J. Phys. Chem. B 2007, 111, 13619-13625
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Single-Molecule Force Spectroscopy Study of Interaction between Transforming Growth Factor β1 and Its Receptor in Living Cells Junping Yu,† Qiang Wang,‡ Xiaoli Shi,† Xinyong Ma,† Huayan Yang,† Ye-Guang Chen,‡ and Xiaohong Fang*,† Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, and State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua UniVersity, Beijing 100084, People’s Republic of China ReceiVed: July 25, 2007; In Final Form: September 18, 2007
Transforming growth factor β1 (TGF-β1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-β type II receptor (TβRII) and type I receptor (TβRI). However, it is not fully understood how the TGF-β1 ligandreceptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-β1/TβRII/TβRI formation. In this study, we have investigated the interaction between TGF-β1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-β1modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-β receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of TβRI with TβRII enhanced the binding force of TGF-β1 with its receptors, whereas the expressed TβRI itself exhibited no binding affinity to TGF-β1. Moreover, the unbinding dynamics of TGF-β1/TβRII and TGF-β1/TβRI/TβRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-β1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-β1 with the receptor after TβRI is recruited and the important contribution of TβRI to the signaling complex formation during TGF-β1 signaling.
1. Introduction Since its invention, atomic force microscopy (AFM) has proven to be a powerful tool to image biological samples under physiologically relevant conditions with high spatial resolution.1,2 In addition, AFM has been developed in recent years to study the binding force governing biomolecular interaction at the single molecule level.3,4 This technique, known as singlemolecule force spectroscopy, has been used to probe the affinity and recognition property of a variety of biomolecular interactions including ligand/receptor interaction,5 antibody/antigen interaction,6,7 protein/DNA8-10 and DNA/DNA interaction.11 As AFM is minimally disruptive to living cells,12 the application of this technique to the direct measuring of the interaction force in living cells is of great interest. Much effort has been focused on the study of cell adhesion with living-cell single-molecule force spectroscopy. For example, the single integrin molecular adhesion forces have been measured13 and the binding strength between individual glycoprotein contact site A (csA) molecules has been studied in intact cells.14 Many other molecules regulating cell adhesion have also been characterized, such as leukocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1),15 R4β1 integrin/vascular cell adhesion molecule-1 (VCAM-1),16 R5β1 integrin/fibronectin,17 selectin/P-selectin glycoprotein ligand-1(PSGL-1),18 etc. AFM * Corresponding author. Phone and Fax: +86 10 62650024. E-mail:
[email protected]. † Chinese Academy of Sciences. ‡ Tsinghua University.
tips attached with the living cells were usually used for the force measurement in these works.14-17 Besides the adhesion study, the single-molecule force experiment was performed utilizing the antibody (e.g., heat shock protein 60 monoclonal antibody) functionalized AFM tip to locate the membrane receptors on the fixed cells with the help of an integrated AFM and fluorescence system.19,20 To explore the wide application of living-cell single-molecule force spectroscopy to the study of biological processes, we carried out the AFM force measurement to investigate the interaction between transforming growth factor β1 (TGF-β1) and its receptors for cell signal transduction study. TGF-β is a family of 25 kDa disulfide-linked homodimeric signal ligands responsible for regulating a remarkable diversity of cellular biological processes, including cell motility, recognition, proliferation, differentiation, and apoptosis.21,22 TGF-β1 signaling is mediated by binding to and bringing together its two receptors, type I TGF-β receptor (TβRI) and type II TGF-β receptor (TβRII). TβRI and TβRII are cell-surface glycoproteins of approximately 55 and 70 kDa, respectively. They belong to serine/threonine kinase family and are structurally conservative with a short cysteine-rich extracellular domain, a single transmembrane domain, and a large kinase-containing intracellular domain. During its signaling process, TGF-β1 first binds to TβRII. The binding allows the subsequent incorporation of TβRI to form a heteromeric complex of TβRI/TβRII. This leads to the phosphorylation of specific serine/threonine residues in TβRI by TβRII and then the phosphorylation of Smad proteins to propagate the signal to the cell nucleus. Since the binding of TGF-β to its receptors is essential for TGF-β signaling, much
10.1021/jp0758667 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/13/2007
13620 J. Phys. Chem. B, Vol. 111, No. 48, 2007 effort has been made to investigate the ligand-receptor interaction and its effect on the overall signaling output.23-26 It has been demonstrated that TGF-β1 does not bind with TβRI in the absence of TβRII. On the other hand, although TGF-β1 is able to bind with TβRII alone, TβRII signaling independent of TβRI is not triggered. The study of the TGF-β1/TβRI/TβRII assembly in vitro with the receptor extracellular domains (ECDs) also indicated that TβRI-ECD binds with a high affinity to the complex of TGF-β1/TβRII-ECD but not to either TGF-β1 or TβRII alone. Previous studies have been mainly accomplished by in vitro biochemical assays, such as gel electrophoresis,23-25 NMR,24 and surface plasmon resonance (SPR),26 with cell lysates or purified recombinant proteins. The molecular mechanism of TGF-β1 ligand-receptor interaction and TGF-β/TβRI/ TβRII signaling complex formation in living cells is not yet fully understood. In this study, we have applied single-molecule force spectroscopy and single-molecule dynamic force spectroscopy to study the interaction between TGF-β1 and its receptors in living cells at the single molecule level. The binding strength of TGFβ1 with TβRII on the cell surface is found to be comparable to that with TβRII extracellular domain modified on AFM silicon substrate. Moreover, our results revealed the cooperative binding of TGF-β receptors to the ligand in living cells. The interaction force of TGF-β1 with TβRI/TβRII complex was stronger than that of TGF-β1 with TβRII alone. To the best of our knowledge, this is the first report of the single-molecule force study of growth factor/receptor interaction in living cells. It provides us a new approach to the better understanding of the molecular mechanism of TGF-β signaling. 2. Materials and Methods Materials. TGF-β1 and TβRII monoclonal antibody (MAb) was obtained from PeproTech EC (London, U.K.) and R&D Systems (U.S.A.), respectively. Poly-D-lysine (MW 70 000150 000) was purchased from Sigma. 3-Amino-propyltriethoxysilane (APTES), (3-mercaptopropyl) trimethoxysilane (MPTMS), and toluene (99.99%, HPLC grade) were obtained from ACRO (U.S.A.). N-Hydroxysuccinimide-polyethylene glycolmaleimide (NHS-PEG-MAL, MW 3400) was purchased from Nektar Therapeutics (Huntsville, AL). Other reagents used in the experiment were all of analytical grade. Milli-Q purified water (18.2 MΩ) was used for all experiments. Plasmids Construction. The N-terminal TβRII extracellular domain (TβRII-ECD) was PCR-amplified from pCMV5-TβRII utilizing the sense primer, 5′-GTTGGATCCGACGCGTATCGCCAGCAC-3′, and antisense primer, 5′- TCGAAGCTTGTCAGGATTGCTGGTGTTA-3′. The PCR product was subcloned into the BamH I and Hind III sites of pET-21b (Novagen, U.S.A.), a vector carrying an optional C-terminal His-tag sequence. The DNA fragments encoding full-length TβRI and TβRII were subcloned into the HindIII and BamHI sites of pGFP-N1 or pDsRed-N1 (Clontech, U.S.A.), yielding the TβRI-GFP, TβRIIGFP, and TβRII-DsRed (RFP) expression vectors.27 All the constructed plasmids were confirmed by sequencing. The fluorescent protein (GFP, RFP) tagged TβRI and TβRII were all tested to be functional as untagged receptors in activating the expression of the TGF-β-responsive reporter CAGAluciferase in the presence of TGF-β. Preparation of TβRII Extracellular Domain. pET-21bTβRII ECD was transformed into E. coli DH-5R. The Nterminal His-tagged TβRII ECD protein was induced by 1 mM IPTG and purified with Ni-NTA agarose (Qiagen, U.S.A.) under denaturing condition. Protein refolding was carried out
Yu et al. according to ref 28. The samples were first diluted with TrisHCl (20 mM, pH 8.0) containing 100 mM NaCl and 0.5-2.0 M urea, then dialyzed against buffer I (0.5 M urea, 20 mM TrisHCl, 1 mM EDTA, 100 mM NaCl, 25% NP-40, 0.2 mM PMSF) and buffer II (20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 0.25% NP-40, 0.2 mM PMSF) for 24 h, respectively. Cell Culture and Plasmid Transfection. HEK293A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, U.S.A.) containing 10% fetal bovine serum (Hyclone, U.S.A.). Sterilized 0.1 mg/mL poly-D-lysine solution was spread on the cell culture dishes to promote cell adhesion. Transient transfection was performed with lipofectamin 2000 (Invitrogen, U.S.A.). An amount of 4 µg/mL plasmids was transfected into the cells. After 12 h of incubation, the cells were washed twice with serum-free medium and the culture solution was changed to serum-free DMEM containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sino-American Biotech., China) for microscopy observation and force measurement. AFM Substrate and Tip Preparation. Chemical modification of AFM substrates for in vitro force measurement was carried out according to the previously reported procedures:8-10 Single-crystal silicon wafers were cut into 1.5 cm × 1.5 cm square. They were first cleaned and hydroxized through the treatment with chloroform, HF acid, alkaline solution (NH4OH/H2O2/H2O ) 1:1:5, v/v) and piranha solution (98% H2SO4/H2O2 ) 7:3, v/v), respectively, to generate SiOH on the wafers. The cleaned wafers were transferred to a solution of 1.0% (v/v) APTES in toluene, incubated for 2 h at room temperature, and then rinsed thoroughly with toluene. In the following steps, glutaraldehyde was used as a cross-linker to immobilize the protein TβRII-ECD to these silanized wafers containing amine groups. The silanized wafer were activated by incubation in a 0.1% (v/v) glutaraldehyde solution in phosphate-buffered saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 140 mM NaCl, pH 7.3) for 0.5 h at room temperature and then rinsed with the buffer. The activated wafers were immersed in a TβRII-ECD PBS solution (about 5 µg/mL) overnight at 4 °C, allowing the protein coupling via the intrinsic amine groups in the protein to the aldehyde groups on the activated wafers. After rinsing with the buffer, the functionalized wafers were stored in PBS at 4 °C until use. AFM silicon nitride (Si3N4) tips (type NP with a radius of 20-60 nm, from Veeco, Santa Barbara, CA) were used in all the experiments. The spring constants of the tips, calibrated by the thermal fluctuation method,29 were in the range of 0.0250.045 N/m. The tips were first cleaned with the same procedure as the silicon wafer to generate Si-OH groups. Then they were transferred to a solution of 1.0% (v/v) MPTMS in toluene, incubated for 2 h at room temperature, and rinsed thoroughly with toluene to be modified with -SH groups.10 After being dried with N2, the tips were activated by incubation in 1 mg/ mL NHS-PEG-MAL, the cross-linker, in dimethyl sulfoxide for 3 h at room temperature,10,30 and then rinsed thoroughly with dimethyl sulfoxide to remove any unbound NHS-PEGMAL. The NHS-PEG-MAL was conjugated to the -SH groups on the AFM tips via its MAL end. These activated tips were immersed into a protein (TGF-β1 or TβRII antibody) solution (3 × 10-8 mol/L in PBS) and incubated at room temperature for 0.5 h. The proteins were bound via their intrinsic amine groups to the NHS end of the PEG derivative. After rinsing with PBS, the protein-modified tips were stored in PBS at 4 °C until use. AFM Force Measurements. AFM force measurements in vitro with the protein-modified tips and substrates were per-
Single-Molecule Force Spectroscopy of TGF-β1/T/βRII
J. Phys. Chem. B, Vol. 111, No. 48, 2007 13621
Figure 1. Schematic diagram of single-molecule force measurement in living cells with a ligand-modified AFM tip. The tip was positioned directly above a cell expressing the desired receptors.
formed on a NanoScope ΙV (Veeco, Santa Barbara, CA) with a liquid cell. The force-distance curves were recorded and analyzed by the Nanoscope 5.30b4 software (Veeco, Santa Barbara, CA). The tip speed was about 800 nm/s with the z range set at 400 nm. The AFM loading rate, determined by multiplying the tip speed (nm/s) and the slope of the forcedistance trace just before the rupture event in force curves (pN/ nm), was (1.0 ( 0.5) × 103 pN/s. The force measurements (shown schematically in Figure 1) of the TGF-β1-modified AFM tip on the living cells expressing TGF-β receptors were carried out on a PicoSPM II with PicoScan 3000 controller and a large scanner (Molecular Imaging, Tempe, AZ). The AFM scanner was mounted on an inverted fluorescence microscope (Olympus IX71, Japan). The fluorescence image of fluorescent proteins (GFP, RFP) was used to locate the AFM tips on the cells expressing TGF-β receptors. The fluorescence microscope was equipped with a Hg lamp and 10×/40× objectives. A U-MWIB2 filter set (Ex, BP460-490 nm; Em, LP510 nm, Olympus, Japan) was used for GFP, and a UMWIY2 filter set (Ex, BP545-580 nm; Em, LP610 nm, Olympus, Japan) was used for RFP. Force measurements at the different loading rates ranging from 1.0 × 102 to 1.3 × 104 pN/s were achieved by changing the cantilever velocity. The force curves measured in living cells were recorded and analyzed by PicoScan 5 software (Molecular Imaging, Tempe, AZ). All forces were measured with contact mode at room temperature. 3. Results and Discussion Binding of TGF-β1 with TβRII Extracellular Domain. It is known that TGF-β1 binds to TβRII with a high affinity (Kd in the picomolar range).21,26 We first took the advantage of recombinant TβRII extracellular domain (TβRII-ECD)26,28 to study the binding strength between TGF-β1 and TβRII in vitro. This was achieved by using the similar strategy of singlemolecule force microscopy as we previously reported.8-10 The measurement was performed with TGF-β1-functionalized AFM tips and TβRII-ECD-modified silicon substrates. The protein density on both tips and substrates was controlled to be low to ensure that only one pair of ligand-receptor molecules could be formed during the force measurement. In addition, a PEG chain was introduced as a spacer to link TGF-β1 to the tip to differentiate the nonspecific interaction and keep the immobilized protein in a more flexible orientation.10,31 The representative force curves measured for TGF-β1/TβRII-ECD are shown in Figure 2A. While the first peaks which appeared randomly were caused by the nonspecific interaction between the AFM tip and substrate, the second peaks, which appeared about 20-40 nm away from separation of the tip and substrate,
Figure 2. (A) Representative force curves obtained from TGF-β1modified AFM tips and TβRII-ECD-modified silicon substrates (a and b) and after the system was blocked with TβRII-ECD solution (c). The X-axis is piezo displacement. The solid arrows marked the rupture of TGF-β1/TβRII-ECD, and the dashed arrows marked the separation of tip and substrate. (B) Histogram of binding forces of TGF-β1/TβRIIECD (dark bars, experimental data; solid line, theoretical Gaussian distribution curve).
represented the specific force of TGF-β1/TβRII-ECD (Figure 2A, curves a and b).10,31 This was confirmed by the blocking experiment as the second peak disappeared if a solution of free TβRII-ECD (about 5 µg/mL) or free TGF-β1 (about 3 × 10-8 mol/L) was introduced (Figure 2A, curve c). Therefore, the interaction force between TGF-β1 and TβRII-ECD was directly extracted from the second peaks in the force curves. The force distribution histogram obtained from 1000 to 1500 force curves is shown as Figure 2B. A single maximum in the histogram by Gaussian fitting indicated that the single-molecule forces were measured.6,9,10 The low frequency of the adhesion events (∼20%) during the measurement also ensured that the forces were mediated by a single ligand-receptor pair (based on Poisson statistics, 85% probability of single-molecule force measurement).14-18 The mean value of the most probable single molecular interaction force (from three histograms obtained in three independent experiments) of TGF-β1/TβRII-ECD was determined as 52.9 ( 5.5 pN at the loading rate of (1.0 ( 0.5) × 103 pN/s. The binding probability was 19.7% ( 4.9% in contrast to that of 5.5% ( 1.8% when the binding was blocked with the TβRIIECD solution. The measured single-molecule force and binding probability all fell in the range of previously reported values for single-molecule force study of noncovalent ligand-receptor binding.5-10,31
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Figure 3. Optical image (A) and corresponding fluorescence images recorded in GFP channel (B) and RFP channel (C) for the cells transfected with TβRII-GFP; optical image (D) and corresponding fluorescence images recorded in GFP channel (E) and RFP channel (F) for the cells cotransfected with TβRI-GFP and TβRII-RFP.
Binding of TGF-β1 with TβRII on the Living-Cell Surface. To directly measure the binding force of TGF-β1 with its highaffinity receptor, TβRII, in living cells, the transfected HEK293A cells expressing TβRII-GFP were adopted in this study. HEK293A cells are derived from 293 cells which have undetectable endogenous TβRII.32,33 Therefore, TβRII expressed on the cell membrane can be observed by the fluorescence signal from GFP. The single-molecule force measurement was carried out with the AFM which was mounted on a fluorescence microscope. Both optical and fluorescence images (e.g., Figure 3, parts A and B) of the transfected cells were taken before the force measurement. The living cells emitting strong fluorescence signals were selected. The TGF-β1-modified AFM tip was then moved to position above such a cell and applied to the cell surface at various locations. The rupture forces were detected when the tip and cell were brought into and out of contact. Optical images of the cell before and after the force measurement were compared to ensure the cell was alive during the experiment. Typical force curves during AFM tip retraction on the selected cells are shown in Figure 4A, curve a and b. Similar to other reported force-distance curves on the soft substrate such as cells,13,14,34 the curves displayed a gradually decrease in the initial stage when the tip was retracted from the cell. The specific rupture forces appeared as the tip moved for a distance of 0.1-3 µm due to the cell elasticity.13,14,34 Occasionally, two rupture peaks (probability
