Journal: PLoS Biology

Article Title: Direct visualization of single-molecule membrane protein interactions in living cells

doi: 10.1371/journal.pbio.2006660

Figure Lengend Snippet: (A) Schematic of the Co-II assay. The interaction between a fluorescently labeled prey protein and a bait protein is specifically probed by the co-immobilized prey produced after antibody-induced immobilization of the bait protein, which is visualized using sptPALM in single living cells. (B) Comparison between a diffusivity-based method (Co-II) and a proximity-based method (e.g., FRET). In the crowded membrane of living cells, Co-II specifically detects genuine interactions between membrane proteins, while the proximity-based methods are vulnerable to producing false positive signals because a prey and a bait are located nearby. Co-II captures membrane protein interactions independent of tag orientation, while the proximity-based methods require a careful design for donor–acceptor orientation. (C) The bait-specific immobilization using a surface-coated antibody in living cells. The immobilized fractions of PMT, EGFR, ErbB2, ErbB3, InsR, and β2-AR in multiple cells before (NT) and after anti-EGFR antibody treatment. Examined membrane proteins were expressed at a level at least 10 times higher than the expression level of EGFR to avoid the specific co-immobilization resulting from the genuine interaction with EGFR. Each dot represents single-cell data, and the red solid lines indicate the average of the immobilized fraction obtained from multiple cells ( n > 10). (D–E) Illustration and trajectory maps for validation of molecule-specific immobilization in the plasma membrane of a living cell. A total of 400 trajectories are shown in each trajectory map. Scale bar, 2 μm. SNAP-EGFR was specifically and almost completely immobilized by anti-EGFR antibody treatment, whereas the immobilized fraction of β2-AR-mEos3.2 was not altered (D). Specific immobilization of β2-AR against EGFR was confirmed vice versa using SNAP-β2-AR and EGFR-mEos3.2 with anti-SNAP antibody (E). β2-AR, beta-2 adrenergic receptor; EGFR, epidermal growth factor receptor; ErbB2, erb-b2 receptor tyrosine kinase 2; ErbB3, erb-b2 receptor tyrosine kinase 3; FRET, fluorescence resonance energy transfer; InsR, insulin receptor; mEos3.2, monomeric Eos fluorescent protein variant 3.2; NT, not treated; PMT, plasma membrane targeting; SNAP, SNAP-tag; sptPALM, single-particle tracking photoactivated localization microscopy.

Article Snippet: The coverslips were treated with anti-phospho EGFR and Alexa 647–labeled secondary antibodies for 30 min. After washing, fluorescence images were obtained to assess the EGFR phosphorylation levels in media supplemented with 5 mM protocatechuic acid (PCA, sc-205818, Santa Cruz), 0.5 U/mL protocatechuate-3,4-dioxygenase (PCD, P8279, Sigma-Aldrich), and 1 mM β-mercaptoethylamine (MEA, 30070, Sigma-Aldrich).

Techniques: Ii Assay, Labeling, Produced, Comparison, Membrane, Expressing, Biomarker Discovery, Clinical Proteomics, Fluorescence, Förster Resonance Energy Transfer, Variant Assay, Single-particle Tracking, Microscopy

Journal: PLoS Biology

Article Title: Direct visualization of single-molecule membrane protein interactions in living cells

doi: 10.1371/journal.pbio.2006660

Figure Lengend Snippet: (A) Schematic representation of the K D measurement of EGFR homodimerization using Co-II. EGFR-mEos3.2 becomes co-immobilized only when interacting with the surface-immobilized SNAP-EGFR by an anti-SNAP antibody; otherwise, it remains in a mobile state. (B) Trajectory map of CF660R-labeled SNAP-EGFR and EGFR-mEos3.2 before and after anti-SNAP antibody treatment in the same single COS7 cell growing with 10% FBS. A total of 200 trajectories are shown in each trajectory map. Scale bar, 3 μm. (C) Diffusion-coefficient distribution of SNAP-EGFR and EGFR-mEos3.2 before (black line) and after anti-SNAP antibody treatment (red line). The immobilization criteria are presented as a blue dashed line. (D) The immobilized fractions of SNAP-EGFR and EGFR-mEos3.2 before and after anti-SNAP antibody treatment. (E) Fluorescence images of total expression and single-molecule–level expression of SNAP-EGFR. Scale bars, 5 μm and 2 μm, respectively. A fluorescence intensity profile of single SNAP-EGFR shows a single bleaching step. (F) K D analysis using a binding curve of prey EGFR to bait EGFR (y-axis) with respect to the density of the antibody-induced immobilized bait EGFR (x-axis). The bound/unbound ratio of the prey with respect to the density is shown (left inset) with a linear fit (red solid line) and a 95% confidence interval (red dashed lines). Scatchard plot for EGFR pre-homodimerization is shown (right inset). The K D was determined in DMEM supplemented with 10% FBS at 37 °C. Each dot indicates data obtained from individual cells. (G) K D of EGFR pre-homodimerization measured in various cell lines. The error bars represent the SEM at the single-cell level ( n > 4). (H) A spatial K D map of EGFR pre-homodimerization and the log-normal distribution of the K D values obtained from different regions of plasma membrane in a single living cell. Scale bar, 5 μm. (I) The K D profiles obtained from the cross sections corresponding to the red dashed lines in panel H. (J) The box plots displaying the distributions of K D values obtained from periphery or center regions of each single cell. n = 10. * p < 0.05 (Student t test). A.U., arbitrary unit; DMEM, Dulbecco's Modified Eagle Medium; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; mEos3.2, monomeric Eos fluorescent protein variant 3.2; SNAP, SNAP-tag.

Article Snippet: The coverslips were treated with anti-phospho EGFR and Alexa 647–labeled secondary antibodies for 30 min. After washing, fluorescence images were obtained to assess the EGFR phosphorylation levels in media supplemented with 5 mM protocatechuic acid (PCA, sc-205818, Santa Cruz), 0.5 U/mL protocatechuate-3,4-dioxygenase (PCD, P8279, Sigma-Aldrich), and 1 mM β-mercaptoethylamine (MEA, 30070, Sigma-Aldrich).

Techniques: Labeling, Diffusion-based Assay, Fluorescence, Expressing, Binding Assay, Clinical Proteomics, Membrane, Modification, Variant Assay

Journal: PLoS Biology

Article Title: Direct visualization of single-molecule membrane protein interactions in living cells

doi: 10.1371/journal.pbio.2006660

Figure Lengend Snippet: (A–B) K D values of EGFR homodimerization measured with and without EGF under the treatment of nonnatural ligands, including a Fab fragment of cetuximab and two types of tyrosine kinase inhibitors, erlotinib and lapatinib, in serum-starved COS7 cells. (C) K D values of homodimerization for EGFR WT (the same data for NT in panel A), EGFRvIII, and EGFR L858R. The error bars represent the SEM at the single-cell level ( n > 10). All the measurements were performed in a serum-free DMEM at 37 °C. * p < 0.05 (Student t test). (D) A scale mapping K D values of EGFR homodimerization under various molecular perturbations. The yellow and green dots indicate the perturbations to EGFR ECD and ICD, respectively, and a black dot indicates no perturbation. Each perturbation site is displayed in the illustration, representing the reaction of EGFR pre-homodimerization with log2 fold change values compared with the K D without perturbation. DMEM, Dulbecco's Modified Eagle Medium; ECD, extracellular domain; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Fab, fragment antigen-binding; ICD, intracellular domain; NT, not treated; WT, wild type.

Article Snippet: The coverslips were treated with anti-phospho EGFR and Alexa 647–labeled secondary antibodies for 30 min. After washing, fluorescence images were obtained to assess the EGFR phosphorylation levels in media supplemented with 5 mM protocatechuic acid (PCA, sc-205818, Santa Cruz), 0.5 U/mL protocatechuate-3,4-dioxygenase (PCD, P8279, Sigma-Aldrich), and 1 mM β-mercaptoethylamine (MEA, 30070, Sigma-Aldrich).

Techniques: Modification, Binding Assay

Journal: PLoS Biology

Article Title: Direct visualization of single-molecule membrane protein interactions in living cells

doi: 10.1371/journal.pbio.2006660

Figure Lengend Snippet: K D values of EGFR and β2-AR homodimerizations were determined by Co-II under the existence of their ligands (EGF and ISO, respectively) and the sequestration of cholesterol in a plasma membrane. The scale mapping K D values for their homodimerizations are displayed for direct comparisons. β2-AR, beta-2 adrenergic receptor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ISO, isoproterenol; NT, not treated; SNAP, SNAP-tag.

Article Snippet: The coverslips were treated with anti-phospho EGFR and Alexa 647–labeled secondary antibodies for 30 min. After washing, fluorescence images were obtained to assess the EGFR phosphorylation levels in media supplemented with 5 mM protocatechuic acid (PCA, sc-205818, Santa Cruz), 0.5 U/mL protocatechuate-3,4-dioxygenase (PCD, P8279, Sigma-Aldrich), and 1 mM β-mercaptoethylamine (MEA, 30070, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane