label endogenous EGFR and CXCR4 on live cells without any loss of activity and to achieve real-time fluorescence imaging of the ligand-induced dynamics of these endogenous receptors, highlighting the utility of this method for functional analyses of endogenous receptors on live cells. To construct DMAP-conjugated cytokines and growth factors without loss of cytokine or growth factor function, we exploited the coordination chemistry between a His tag fused to the cytokine or growth factor and Ni(II)-NTA with DMAP (DMAP-Ni(II)-NTA) (Figure 1A). Because His tags are widely used for purification of cytokines and growth factors, and various His tag-fused cytokines and growth factors are commercially available, this strategy was considered likely to exert minimal detrimental effects on the cytokine and growth factor structure and function. As a Ni(II)-NTA moiety, two types of dimeric Ni(II)-NTA were designed and synthesized: a conventional flexibly branched lysine (DMAP-Ni(II)-NTA 1–6) and a rigidly branched xylene (DMAP-Ni(II)-NTA 7–9) (Figure 1B and Scheme S1–S6) to achieve a high labeling yield of the target receptor with suppression of labeling of the ligand itself (self-labeling). The dissociation constant of the rigid-type Ni(II)-NTA dimer toward the His tag peptide was determined to be Kd = 29 nM with fluorescein (Fl)-appended rigid-Ni(II)-NTA 10 (Figure S1 and Scheme S7). This Kd value is similar to that reported previously for the flexible Ni(II)-NTA dimer (Kd = 68 nM).17Lata S. Reichel A. Brock R. Tampe R. Piehler J. High-affinity adaptors for switchable recognition of histidine-tagged proteins.J. Am. Chem. Soc. 2005; 127: 10205-10215Crossref PubMed Scopus (325) Google Scholar The catalytic moiety was constructed with one, four, or six DMAP units to test acceleration of the labeling rate by the multivalent effect of the catalysts.18Koshi Y. Nakata E. Miyagawa M. Tsukiji S. Ogawa T. Hamachi I. Target-specific chemical acylation of lectins by ligand-tethered DMAP catalysts.J. Am. Chem. Soc. 2008; 130: 245-251Crossref PubMed Scopus (116) Google Scholar, 19Wang H. Koshi Y. Minato D. Nonaka H. Kiyonaka S. Mori Y. Tsukiji S. Hamachi I. Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts.J. Am. Chem. Soc. 2011; 133: 12220-12228Crossref PubMed Scopus (86) Google Scholar, 20Sun Y. Takaoka Y. Tsukiji S. Narazaki M. Matsuda T. Hamachi I. Construction of a 19F-lectin biosensor for glycoprotein imaging by using affinity-guided DMAP chemistry.Bioorg. Med. Chem. Lett. 2011; 21: 4393-4396Crossref PubMed Scopus (16) Google Scholar The Ni(II)-NTA and DMAP groups were connected via a flexible oligoethylene glycol linker or rigid oligoproline linker ((Pro)n, n = 6, 3, 0). The latter was expected to extend the DMAP moiety away from the His tag-fused cytokine or growth factor, so that the catalytic DMAP would be able to interact efficiently with the receptor.16Hayashi T. Yasueda Y. Tamura T. Takaoka Y. Hamachi I. Analysis of cell-surface receptor dynamics through covalent labeling by catalyst-tethered antibody.J. Am. Chem. Soc. 2015; 137: 5372-5380Crossref PubMed Scopus (49) Google Scholar, 21Hayashi T. Sun Y. Tamura T. Kuwata K. Song Z. Takaoka Y. Hamachi I. Semisynthetic lectin-dimethylaminopyridine conjugates for labeling and profiling glycoproteins on live cell surfaces.J. Am. Chem. Soc. 2013; 135: 12252-12258Crossref PubMed Scopus (44) Google Scholar EGFR was selected as the initial target protein because it is one of the most important RTKs in the regulation of various cell signaling cascades for cellular growth, differentiation, migration, and tumorigenesis.22Lurje G. Lenz H.J. EGFR signaling and drug discovery.Oncology. 2009; 77: 400-410Crossref PubMed Scopus (342) Google Scholar EGF, a growth factor known to bind to EGFR (Kd = 300 pM and 2 nM for high- and low-affinity EGFR, respectively),23Lemmon M.A. Ligand-induced ErbB receptor dimerization.Exp. Cell Res. 2009; 315: 638-648Crossref PubMed Scopus (161) Google Scholar was fused to a His6 tag at the C terminus (EGF-His). First, construction of the DMAP-EGF supramolecular conjugate was confirmed by fluorescence titration using fluorescein-modified EGF-His (Fl-EGF-His). When DMAP-Ni(II)-NTA 5 or 8 was added to the solution containing Fl-EGF-His, decreasing fluorescence was clearly observed in a typical saturation manner with increasing DMAP-Ni(II)-NTA concentrations (Figures 2A and S2A). This quenched fluorescence could be recovered by addition of excess EDTA (Figures 2B and S2B). These results indicated that the Fl-EGF-His fluorescence was quenched by binding of the Ni(II)-NTA moiety of 5 or 8, and that the interaction between DMAP-Ni(II)-NTA and Fl-EGF-His was mediated by the coordination chemistry between Ni(II)-NTA and the His tag. Curve-fitting analysis of the titration plots gave Kd values of 168 nM and 131 nM for the binding of 5 and 8, respectively, to Fl-EGF-His, revealing that EGF-His was tightly bound to DMAP-Ni(II)-NTA through the non-covalent interaction.17Lata S. Reichel A. Brock R. Tampe R. Piehler J. High-affinity adaptors for switchable recognition of histidine-tagged proteins.J. Am. Chem. Soc. 2005; 127: 10205-10215Crossref PubMed Scopus (325) Google Scholar, 24Park S.J. Ryu K. Suh C.W. Chai Y.G. Kwon O.B. Park S.K. Lee E.K. Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin.Biotechnol. Bioprocess Eng. 2002; 7: 1-5Crossref Google Scholar After confirming construction of the supramolecular DMAP-EGF conjugates, we subsequently optimized the number of DMAP units, the structure of the Ni(II)-NTA moiety, and the linker structure between DMAP-Ni(II)-NTA 1–9. We used HEK293T cells transiently expressing EGFR fused to an HA tag (HA-EGFR). After a short pre-incubation (5 min) of the HEK293T cells with EGF-His and DMAP-Ni(II)-NTAs for the construction of DMAP-EGF in an in situ manner, non-cell-permeable acyl donor 11 bearing Fl was added, and then the mixture was incubated for 60 min. The cells were subsequently washed with buffer, lysed, and subjected to western blot analysis using anti-Fl and anti-HA antibodies. Dependence of the labeling efficiency on the number of DMAP units was initially investigated with DMAP-Ni(II)-NTA 1, 2, and 3, which comprise one, four, and six DMAP units, respectively. As shown in Figure S3A, two clear bands were detected for 2 in the range of 150–250 kDa by the anti-Fl antibody, which was in agreement with the bands detected by the anti-HA antibody, indicating that HA-EGFR was covalently labeled with high selectivity. On the basis of the relative band intensities (IFl/IHA), it was clear that DMAP-Ni(II)-NTA 2 exhibited the highest labeling efficiency among constructs 1–3 toward HA-EGFR (Figures S3A and S3B, lanes 1–3). Conversely, the construct comprising six DMAP units (3) exhibited substantial non-specific labeling toward proteins other than EGFR, suggesting that there is an optimal number of DMAP units for selective and efficient labeling. We next compared the effect of the linker between 2 and 4, which revealed that 4 produced a higher labeling efficiency, as shown by the more intensely labeled EGFR band for 4 (rigid proline linker) compared with 2 (flexible PEG linker; Figure S3A, lanes 2 and 4). We performed the next steps to optimize the length of the proline linker and the structure of the Ni(II)-NTA moiety by using 4–9. As shown in Figures 3A and S3C, the most intense band was obtained for 8, which contained a tri-Pro linker and rigid-branched xylene-type Ni(II)-NTA (lane 5), whereas the other tested DMAP-Ni(II)-NTAs showed lower labeling efficiencies. Using the optimized DMAP-Ni(II)-NTA 8 for EGFR labeling, we carefully examined the specificity of this method by various negative control experiments. As discussed above, two bands corresponding to HA-EGFR were clearly detected by the anti-Fl antibody when labeled with EGF-His and 8 in the presence of acyl donor 11 (Figure 3B, lane 1). In contrast, negligible bands were observed in the absence of EGF-His (lane 2), without NiCl2 (lane 3), without EGF-His or 8 (lane 4), or when no transfection of HA-EGFR occurred (lane 5). These results clearly demonstrate that a supramolecular DMAP-conjugated EGF was formed through non-covalent interactions between Ni(II)-NTA and the His tag even under crude cellular conditions, and this assembly could then in turn successfully catalyze the selective acylation of HA-EGFR driven by the specificity of EGF-EGFR recognition. These results were supported by live-cell imaging of Fl-labeled EGFR by confocal laser scanning microscopy (CLSM). As shown in Figure S3D, a strong fluorescence signal attributed to Fl was detected only from the cell membrane. EGFR expression in HEK293T cells was separately confirmed by staining with commercially available tetramethylrhodamine (TMR)-modified EGF (TMR-EGF). Co-staining experiments showed a clear overlap between the fluorescence signals of Fl and TMR. In contrast, negligible Fl fluorescence was observed from the cell membrane in the absence of EGF-His, indicating that the supramolecular DMAP-EGF conjugate was crucial for selective labeling of EGFR on live-cell surfaces, which is consistent with the results obtained by western blotting (Figures S3D and S3E). Moreover, no significant change in cell morphology was detected during the labeling process, implying negligible detrimental effects of this method. Because this methodology is modular, various membrane receptors can be covalently labeled simply through the replacement of the EGF-His with other His tag-fused cytokines and growth factors. We next chose two other receptors—CXCR4, a GPCR that acts as a chemokine receptor,25Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. 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Cascieri M.A. et al.The CXCR4 agonist ligand stromal derived factor-1 maintains high affinity for receptors in both Gαi-coupled and uncoupled states.Eur. J. Pharmacol. 2009; 409: 143-154Crossref Scopus (23) Google Scholar, 29Crump M.P. Gong J.-H. Loetscher P. Rajarathnam K. Amara A. Arenzana-Seisdedos F. Virelizier J.-L. Baggiolini M. Brian D.S. Clark-Lewis I. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.EMBO J. 1997; 6: 6996-7007Crossref Scopus (631) Google Scholar) and transiently expressed CXCR4 fused to monomeric Cherry (CXCR4-mCherry) in HEK293T cells. As shown in Figure S4A, the combination of DMAP-Ni(II)-NTA 6 and acyl donor 11 produced a single band at ∼70 kDa in western blotting with detection by the anti-Fl antibody (lane 3), which coincided with a part of the expected molecular weight band for CXCR4-mCherry as detected by an anti-mCherry antibody. This band was negligible in control experiments in which cells were labeled in the absence of His-SDF1α (lane 2), in the presence of AMD-3100, a strong inhibitor of CXCR4 (lane 4),30Donzella G.A. Schols D. Lin S.W. Esté J.A. Nagashima K.A. Maddon P.J. Allaway G.P. Sakmar T.P. Henson G. De Clercq E. et al.AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor.Nat. Med. 1998; 4: 72-77Crossref PubMed Scopus (684) Google Scholar or without transfection of the CXCR4-mCherry plasmid (lane 5). These results indicated that CXCR4 was selectively labeled and that the reaction was driven by the specificity of SDF1α and CXCR4 recognition. It was notable that DMAP-Ni(II)-NTA 6 provided optimal results for CXCR4 labeling in contrast to 8 for EGFR labeling, implying that the optimal Pro linker may depend on the target receptor (data not shown). CLSM analyses confirmed specific CXCR4 labeling on live-cell surfaces, as shown in Figures S4B and S4C. Neuropilin 1 was labeled next by a similar strategy. In this case, VEGF165 was fused to a His6 tag at the N terminus (His-VEGF165, Kd = 0.93 nM31Gu C. Limberg B.J. Whitaker G.B. Perman B. Leahy D.J. Rosenbaum J.S. Ginty D.D. Kolodkin A.L. Structural features that confer binding to semaphorin 3A and vascular endothelial growth factor.J. Biol. Chem. 2002; 277: 18069-18076Crossref PubMed Scopus (199) Google Scholar). Neuropilin 1 was fused to mCherry (Neuropilin 1-mCherry) and transiently expressed in HEK293T cells. As shown in Figure S5A, Neuropilin 1 underwent selective labeling only in the presence of both His-VEGF165 and 6. These results clearly demonstrate that this method can be applied to any cytokine or growth factor receptor simply through the appropriate choice of His tag-fused cytokines and growth factors. We also confirmed no cell cytotoxicity of the labeling reagents under these conditions (Figure S5B), indicating the high biocompatibility of our labeling method. In principle, the present method does not require any tags for covalent labeling, which potentially enables the modification of endogenous membrane receptors on the surface of live cells. Compared with proteins exogenously expressed by transfection, labeling endogenous proteins in live cells is preferable for analysis, despite being far more difficult. Therefore, in our next experiment, we carried out labeling and imaging of endogenous EGFR on the surface of live A431 cells, a type of tumor cell that naturally expresses EGFR. After pretreatment of the A431 cells with EGF-His and 8, acyl donor 11 was added, followed by incubation for 30–90 min. Western blot analyses using anti-Fl antibody and anti-EGFR antibody confirmed the high specificity of this labeling (Figure 4A and S6A–S6C). As shown in lanes 1–3, a single band was clearly detected by the anti-Fl antibody at ∼200 kDa, which is in agreement with the EGFR band detected by the anti-EGFR antibody. This band did not appear in any of the control experiments (Figures 4A and S6A, lanes 4–12). As shown in Figure 4B, strong fluorescence was detected on the cell membrane by CLSM in the presence of EGF-His and 8, whereas negligible fluorescence was observed in the control experiments, in which A431 cells were incubated in the absence of EGF-His. The EGFR labeling yield was 55% ± 6.7% on the live-cell surface (Figure S6D), indicating that efficient covalent modification of endogenous EGFR was carried out with the DMAP-EGF supramolecular conjugate. In addition, various other fluorophores such as Alexa Fluor 488 and 568 could be attached to endogenous EGFR with acyl donors 12 or 13 (Figure S7). With the development of this method for highly selective and traceless labeling of endogenous EGFR under live-cell conditions, we were able to fluorescently visualize endogenous EGFR dynamics. It is well known that EGFR internalizes into the cytosol through endocytosis upon stimulation with a growth factor, such as EGF or transforming growth factor α (TGF-α), which regulates EGFR signaling for cell growth and migration.32Waterman H. Yarden Y. Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases.FEBS Lett. 2001; 490: 142-152Crossref PubMed Scopus (271) Google Scholar After labeling according to the above protocol, EGFR on the A431 cell surface was stimulated with EGF-His, followed by CLSM analysis. As shown in Figure S8, fluorescent endosome particles were clearly observed in the cytosol after 1 hr. These fluorescent particles were less observed in the absence of EGF stimulation (Figure S8B), indicating successful imaging of the internalization of endogenous EGFR into live A431 cells without any loss of activity of the receptor. Moreover, pulse-chase multi-color fluorescent imaging of EGFR internalization was performed with two kinds of acyl donors with different fluorophores (Figures 4C and S9). EGFR expressed on A431 cells was first labeled with Alexa-Fluor-488-conjugated acyl donor 12 (Scheme S8) in the presence of EGF-His6 and 8 (Figure S9A, step II), followed by incubation for 30 min at 37°C to induce the first internalization (step III). The second labeling of EGFR was then conducted by incubation with Alexa-Fluor-568-conjugated acyl donor 13 (Scheme S9) for 90 min at 4°C (Figure 4C, step IV), followed by additional incubation at 37°C for the second EGFR internalization. Immediately after the second labeling, only green particles were detected in the cytosol, and most of the yellow color derived from both green and red fluorophores remained on the membrane. Yellow particles in the cytosol appeared gradually after 1 hr of incubation (Figure S9B, step V). After 4 hr of the second internalization (Figure 4C, step VI), the fluorescence on the cell membrane mainly became reddish, whereas orange-colored particles were observed in the cytosol. These results indicated that each EGFR internalized at steps IV, V, and VI could be independently visualized by pulse-chase imaging. We next examined whether the EGFR dynamics are dependent on the ligand type through real-time imaging.33Well A. EGF receptor.Int. J. Biochem. Cell Biol. 1999; 31: 637-643Crossref PubMed Scopus (893) Google Scholar, 34Roepstorff K. Grandal M.V. Henriksen L. Knudsen S.L. Lerdrup M. Grøvdal L. Willumsen B.M. Van Deurs B. Differential effects of EGFR ligands on endocytic sorting of the receptor.Traffic. 2009; 10: 1115-1127Crossref PubMed Scopus (233) Google Scholar, 35McClintock J.L. Ceresa B.P. Transforming growth factor-α enhances corneal epithelial cell migration by promoting EGFR recycling.Invest. Ophthalmol. Vis. Sci. 2010; 51: 3455-3461Crossref PubMed Scopus (62) Google Scholar After labeling EGFR with acyl donor 12, we washed the treated A431 cells with acidic buffer to remove EGF-His. Thereafter, the cells were stimulated with various ligands (100 nM), such as EGF, TGF-α, or heparin-binding (HB)-EGF, for 5 min at 4°C. After excess ligands were washed away, the cells were incubated at 37°C. CLSM observation revealed that the relative intensity of fluorescence on the cell membrane (I/I0) decreased rapidly after EGF or HB-EGF stimulation until a plateau was reached at a relative i