The spatiotemporal organization of ErbB receptors: insights from microscopy

Abstract

Signal transduction is regulated by protein-protein interactions. In the case of the ErbB family of receptor tyrosine kinases (RTKs), the precise nature of these interactions remains a topic of debate. In this review, we describe state-of-the-art imaging techniques that are providing new details into receptor dynamics, clustering, and interactions. We present the general principles of these techniques, their limitations, and the unique observations they provide about ErbB spatiotemporal organization.

Figure 1.

Summary of imaging techniques for quantifying receptor clustering, dynamics, and interactions.

Figure 2.

High-resolution imaging of receptor distribution. (A,B) Transmission electron microscopy images of ErbB1/ErbB2 distribution on SKBR3 membrane sheets. Dual labeling (10-nm and 5-nm gold) allows for mapping of ErbB1 and ErbB2 proximity. Note the localization of ErbB1 to a clathrin-coated pit in (B). Images courtesy of Bridget Wilson created from data based on Yang et al. (2007). (C)–(H) Two-color stochastic optical reconstruction microscopy (STORM) imaging of ErbB1 with respect to actin on Chinese hamster ovary cells stably expressing ErbB1. Diffraction-limited images of actin (C) and ErbB1 (D) as imaged on the basal cell surface using TIRF microscopy. Super-resolution reconstruction overlay (E) of actin (red, phalloidin-AlexaFluor647) and ErbB1 (green, anti-ErbB1 antibody conjugated to Cy3b) showing the subdiffraction resolution attained using STORM and the localization of ErbB1 within the actin cytoskeleton. The reconstruction overlay is shown at increasing zoom (white boxes) to highlight imaging resolution (F,G,H).

Figure 3.

SPT captures dimerization of EGF-bound ErbB1. (A) Images from a time series in which EGF-QD585 (green) and EGF-QD655 (red) are simultaneously tracked. The pixelated image shows the raw data, and the circles indicate the subdiffraction localization of individual receptor molecules. (B) Plot showing the separation over time of the two receptors in (A). Particle trajectories are analyzed using a three-state hidden Markov model based on interparticle distance to distinguish between free (red line), confined (purple line), and dimeric (blue line) receptor populations. In this example, the EGF-bound receptors are seen to undergo repeated (four) dimerization events (see also Low-Nam et al. 2011). (C) Hyperspectral imaging of eight spectrally distinct QDs bound to EGF. Pseudocoloring of the data is generated based on each quantum dot’s (QD) peak spectral wavelength (λ). (D) Three-dimensional particle trajectories (x,y,t) of the corresponding boxed regions in (C). Correlated motion of dimerized receptors can be observed. Color map (right) indicates QD emission peak. (Images from Cutler et al. 2013; reprinted, with permission, from the investigators.)