Single Mol. 1 (2000) 2, 159-163 RESEARCH PAPER MoleculesSingle
Single-Molecule Imaging of Signaling Molecules inLiving Cells
Yasushi Sakoa), Kayo Hibinoc), Takayuki Miyauchia), Yoshikazu Miyamotoa,b),Masahiro Uedaa), and Toshio Yanagidaa,c,d).
a)Department of Physiology and Biosignaling, and b)
Department of Anesthesiology, Graduate School ofMedicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan
c) Department of Systems and Human Science, GraduateSchool of Engineering Science, Osaka University, 1-3Machikaneyama, Toyonaka 560-8531, Japan
d)Single Molecule Processes Project, ICORP, JST, 2-4-14,
Senba-Higashi, Mino 562-0035, Japan
Corresponding toYasushi SakoDepartment of Physiology and Biosignaling, Graduate Schoolof Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, JapanPhone +81-6-6879-3621Fax +81-6-6879-3628e-mail firstname.lastname@example.org,jp
submitted 01 May 2000published 23 Jun 2000
Single-molecule imaging is an ideal technology to studymolecular mechanisms of biological reactions in vitro.Recently, we extended this technology to real-timeobservation of complexes of fluorescent dye-labeledepidermal growth factor (EGF) and its receptor (EGFR).Detection of single molecules was confirmed by single-stepphotobleaching. Using the single-molecule technique, theprocess of dimerization, a key step of EGFR signaltransduction was observed. In addition to EGFR, single-molecules of fluorescently labeled nerve growth factor, GFPfusion forms of small G proteins and Raf1 kinase, and afluorescent analogue of cAMP were observed in living cells.
Single-molecule imaging enables us to observe localization,movement, oligomerization and turnover of individualsignaling molecules in the plasma membrane of living cells.
The detection of intracellular molecules with single-molecular sensitivity and resolution has been sought toelucidate the mechanism of cellular functions [1,2].Especially, in cell signaling, molecules transducing andprocessing signals are small in number, and the extent oftheir activity may be diverse depending on the position ofcells. Therefore, it is very important to develop techniquesto measure the reactions of individual cell-signalingmolecules in living cells with spatio-temporal resolution.We have previously reported single-molecule imaging offluorescently-labeled biomolecules in solution [3,4] and inartificial lipid membrane  by using total internal reflectionmicroscopy . Recently, we extended this technique toobserve single-molecules of fluorescently-labeled epidermalgrowth factor (EGF) on the surface of living cells to study thesignal transduction of EGF receptor (EGFR) . Thedetection of single molecules on the surface of living cellswas also reported by another group [8,9].
In this study, we observed the dimerization process ofEGFR, which is an early event in signal transduction of EGF,in the plasma membrane of A431 carcinoma cells. Singlemolecule tracking revealed that the predominantmechanism of the dimerization was direct arrest of an EGFmolecule in solution onto the position just beside a cellsurface complex of EGF/EGFR, suggesting the presence ofpre-formed dimers of EGFR. In addition to EGF, single-molecules of other cell signaling molecules, a peptidehormone that induces nerve cell differentiation (nervegrowth factor: NGF), small G proteins (Ras, Rho, Rac andCdc42), a cytoplasmic serine/threonine kinase (Raf1), anda chemoattractant for cellular slime mold (cAMP) wereobserved in living cells.
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Human epithelial cell lines A431 and HeLa, and a canineepithelial cell line MDCK were cultured in MEMsupplemented with 10% fetal calf serum. A mousephenochromocytoma PC12D  was cultured in MEMsupplemented with 10% fetal calf serum and 10% horseserum. The culture medium was changed to that withoutserum and phenol red one day prior to the experiments.
D. discoidium (strain AX2) was cultured according to. Prior to microscopic observation, cells were harvested,suspended in the development buffer  and starved for6hr.
F l u or es c e nc e Lab el i ng of Si g nal i ng Mol ec ul es
Cy3-EGF was prepared and purified as described before .Amino-NGF was prepared according to , and labeled withamino-reactive Cy3 (Amasham) as Cy3-EGF. Synthesis offluorescent cAMP analog (Cy3-cAMP) will be described indetail elsewhere. To add a fluorescent label to cAMP in amanner that does not interfere with its bioactivity, Cy3 dyewas conjugated to 2-OH of ribose moiety of cAMP.
The same apparatus of an objective-type TIR fluorescencemicroscope  used in  was used for all experiments.
Results and Discussion
Single Molecule Imaging of Cy3-EGF on theSurface of Living Cells
Epidermal growth factor (EGF) is a small peptide hormonethat induces cell proliferation. Since mouse EGF has onlyone reactive amino residue (at the amino terminus), it canbe labeled with amino-reactive Cy3 dye with a dye/proteinratio of exactly 1 to 1 (Fig. 1A). Binding of Cy3-labeled EGF(Cy3-EGF) on the surface of A431 cells was observed usingan objective-type total internal reflection (TIR) fluorescencemicroscope  (Fig. 1B). This binding was completelyinhibited by the addition of an excess amount of non-labeledEGF at the same time as the addition of Cy3-EGF (Fig. 1Binset), indicating specific binding of Cy3-EGF to cell surfaceEGFR. By carefully adjusting the incident angle of theexcitation laser beam, Cy3-EGF on both apical andbasolateral plasma membrane was visualized by TIRmicroscopy . A slight difference in refractive indices ofthe cytoplasm and the culture medium could result inimaging of the apical surface of the cell membrane.
In many cases, Cy3-EGF spots on the cell surface werephotobleached in a single-step (Fig. 1C), strongly suggesting
that the fluorescent spots represented single Cy3-EGFmolecules . Single-molecule detection was alsoconfirmed by the quantum nature of the intensitydistribution of fluorescent spots . Single molecules ofEGF labeled with other fluorescent dyes, Cy5 andtetramethylrhodamine, were visualized by the sametechnique (Fig. 1C).
Fig. 1. Visualization of Cy3-EGF on the plasma membrane ofliving A431 cells. (a) Mouse EGF was conjugated with afluorophore Cy3 at the amino terminus. (b) Cy3-EGF (0.2ng/ml) was added to the culture medium of living A431cells under a TIR fluorescence microscope. Images wereacquired 1 min after the addition of Cy3-EGF. Binding ofCy3-EGF was completely inhibited in the presence of 200ng/ml of EGF (inset). (c) The fluorescence intensity changein single Cy3 (Cy5, and tetramethylrhodamine)-EGF spots onliving cells was plotted against time. The spots werephotobleached in a single step, indicating that they weresingle molecules. (d) Dimerization of EGF/EGFR complexeswas observed. In the upper case, two spots collided at time0:40 s and then moved together. In the middle case, afluorescence spot with single molecule intensity wasobserved until 1:00 s. Intensity of this spot increasedsuddenly between 1:00 - 2:00 s. Quantitation of thefluorescence intensity change in the middle case is shownin the bottom.
Single-Molecule Observation of EGFRDimerization
Binding of EGF to EGFR induces dimerization of EGFR, whichis a key step of EGFR signal transduction [14, 15]. Theprocess of EGFR dimerization was observed using thesingle-molecule technique. In a few cases (3/26), two spotsdiffusing laterally along the plasma membrane collided and
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Single-Molecule Imaging of Signaling Molecules in Living Cells
then moved together (Fig. 1D upper). However, in mostcases (23/26) fluorescence intensity of a spot increasedsuddenly by a factor of about two, and then bleached in twosteps (Fig. 1D middle). The fluorescence intensity change ofthe latter case shows two-step buildup and bleaching(Fig.1D bottom). This case is probably caused by the directbinding of EGF molecules in solution to clusters of EGF-bound EGFR and EGF-free EGFR in the plasma membrane.This result suggests that dimers of EGFR were preformed.
Binding of Single-Molecules of NGF to theCell Surface
Nerve growth factor (NGF) is another kind of peptidehormone that induces differentiation of nerve cells . Thenative form of NGF has a subunit structure of a2bg2, and theb-subunit is responsible for biological activity of NGF. As theb-subunit is a dimer of 118-amino acid polypeptide (half bsubunit), it was a question whether single half b subunitbinds to NGFR and induces cellular response. (In aqueoussolution, two half b-subunits dimerize spontaneously (Fig.2B).) We made half b subunit labeled with Cy3. Half bbound to the cell surface as single-molecules (Fig. 2), andthe binding induced neurite outgrowth (data not shown).These results are important when considering themechanism of dimerization and activation of NGF receptor.
Fig. 2. (A) Binding of half b subunits of NGF conjugated withCy3 (Cy3-NGF) on the cell surface. (B) Histograms of thefluorescence intensity of Cy3-NGF (half b) spots. Monomersof Cy3-NGF were purified by reversed phasechromatography. Fluorescence intensity distribution of Cy3-EGF just after purification was examined (top). After 6 days,Cy3-NGF dimerized spontaneously (middle). Cy3-NGF justafter purification was applied to PC12D cells as shown in(A). Fluorescence intensity distribution indicates that mostof the Cy3-NGF bound to the cell surface as monomers(bottom).
Imaging of Small G Proteins and Raf1 KinaseBeneath the Plasma Membrane
A small GTP-binding protein Ras is a key molecule in manysignal transduction pathways including EGF and NGF.
Activation of Ras induces translocalization of a MAP kinasekinase kinase Raf1 from cytoplasm to the plasmamembrane . We observed this pathway in living cells bymaking GFP (YFP)-fusion of Ras and Raf1 (Fig. 3A-C). UsingTIR microscopy, GFP (YFP)-fusion forms of Ras and Raf1were observed as fluorescent spots (Figs. 3D and E). Bothspots of Ras and Raf1 were diffusing very rapidly beneaththe plasma membrane (data not shown). Fluorescent spotsof YFP-Raf1 were photobleached in discrete stepssuggesting that single molecules of GFP can be observedunder the plasma membrane (Fig. 3F).
Fig. 3. Imaging of small G protein Ras and Raf1 kinase.HeLa cells were transfected with cDNA of YFP-Ras (A) orGFP-Raf1 (B, C). YFP-Ras located at the plasma membrane(A). GFP-Raf1, which localized in the cytoplasm of a cell inthe resting state (B), translocated to the plasma membraneafter 10 min of stimulation with a 10-s pulse of 100 ng/mlof EGF (C). (The cell in B and C was cotransfected with Rasand GFP-Raf1.) Fluorescent spots of YFP-Ras (D) and YFP-Raf1 (after stimulation with EGF: E) beneath the plasmamembrane were visualized by TIR microscopy (arrowheads).Fluorescence intensity of these spots was similar as that forsingle YFP molecules. Single- or two-step(s) photobleachingof these spots suggests that these were a single and adimer of YFP-fusion proteins (F).
The Rho family small G-proteins (Rho, Rac and Cdc42) arekey molecules to control the organization of thecytoskeleton. YFP-fusion forms of Rho, Rac and Cdc42 werealso observed as individual diffusion units on the plasmamembrane of living cells (Fig. 4A). Even though individualspots of YFP-Rac were observed for only 2 s on the plasmamembrane (Fig. 4B), overall density of the spots of YFP-Racwas not changed for several minutes (Fig. 4C). This resultsuggests the rapid turnover of Rac between cytoplasm andthe plasma membrane.
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Fig. 4. Turnover of a Rho family small G protein Racbetween cytoplasm and the plasma membrane. (A) Imagesof YFP-Rho, Rac and Cdc42 on the plasma membrane ofMDCK cells. (B) Histogram of the duration during whichindividual spots of YFP-Rac were observed on the cellsurface. A solid line is the result of single exponentialfitting. Duration less than 0.3 s was not counted to avoidcontamination of shot noise. (C) Density of the fluorescentspots of YFP-Rac was measured repeatedly in the samefield (460 mm2) for 3 min.
Visualization of Cy3-cAMP on the CellSurface
Dictiostelium amoebae performs chemotaxis to adenosine3,5-monophosphate (cAMP), which is mediated by Gprotein-coupled cAMP receptors . To elucidate how cellssense gradients of cAMP, we synthesized Cy3-labeled cAMPand observed the binding of Cy3-cAMP to its receptor on thesurface of cells undergoing chemotactic migration (Fig. 5A).Tracking of individual Cy3-cAMP spots revealed that cAMPreceptors have a highly dynamic property in their lateralmobility along the plasma membrane (Fig. 5C). Thebiological significance of this mobility in signal transductionfor chemotaxis remains to be solved.
Conclusion and Perspective
By extending single-molecule techniques in aqueoussolution, we now can observe single molecules in livingcells. Here we demonstrated the detection of location,movement, recognition and local turnover of various kinds ofcell signaling molecules. Peptides and small molecules fromextracellular solution as well as cytoplasmic proteins can beobserved in single molecules on the plasma membrane.Expression as GFP-fusion forms potentially makes itpossible to observe every kind of signaling protein as singlemolecules in living cells. Observation of reactions of singlemolecules in living cells with spatio-temporal resolution willbe indispensable in understanding the mechanism of cellsignaling.
Fig. 5. Cy3-cAMP on the surface of a chemotactic amoeba.(A) Visualization of single-molecules of Cy3-cAMP bound toits receptor on a D. discoidium amoebae under chemotacticmovement along a gradient of Cy3-cAMP. Single-stepphotobleacing of a Cy3-cAMP spot suggests that single-molecule can be visualized (inset). Bright spots of Cy3-cAMPmay be clusters of cAMP/receptor complexes. (B)Quantized photobleaching of a fluorescent spot indicated bythe arrow in (A) suggests that the spot was produced froma single Cy3-cAMP molecule. (C) Typical trajectories oflateral movement of individual Cy3-cAMP/receptorcomplexes on the cell surface shown in (A). Centroids ofindividual Cy3-cAMP molecules at 33-msec intervals werelinked to show the trajectory. The trajectories were plottedagainst the substrate.
Acknowledgement.: We thank Mamoru Sano for PC12Dcells, Toru Kataoka for plasmids containing ras and raf1,Kozo Kaibuchi for plasmids containing rho, rac and cdc42,Atsuko Iwane-Hikikoshi for technical advice on constructionof cDNAs, and Keiji Tanaka for technical advisce on cAMPsynthesis.
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