Abstract
To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of = |0.0054±0.0064| µm2/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions.
Highlights
The signaling activity of members of the epidermal growth factor receptor family, which comprises the receptors ErbB1-4 [1,2], does depend on the association of the receptors into discrete molecular species, such as dimers [3,4] and potentially higher order oligomers [5,6,7], and on the self-organization of the receptors on longer spatial length scales [8]
The average separation of multiple NPs co-confined in one membrane domain depends on the size and shape of the confinement, and we show that the ability to monitor the configurational dynamics of the NP clusters within the membrane domains provides detailed structural information as function of time and location on the cell surface
Optimization of NP Binding Affinity on Living Cells Our rational for using plasmon coupling to localize plasma membrane domains enriched in ErbB1 receptors on epidermoid carcinoma cells (A431) is that areas of high local receptor concentration exhibit a higher binding affinity for immunolabels than adjacent areas with lower receptor concentration [11,12]
Summary
The signaling activity of members of the epidermal growth factor receptor family, which comprises the receptors ErbB1-4 [1,2], does depend on the association of the receptors into discrete molecular species, such as dimers [3,4] and potentially higher order oligomers [5,6,7], and on the self-organization of the receptors on longer (i.e. tens to hundreds of nanometer) spatial length scales [8]. The exact relationship between the topography of the ErbB receptor enrichment and the signaling activity is not accurately understood, it is clear that the geometric size, shape, and number of receptors of individual signaling domains determine the local receptor density. The latter will influence the receptor collision rate and could, have direct implications for the signaling activity, for instance, by shifting the local receptor association levels. It has been reported that the ErbB family members are enriched in spontaneously formed membrane compartments (‘‘lipid rafts’’) [25–
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