Single-molecule imaging of EGFR signalling on the surface of living cells

Sako, Yasushi; Minoghchi, Shigeru; Yanagida, Toshio

doi:10.1038/35004044

Cited by 832 publications

(669 citation statements)

References 26 publications

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“…Previous studies have indicated that EGFR might exist as preformed dimers in the absence of ligands [43][44][45]. From our data analyses, we also find that EGFR form clusters without ligand engagement ( Figures 1A and 3C) and the clusters contain from a few to a dozen of EGFR proteins ( Figures 1F and 3F).…”

Section: Discussionsupporting

confidence: 64%

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

et al. 2014

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The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

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Section: Discussionsupporting

confidence: 64%

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

et al. 2014

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“…Gefitinib, an EGFR tyrosine kinase inhibitor, is a useful targeted therapeutic agent for non-small cell lung cancer (Gandara et al, 2004). EGFR bound its ligands, was dimerized and phosphorylated, which activates EGFR signaling cascade, leading to the induction of cell proliferation (Sako et al, 2000). Gefitinib blocks the signal transduction via EGFR by inhibiting its autophosphorylation and thus blocking downstream intracellular signaling.…”

Section: Discussionmentioning

confidence: 99%

Potential use of an anticancer drug gefinitib, an EGFR inhibitor, on allergic airway inflammation

Hur

Lee²,

Lee³

et al. 2007

Exp Mol Med

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The EGFR plays an essential role in goblet cell hyperplasia and mucus hypersecretion. EGFR has an intrinsic tyrosine kinase activity that, when activated, induces the production of MUC5AC through the signaling kinase cascade in the airway epithelium. We have investigated the effects of an EGFR tyrosine kinase inhibitor, gefitinib, on ovalbumin (OVA)-induced, allergic inflammation in airway epithelia of mice. OVA-sensitized mice were pretreated with gefitinib at two different doses (12.5 and 50 mg/kg) and then challenged with OVA. The OVA challenge increased the total cell count and eosinophil count in bronchoalveolar lavage fluid (BALF), as well as the concentrations of T-helper2 (Th2) cytokines, such as IL-4 and IL-13, overall eosinophil recruitment in the lung tissue and airway hyperresponsiveness (AHR). Pretreatment with gefitinib reduced the inflammatory cell counts and released cytokine concentrations (IL-4 and IL-13) in BALF, as well as eosinophil recruitment in the lungs and AHR, in a dose-dependent manner. This was associated with decreased EGFR and Akt phosphorylation. We showed that gefitnib inhibits EGFR and phosphoinositol 3'-kinase (PI3K)/Akt activation which were activated in OVA sensitized mice. These findings suggest that inhibitors of the EGFR cascade may have a role in the treatment of asthma.

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“…For the sake of completeness, we recall here the definition of this ratio, used in alternating laser configurations (8) In this work, F FRET will be replaced in eq 8 by F D A (eq 2) for simplicity.…”

Section: )mentioning

confidence: 99%

Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and Experiments

et al. 2006

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We describe a simple approach and present a straightforward numerical algorithm to compute the best fit shot-noise limited proximity ratio histogram (PRH) in single-molecule fluorescence resonant energy transfer diffusion experiments. The key ingredient is the use of the experimental burst size distribution, as obtained after burst search through the photon data streams. We show how the use of an alternated laser excitation scheme and a correspondingly optimized burst search algorithm eliminates several potential artifacts affecting the calculation of the best fit shot-noise limited PRH. This algorithm is tested extensively on simulations and simple experimental systems. We find that dsDNA data exhibit a wider PRH than expected from shot noise only and hypothetically account for it by assuming a small Gaussian distribution of distances with an average standard deviation of 1.6 Å. Finally, we briefly mention the results of a future publication and illustrate them with a simple two-state model system (DNA hairpin), for which the kinetic transition rates between the open and closed conformations are extracted.

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Single-molecule imaging of EGFR signalling on the surface of living cells

Cited by 832 publications

References 26 publications

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

Potential use of an anticancer drug gefinitib, an EGFR inhibitor, on allergic airway inflammation

Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and Experiments

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