Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling

<div><p>Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca²⁺ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization.</p></div

Participation of nPKC and PKD1 in the feedback monomerization of the EGFR.

<p>(A) Time traces of diffusion coefficient of EGFR^wt in the presence of the PKC activator PMA (upper) or under nominally Ca²⁺-free conditions (lower panel). In red, the time trace of cytosolic Ca²⁺ concentration monitored with Fura Red is shown. (B) Time traces of diffusion coefficient of eGFP-EGFR^wt in the cell coexpressing constitutively active mutant, mCherry-PKD1^S738/742E (upper), in the cell pre-treated with 10 μM CID755673, PKD1 inhibitor (middle), or in the cell coexpressing kinase deficient mutant, mCherry-PKD1^K612W (lower panel).</p

Mutational study of the EGFR monomer-dimer equilibrium at the JM domain phosphorylation sites.

<p>^a A and E stand for the phosphodeficient and phosphomimic mutation (alanine and glutamate substitution, respectively).</p><p>^b The state towards which the monomer-dimer equilibrium is shifted after EGF addition.</p><p>^{c, d} Minimum (D_min) and maximum (D_max) level of the diffusion coefficient.</p><p>^e Not determinable.</p><p>Mutational study of the EGFR monomer-dimer equilibrium at the JM domain phosphorylation sites.</p

Repetitive change in the monomer to dimer ratio of EGFR revealed by RICS analysis.

<p>(A) (left) Plasma membrane localization of eGFP-EGFR^wt in CHO-K1 cells. Confocal images of the basal plasma membrane before EGF addition. The bottom and right panels show cross sections of the cell. The scale bar indicates 10 μm. (right) Example of 50 frames of eGFP-EGFR in CHO-K1 basal plasma membrane used to calculate the RICS auto-correlation. The scale bar indicates 5 μm. (B) RICS auto-correlation (left) of eGFP-EGFR^wt in CHO-K1, calculated for a time bin of 50 frames (for ξ and ψ from -16 to 16 pixels, respectively), fitted to the 2D diffusion model with residual plot (right). The residuals of the fitting being less than 10% of the magnitude of the function indicate the appropriate fit. (C) Reproducible time trace of eGFP-EGFR^wt diffusion coefficient. At the t = 0 EGF was added. Each point represents one time bin in RICS analysis, consisted of 50 frames which corresponds to 83 s, with 5 frame (8.3 s) interval. The EGFR monomer to dimer ratio at the regions indicated by arrows was calculated by N&B analysis. The experiment was repeated 20 times with essentially the same result. (D) Schematic structure of eGFP-EGFR monomer and dimer based on following PDB files: 3EVP, 1EGF, 2JWA, 1M17, 2M20, 2GS2 and 3GOP. EGF and eGFP are shown in red and green, respectively. 2JWA structure based on ErbB2 was used to display transmembrane helix dimer. (E) N&B analysis of eGFP-EGFR^wt before (left) and after (right) EGF addition (indicated with red boxes in C). Histograms of apparent molecular brightness are shown. Green and blue lines represent the distribution of the EGFR monomer (ε ≈ 0.1, B ≈ 1.1) and dimer (ε ≈ 0.2, B ≈ 1.2), respectively; red line shows the cumulative distribution of both monomer and dimer.</p

Thermocapillary Fingering in Surfactant-Laden Water Droplets

The drying of sessile droplets represents an intriguing problem, being a simple experiment to perform but displaying complexities that are archetypical for many free surface and coating flows. Drying can leave behind distinct deposits of initially well dispersed colloidal matter. For example, in the case of the coffee ring effect, particles are left in a well-defined macroscopic pattern with particles accumulating at the edge, controlled by the internal flow in the droplet. Recent studies indicate that the addition of surfactants strongly influences this internal flow field, even reversing it and suppressing the coffee ring effect. In this work, we explore the behavior of droplets at high surfactant loadings and observe unexpected outward fingering instabilities. The experiments start out with droplets with a pinned contact line, and fast confocal microscopy is used to quantify a radially outward surfactant-driven Marangoni flow, in line with earlier observations. However, the Marangoni flows are observed to become unstable, and local vortex cells are now observed in a direction along the contact line. The occurrence of these vortices cannot be explained on the basis of the effects of surfactants alone. Thermal imaging shows that thermocapillary effects are superimposed on the surfactant-driven flows. These local vortex cells acts as little pumps and push the fluid outward in a fingering instability, rather than an expected inward retraction of the drying droplet. This leads to a deposition of colloids in a macroscopical flower-shaped pattern. A scaling analysis is used to rationalize the observed wavelengths and velocities, and practical implications are briefly discussed

ERK activity correlates with the level of EGFR dimerization.

<p>ERK activity was monitored with EKAREV. (A) Time traces of the normalized fluorescence intensity ratio of the acceptor over the donor channel (dots) fitted to the sigmoid function (lines). Sample curves, showing the median values of sigmoid gain for EGFR^T654/669A (blue), EGFR^{Y992/1148/1173F} (grey), EGFR^wt (red), and EGFR^T654/669E (green) are shown. (B) Sigmoid gains as indices of the ERK activity. The data are means ± SD. The symbol * indicates significant differences with p < 0.005 calculated using a two sided T-test with unequal variance.</p

Mutational study of the EGFR feedback monomerization at the PLCγ binding sites.

<p>^a F stands for the phosphodeficient mutation (phenylalanine substitution).</p><p>^b The strength of negative feedback scaled from as strong as for the EGFR^wt (+ +) to none (—).</p><p>^{c, d} Minimum (D_min) and maximum (D_max) level of the diffusion coefficient.</p><p>^e The diffusion coefficient averaged over the whole measurement time after EGF addition (D_avg).</p><p>^f Not determinable.</p><p>Mutational study of the EGFR feedback monomerization at the PLCγ binding sites.</p

Excitation Polarization Sensitivity of Plasmon-Mediated Silver Nanotriangle Growth on a Surface

In this contribution, we report an effective and relatively simple route to grow triangular flat-top silver nanoparticles (NPs) directly on a solid substrate from smaller NPs through a wet photochemical synthesis. The method consists of fixing small, preformed nanotriangles (NTs) on a substrate and subsequently irradiating them with light in a silver seed solution. Furthermore, the use of linearly polarized light allows for exerting control on the growth direction of the silver nanotriangles on the substrate. Evidence for the role of surface plasmon resonances in governing the growth of the NTs is obtained by employing linear polarized light. Thus, this study demonstrates that light-induced, directional synthesis of nanoparticles on solid substrates is in reach, which is of utmost importance for plasmonic applications

Proposed mechanism of EGFR feedback monomerization upon EGF stimulation.

<p>After EGF challenging, two EGFR monomers associate on the plasma membrane to form an asymmetric dimer. The asymmetric dimer formation activates the kinase domain which transphosphorylates tyrosine residues at the C-termini of the receptor, three of which (pY1173, pY1148, pY992) recruit PLCγ1. EGFR phosphorylates PLCγ1 on Y783 for activation. PLCγ1 hydrolyses PIP₂ forming DAG. DAG activates nPKCs, which phosphorylates PKD at S744 and S748 for activation. PKD causes EGFR phosphorylation at T654 and/or T669 to shift the monomer-dimer equilibrium of liganded EGFR back towards the monomer. The insets show the close-up of the JM part. The two PDB files (2M20, 3GOP) were superimposed and aligned in the JM-A region to obtain presented images.</p

Participation of PLCγ1 in the feedback monomerization of the EGFR.

<p>(A) ccRICS analysis of PLCγ1 binding to EGFR upon EGF challenge. 2D cross-correlation of eGFP-EGFR^wt and mCherry-PLCγ1^wt in CHO-K1 cells before (left) and after (right) EGF addition. (B) Cross-correlation index (CC_i) between mCherry-PLCγ1, eGFP-EGFR, and their mutants before (white) and after (grey) EGF addition. Lyn-mCherry + eGFP (1) and lyn-mCherry + lyn-eGFP (2) were used as negative controls (CTRL). Data are means ± SD. Symbol * indicates significant difference with p < 0.005 calculated using a two sided T-test with unequal variance. (C) Time traces of diffusion coefficient of eGFP-EGFR^{Y992/1148/1173F}, lacking PLCγ binding sites in the upper panel, and eGFP-EGFR^wt coexpressed with mCherry-PLCγ1^Y783F (inactive mutant, lower panel).</p