Inhee Chung

Spatial control of EGF receptor activation by reversible dimerization on living cells

Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.

Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants

High-affinity transferrin receptor (TfR) bispecific antibodies facilitate trafficking of TfR to lysosomes and induce TfR degradation to decrease the ability of TfR to mediate BBB transcytosis.

Room temperature measurements of the 3D orientation of single CdSe quantum dots using polarization microscopy

Simple far-field emission polarization microscopy reveals that the emission transition dipole of CdSe colloidal quantum dots (QDs) is twofold degenerate at room temperature. We measure, model, and compare polarization anisotropy statistics of CdSe QDs and DiI (a one-dimensional emitter). We find excellent agreement between experiment and theory if the transition dipole of CdSe QDs is assumed to be twofold degenerate. This implies that the three-dimensional orientation of the unique crystal axis in QDs can be determined at room temperature with polarization microscopy. We describe an optical setup to measure four polarization angles of multiple single QDs simultaneously and in real time (≈16 Hz). We use this setup in a proof-of-concept experiment to demonstrate that the rotational motion of QDs can be monitored in various host matrices.

High cell-surface density of HER2 deforms cell membranes.

Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell–substrate and cell–cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner.

Optical measurement of receptor tyrosine kinase oligomerization on live cells

Receptor tyrosine kinases (RTK) are important cell surface receptors that transduce extracellular signals across the plasma membrane. The traditional view of how these receptors function is that ligand binding to the extracellular domains acts as a master-switch that enables receptor monomers to dimerize and subsequently trans-phosphorylate each other on their intracellular domains. However, a growing body of evidence suggests that receptor oligomerization is not merely a consequence of ligand binding, but is instead part of a complex process responsible for regulation of receptor activation. Importantly, the oligomerization dynamics and subsequent activation of these receptors are affected by other cellular components, such as cytoskeletal machineries and cell membrane lipid characteristics. Thus receptor activation is not an isolated molecular event mediated by the ligand-receptor interaction, but instead involves orchestrated interactions between the receptors and other cellular components. Measuring receptor oligomerization dynamics on live cells can yield important insights into the characteristics of these interactions. Therefore, it is imperative to develop techniques that can probe receptor movements on the plasma membrane with optimal temporal and spatial resolutions. Various microscopic techniques have been used for this purpose. Optical techniques including single molecule tracking (SMT) and fluorescence correlation spectroscopy (FCS) measure receptor diffusion on live cells. Receptor-receptor interactions can also be assessed by detecting Förster resonance energy transfer (FRET) between fluorescently-labeled receptors situated in close proximity or by counting the number of receptors within a diffraction limited fluorescence spot (stepwise bleaching). This review will describe recent developments of optical techniques that have been used to study receptor oligomerization on living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Single-Molecule Optical Methods Analyzing Receptor Tyrosine Kinase Activation in Living Cells

Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors.

Abstract 3330: HER2 overexpression induces membrane deformation that increases cell motility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Approximately 20% of breast cancers (BC) are characterized by the gene amplification and overexpression of HER2, a member of the ErbB/HER receptor family. While targeted therapies against HER2 effectively delay disease progression in this BC subtype, details of how overexpressed HER2s drive these tumors to malignancy are still unclear. To better understand this process, we investigated various cellular responses to HER2 overexpression in individual live cells. We developed novel single receptor diffusion analyses that determine the activation status of HER2 by diffusivity, to estimate average HER2 activity per single cell. Surprisingly, we found HER2 overexpression induces membrane deformation, which depends only on the HER2 density, but not the receptor activation status. Moreover, this membrane deformation lowers the available surface area for formation of focal adhesion sites, resulting in reduced cell adhesion and increased cell motility. These findings suggest there are signaling-independent roles of HER2 overexpression in disease progression of HER2 positive BCs. Citation Format: Inhee Chung, Mike Reichelt, Donald Dowbenko, Ira Mellman, Mark Sliwkowski. HER2 overexpression induces membrane deformation that increases cell motility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2014-3330