Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling. The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling. IntroductionThe plasma membrane of the cell is a complex, carefully regulated, dynamic structure that is compartmentalized into cell surface domains such as lipid rafts, caveolae, and clathrin-coated pits (1Hancock J.F. Nat. Rev. Mol. Cell Biol. 2006; 7: 456-462Crossref PubMed Scopus (666) Google Scholar). Lipid rafts are postulated to be dynamic nanodomains ranging in size from 10 to 200 nm that are enriched in cholesterol, sphingolipids, and certain proteins and that play an important role in assembling signaling complexes (2Mayor S. Rao M. Traffic. 2004; 5: 231-240Crossref PubMed Scopus (333) Google Scholar, 3Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar, 4Pike L.J. J. Lipid Res. 2006; 47: 1597-1598Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar). Caveolae are invaginated lipid raft domains (50–150 nm) whose stability at the plasma membrane is attributable to the formation of stable oligomers of their coat protein, caveolin-1 (5Parton R.G. Hanzal-Bayer M. Hancock J.F. J. Cell Sci. 2006; 119: 787-796Crossref PubMed Scopus (228) Google Scholar). Caveolae are thought to serve as concentrators of various signal transduction machineries. Clathrin-coated pits (100–150 nm) internalize rapidly upon formation at the plasma membrane, and their lateral cell surface mobility is enhanced by actin cytoskeleton depolymerization (6Gaidarov I. Santini F. Warren R.A. Keen J.H. Nat. Cell Biol. 1999; 1: 1-7Crossref PubMed Scopus (355) Google Scholar).The epidermal growth factor receptor (EGFR), 3The abbreviations used are: EGFRepidermal growth factor receptor(s)MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseNSOMnear field scanning optical microscopyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinemAbmonoclonal antibodyFRETfluorescence resonance energy transferGFPgreen fluorescent protein. a 170-kDa transmembrane glycoprotein, is one of four members of the ErbB family of receptor tyrosine kinases involved in oncogene signaling. The initial step in receptor activation involves binding of the EGF peptide to the extracellular domain leading to dimerization or activation of pre-existing dimers (7Citri A. Yarden Y. Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1578) Google Scholar, 8Ferguson K.M. Annu. Rev. Biophys. 2008; 37: 353-373Crossref PubMed Scopus (232) Google Scholar). Following ligand binding, the EGFR is auto-phosphorylated in several tyrosine residues of the intracellular domain, creating high affinity sites for various adaptor molecules that transmit the mitogenic signal to the Ras/MAPK signal transduction pathway (9Ciardiello F. Tortora G. Clin. Cancer Res. 1998; 4: 821-828PubMed Google Scholar). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2, which regulate transcription of molecules that are linked to cell proliferation, survival, and transformation. Enhanced EGFR expression has been detected in glioblastoma, breast, lung, ovarian, colorectal, and renal carcinomas (10El-Rayes B.F. LoRusso P.M. Brit. J. Cancer. 2004; 91: 418-424Crossref PubMed Scopus (149) Google Scholar). Overexpression of EGFR is associated with poor prognosis in many tumors including brain and breast cancer. Two main anti-EGFR strategies are currently exploited in clinical development: chimeric anti-receptor antibodies such as 225 (cetuximab), which binds directly to the ligand-binding site on the extracellular domain and has similar affinity to EGF peptide, and small molecule tyrosine kinase inhibitors such as tryphostin AG1478, gefitinib (ZD1839), or erlotinib (OSI-774) that compete with ATP for binding to the EGFR intracellular tyrosine kinase domain. Both classes of therapeutics have shown encouraging preclinical and clinical activity in a variety of tumors (11Mendelsohn J. Baselga J. J. Clin. Oncol. 2003; 21: 2787-2799Crossref PubMed Scopus (1175) Google Scholar, 12Sebastian S. Settleman J. Reshkin S.J. Azzariti A. Bellizzi A. Paradiso A. Biochim. Biophys. Acta. 2006; 1766: 120-139PubMed Google Scholar). Nevertheless, the development of resistance to some therapies illustrates the need for a more detailed understanding of the factors that regulate EGFR signaling (12Sebastian S. Settleman J. Reshkin S.J. Azzariti A. Bellizzi A. Paradiso A. Biochim. Biophys. Acta. 2006; 1766: 120-139PubMed Google Scholar, 13Yu C. Hale J. Ritchie K. Prasad N.K. Irudayaraj J. Biochem. Biophys. Res. Commun. 2009; 378: 376-382Crossref PubMed Scopus (21) Google Scholar).The nanometer scale clustering of receptors in higher order assemblies has attracted increased attention in studies of receptor biology and signal transduction. The functional consequences of receptor assemblies include their potential for inter-receptor communication and increased signaling efficiency, as well as their information processing capacity (14Irvine D.J. Hue K.A. Mayes A.M. Griffith L.G. Biophys. J. 2002; 82: 120-132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Membrane domains such as lipid rafts, caveolae, and clathrin-coated pits provide one mechanism for spatial and temporal control of the assembly of nanoscale signaling domains (15Pontier S.M. Percherancier Y. Galandrin S. Breit A. Galés C. Bouvier M. J. Biol. Chem. 2008; 283: 24659-24672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, progress in understanding the assembly of signaling complexes and their association with membrane domains has so far been hampered by the limited availability of methods for the direct visualization of proteins and complexes with high spatial resolution in the membranes of intact cells.A mechanistic understanding of EGFR activation in a tumor cell environment requires knowledge of the spatial organization of the receptor on the cell surface. Recently single molecule fluorescence and single particle tracking have provided insight on the role of EGFR dimers and oligomers but in most cases do not visualize the entire population of receptors (16Saffarian S. Li Y. Elson E.L. Pike L.J. Biophys. J. 2007; 93: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Sergé A. Bertaux N. Rigneault H. Marguet D. Nat. Methods. 2008; 5: 687-694Crossref PubMed Scopus (410) Google Scholar). We report the use of near field scanning optical microscopy (NSOM) to probe the distribution and nanometer scale associations of EGFR in HeLa cells and to determine the influence of the tyrosine kinase inhibitor AG1478 and 225 EGFR-blocking antibody on the distribution of EGFR. NSOM is one of an emerging group of super-resolution methods for fluorescence imaging with subdiffraction resolution (18Hell S.W. Nat. Methods. 2009; 6: 24-32Crossref PubMed Scopus (821) Google Scholar, 19Lippincott-Schwartz J. Manley S. Nat. Methods. 2009; 6: 21-23Crossref PubMed Scopus (145) Google and is based on an that has Chem. Rev. 1999; PubMed Scopus Google Scholar, A. H. A. A. E. Nat. 2003; 21: PubMed Scopus Google to the NSOM has been used for nanoscale fluorescence imaging of membrane protein clusters with spatial resolution nm in cells A. M. D. J. L.J. Biophys. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. M. L.J. Nat. Chem. Biol. 1: PubMed Scopus Google Scholar, M. A. 2004; PubMed Scopus Google Scholar, C. A. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, C. 2008; PubMed Scopus Google Scholar, F. A. 2007; PubMed Scopus Google Scholar, A. G. S. A. J. Cell Sci. 2008; PubMed Scopus Google Scholar). that EGFR is in clusters in the plasma membrane and that association with lipid raft and domains receptor and in to activation in HeLa clinical results the of for colorectal, and and use of for However, a fraction of to and of are is to understanding of the of signaling a understanding of the mechanism of in cells high of EGFR is to understanding EGFR overexpression tumor is also for therapeutics that signaling of proteins in signaling complexes a mechanism for protein and modulating the of signal transduction. the size and of signaling complexes and their to membrane domains is an of significant methods have been used to the dimerization of but the assembly of individual receptors or small oligomers into complexes is a more requires methods for proteins on a scale that is the by fluorescence resonance energy and the spatial resolution with have near field microscopy to examine the in EGFR signal with the of understanding the dynamic organization of membrane receptors is by receptor or that EGFR in cells is organized in clusters with to nm in and more than of clusters have is a small fraction of clusters with greater than The clustering of EGFR in the of ligand is with studies of ErbB receptors (16Saffarian S. Li Y. Elson E.L. Pike L.J. Biophys. J. 2007; 93: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Sergé A. Bertaux N. Rigneault H. Marguet D. Nat. Methods. 2008; 5: 687-694Crossref PubMed Scopus (410) Google Scholar, 2007; PubMed Scopus Google Scholar, F. C. D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. J. Cell Sci. 2007; PubMed Scopus Google Scholar, A. J. S. J. Cell Sci. 1999; PubMed Google Scholar). 2007; PubMed Scopus Google have used microscopy to an average EGFR cluster density of with in HeLa cells. and fluorescence imaging the of higher order with most in clusters organization of receptors into nanoscale complexes was to the of EGFR to in of EGF peptide and to to that be or the receptors as or dimers at low density on the cell 2007; PubMed Scopus Google Scholar). and the of provide a detailed of the distribution of EGFR on the plasma membrane of HeLa cells. The EGFR is organized in clusters with a of and receptor and with a significant fraction of receptor oligomers in the receptor has been for a variety of membrane proteins (15Pontier S.M. Percherancier Y. Galandrin S. Breit A. Galés C. Bouvier M. J. Biol. Chem. 2008; 283: 24659-24672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, A. Y. M. L.J. Nat. Chem. Biol. 1: PubMed Scopus Google Scholar, A. G. S. A. J. Cell Sci. 2008; PubMed Scopus Google cells a fraction of small clusters nm) with fewer with control cells. The in cluster size and density EGF stimulation that receptors are in nanoscale domains on the cell surface and that for organization of the of ligand The results microscopy of several ErbB receptors in cells S. J. J. Cell Sci. 2007; PubMed Scopus Google Scholar). clustering was at low expression with from to in cluster size was for EGFR or ligand cluster increased EGF The of of the ErbB receptors to the that receptor a mechanism for Changes in clustering as a of ligand activation are for EGFR family an a cluster diameter activation of receptors in cells A. J. S. J. Cell Sci. 1999; PubMed Google and the studies have protein clustering in a of the dynamic of the However, a similar from studies in cells. receptor for EGFR in one that one that is in nm diameter and one that is A. Bertaux N. Rigneault H. Marguet D. Nat. Methods. 2008; 5: 687-694Crossref PubMed Scopus (410) Google Scholar). fluorescence for in cells has that EGFR is a of and in control with an average of to activation with EGF peptide (16Saffarian S. Li Y. Elson E.L. Pike L.J. Biophys. J. 2007; 93: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). By contrast, a single molecule fluorescence of in HeLa cells that dimers EGF peptide Y. Biochem. Biophys. Res. Commun. 2008; PubMed Scopus Google in the nanoscale distribution of EGFR are with the two inhibitors that have the cluster size decreases and the cluster density by a factor of AG1478 similar increase in cluster density with a concomitant in the of was by 2007; PubMed Scopus Google leading to the that of tyrosine phosphorylation leads to the loss of EGFR with of EGFR with the tyrosine inhibitor clustering C. S. 2007; PubMed Scopus Google Scholar). However, NSOM have the in cluster distribution that AG1478 leads to of many small individual EGFR clusters in illustrates the of receptor organization on both and nanometer is that inhibitors that with ligand binding to the extracellular domain or phosphorylation of intracellular residues have similar on the nanoscale clustering of EGFR. By contrast, ligand activation leads to in the nanoscale organization of the Both The of understanding the of the EGFR in the of AG1478 is by the that AG1478 the of EGFR the binding of an anti-EGFR antibody with enhanced therapeutic potential F. A. A.M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the of tyrosine kinase inhibitors with anti-EGFR antibodies to is an of clinical F. G. S. J. M. S. J. J. Baselga J. Clin. Cancer Res. 2004; PubMed Scopus Google peptide binding the and of the the cells for mitogenic A. 1998; PubMed Google Scholar). a few studies have EGFR EGF J. Cancer. PubMed Scopus Google Scholar, Y. S. Y. Biochem. J. PubMed Scopus Google Scholar, J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google to the of knowledge is the of in the plasma We have shown that of EGF to HeLa cells a of receptor at the plasma membrane and a increase in the protein. The EGFR at and have been in and was that the and proteins of the EGFR N. Cancer Res. Google Scholar). that the enhanced receptor in HeLa cells M. PubMed Scopus Google was by AG1478 and to a by 225 antibody We have that 225 EGFR and in a in surface EGFR S. A. M. Cell Res. 2006; PubMed Scopus Google determine the of EGFR to the fluorescence detected NSOM and microscopy the antibody used binds to EGFR However, be to visualize the EGFR and their spatial be by antibodies and for the individual EGFR products. also be of to determine the EGFR signaling to and signaling molecules in plasma membrane for their and signaling M. Res. PubMed Scopus Google Scholar). that to the plasma membrane EGFR is in clusters that caveolin-1 in both control and cells. The of EGFR in clusters with caveolin-1 has potential for the of cell signaling. clusters also provide a high density of EGFR molecules in the of the plasma membrane, which be for to higher order EGFR S. C. E. C. S. Sci. PubMed Scopus Google that in HeLa cells EGFR and to as a of EGF ligand and receptor low EGF the EGFR is for and clathrin-coated at high EGF used EGFR and is S. C. E. C. S. Sci. PubMed Scopus Google have shown an of caveolin-1 and protein expression in cells of AG1478 was not in cell cells A. S. A. M. D. 2004; PubMed Scopus Google Scholar). the plasma membrane caveolin-1 protein expression was to of AG1478, that cell have to tyrosine kinase the are to the or are by in the the of caveolin-1 and with is to be of EGFR with lipid rafts was similar to in both control and cells The results of with both raft and EGFR as well as a significant population that not in membrane domain. The average diameter for the raft domains was nm, the for raft L.J. J. Lipid Res. 2006; 47: 1597-1598Abstract Full Text Full Text PDF PubMed Scopus (1111) Google and in with NSOM studies nanoscale domains for and of and nm, in cells and for in HeLa cells nm) A. E. D. S. L.J. J. 2008; PubMed Scopus Google Scholar, Y. J. J. Lipid Res. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). are than from of of or proteins in the plasma membrane of cells. fluorescence provided for raft domains nm in size C. C. G. K. S. C. A. S.W. Nature. 2009; PubMed Scopus Google Scholar). fluorescence of with the of nanodomains nm in in the plasma membrane of cells F. Y. A.M. S.M. Rigneault H. D. G. Marguet D. Nat. Chem. Biol. 2008; 4: PubMed Scopus (228) Google Scholar). The of methods to dynamic nanodomains in cells has to the and the of membrane However, in super-resolution are to the limited so far the of a of raft L.J. J. Lipid Res. 2006; 47: 1597-1598Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar). that based on a in cells than do dynamic on cells.A has that EGF of two of raft one in which EGFR is by a lipid of and a and several molecules D. J. P.M. J. Cell Sci. 2008; PubMed Scopus Google Scholar). NSOM for a small fraction of EGFR domains for EGF-treated cells but increase in the size of raft domains in the of is that in HeLa the individual raft domains diameter of nm) by be by with NSOM the lateral organization of membrane receptors is emerging as an important factor in and the assembly of signaling that EGFR is organized in nanoscale clusters with a of and receptor on the surface of HeLa cells and that with two used inhibitors both the of receptor clusters and the EGFR of methods with high spatial resolution is for understanding receptor clustering as a of and signaling. on the distribution of dimers and small oligomers individual clusters and to signaling understanding the receptor organization on the cell surface and the of signaling to strategies for therapeutic in EGFR signaling and have in IntroductionThe plasma membrane of the cell is a complex, carefully regulated, dynamic structure that is compartmentalized into cell surface domains such as lipid rafts, caveolae, and clathrin-coated pits (1Hancock J.F. Nat. Rev. Mol. Cell Biol. 2006; 7: 456-462Crossref PubMed Scopus (666) Google Scholar). Lipid rafts are postulated to be dynamic nanodomains ranging in size from 10 to 200 nm that are enriched in cholesterol, sphingolipids, and certain proteins and that play an important role in assembling signaling complexes (2Mayor S. Rao M. Traffic. 2004; 5: 231-240Crossref PubMed Scopus (333) Google Scholar, 3Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar, 4Pike L.J. J. Lipid Res. 2006; 47: 1597-1598Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar). Caveolae are invaginated lipid raft domains (50–150 nm) whose stability at the plasma membrane is attributable to the formation of stable oligomers of their coat protein, caveolin-1 (5Parton R.G. Hanzal-Bayer M. Hancock J.F. J. Cell Sci. 2006; 119: 787-796Crossref PubMed Scopus (228) Google Scholar). Caveolae are thought to serve as concentrators of various signal transduction machineries. Clathrin-coated pits (100–150 nm) internalize rapidly upon formation at the plasma membrane, and their lateral cell surface mobility is enhanced by actin cytoskeleton depolymerization (6Gaidarov I. Santini F. Warren R.A. Keen J.H. Nat. Cell Biol. 1999; 1: 1-7Crossref PubMed Scopus (355) Google Scholar).The epidermal growth factor receptor (EGFR), 3The abbreviations used are: EGFRepidermal growth factor receptor(s)MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseNSOMnear field scanning optical microscopyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinemAbmonoclonal antibodyFRETfluorescence resonance energy transferGFPgreen fluorescent protein. a 170-kDa transmembrane glycoprotein, is one of four members of the ErbB family of receptor tyrosine kinases involved in oncogene signaling. The initial step in receptor activation involves binding of the EGF peptide to the extracellular domain leading to dimerization or activation of pre-existing dimers (7Citri A. Yarden Y. Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1578) Google Scholar, 8Ferguson K.M. Annu. Rev. Biophys. 2008; 37: 353-373Crossref PubMed Scopus (232) Google Scholar). Following ligand binding, the EGFR is auto-phosphorylated in several tyrosine residues of the intracellular domain, creating high affinity sites for various adaptor molecules that transmit the mitogenic signal to the Ras/MAPK signal transduction pathway (9Ciardiello F. Tortora G. Clin. Cancer Res. 1998; 4: 821-828PubMed Google Scholar). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2, which regulate transcription of molecules that are linked to cell proliferation, survival, and transformation. Enhanced EGFR expression has been detected in glioblastoma, breast, lung, ovarian, colorectal, and renal carcinomas (10El-Rayes B.F. LoRusso P.M. Brit. J. Cancer. 2004; 91: 418-424Crossref PubMed Scopus (149) Google Scholar). Overexpression of EGFR is associated with poor prognosis in many tumors including brain and breast cancer. Two main anti-EGFR strategies are currently exploited in clinical development: chimeric anti-receptor antibodies such as 225 (cetuximab), which binds directly to the ligand-binding site on the extracellular domain and has similar affinity to EGF peptide, and small molecule tyrosine kinase inhibitors such as tryphostin AG1478, gefitinib (ZD1839), or erlotinib (OSI-774) that compete with ATP for binding to the EGFR intracellular tyrosine kinase domain. Both classes of therapeutics have shown encouraging preclinical and clinical activity in a variety of tumors (11Mendelsohn J. Baselga J. J. Clin. Oncol. 2003; 21: 2787-2799Crossref PubMed Scopus (1175) Google Scholar, 12Sebastian S. Settleman J. Reshkin S.J. Azzariti A. Bellizzi A. Paradiso A. Biochim. Biophys. Acta. 2006; 1766: 120-139PubMed Google Scholar). Nevertheless, the development of resistance to some therapies illustrates the need for a more detailed understanding of the factors that regulate EGFR signaling (12Sebastian S. Settleman J. Reshkin S.J. Azzariti A. Bellizzi A. Paradiso A. Biochim. Biophys. Acta. 2006; 1766: 120-139PubMed Google Scholar, 13Yu C. Hale J. Ritchie K. Prasad N.K. Irudayaraj J. Biochem. Biophys. Res. Commun. 2009; 378: 376-382Crossref PubMed Scopus (21) Google Scholar).The nanometer scale clustering of receptors in higher order assemblies has attracted increased attention in studies of receptor biology and signal transduction. The functional consequences of receptor assemblies include their potential for inter-receptor communication and increased signaling efficiency, as well as their information processing capacity (14Irvine D.J. Hue K.A. Mayes A.M. Griffith L.G. Biophys. J. 2002; 82: 120-132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Membrane domains such as lipid rafts, caveolae, and clathrin-coated pits provide one mechanism for spatial and temporal control of the assembly of nanoscale signaling domains (15Pontier S.M. Percherancier Y. Galandrin S. Breit A. Galés C. Bouvier M. J. Biol. Chem. 2008; 283: 24659-24672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, progress in understanding the assembly of signaling complexes and their association with membrane domains has so far been hampered by the limited availability of methods for the direct visualization of proteins and complexes with high spatial resolution in the membranes of intact cells.A mechanistic understanding of EGFR activation in a tumor cell environment requires knowledge of the spatial organization of the receptor on the cell surface. Recently single molecule fluorescence and single particle tracking have provided insight on the role of EGFR dimers and oligomers but in most cases do not visualize the entire population of receptors (16Saffarian S. Li Y. Elson E.L. Pike L.J. Biophys. J. 2007; 93: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Sergé A. Bertaux N. Rigneault H. Marguet D. Nat. Methods. 2008; 5: 687-694Crossref PubMed Scopus (410) Google Scholar). We report the use of near field scanning optical microscopy (NSOM) to probe the distribution and nanometer scale associations of EGFR in HeLa cells and to determine the influence of the tyrosine kinase inhibitor AG1478 and 225 EGFR-blocking antibody on the distribution of EGFR. NSOM is one of an emerging group of super-resolution methods for fluorescence imaging with subdiffraction resolution (18Hell S.W. Nat. Methods. 2009; 6: 24-32Crossref PubMed Scopus (821) Google Scholar, 19Lippincott-Schwartz J. Manley S. Nat. Methods. 2009; 6: 21-23Crossref PubMed Scopus (145) Google and is based on an that has Chem. Rev. 1999; PubMed Scopus Google Scholar, A. H. A. A. E. Nat. 2003; 21: PubMed Scopus Google to the NSOM has been used for nanoscale fluorescence imaging of membrane protein clusters with spatial resolution nm in cells A. M. D. J. L.J. Biophys. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. M. L.J. Nat. Chem. Biol. 1: PubMed Scopus Google Scholar, M. A. 2004; PubMed Scopus Google Scholar, C. A. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, C. 2008; PubMed Scopus Google Scholar, F. A. 2007; PubMed Scopus Google Scholar, A. G. S. A. J. Cell Sci. 2008; PubMed Scopus Google Scholar). that EGFR is in clusters in the plasma membrane and that association with lipid raft and domains receptor and in to activation in HeLa cells.

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