Exploring in vivo cholesterol-mediated interactions between activated EGF receptors in plasma membrane with single-molecule optical tracking

Abstract

Background: The first step in many cellular signaling processes occurs at various types of receptors in the plasma membrane. Membrane cholesterol can alter these signaling pathways of living cells. However, the process in which the interaction of activated receptors is modulated by cholesterol remains unclear.

Methods: In this study, we measured single-molecule optical trajectories of epidermal growth factor receptors moving in the plasma membranes of two cancerous cell lines and one normal endothelial cell line. A stochastic model was developed and applied to identify critical information from single-molecule trajectories.

Results: We discovered that unliganded epidermal growth factor receptors may reside nearby cholesterol-riched regions of the plasma membrane and can move into these lipid domains when subjected to ligand binding. The amount of membrane cholesterol considerably affects the stability of correlated motion of activated epidermal growth factor receptors.

Conclusions: Our results provide single-molecule evidence of membrane cholesterol in regulating signaling receptors. Because the three cell lines used for this study are quite diverse, our results may be useful to shed light on the mechanism of cholesterol-mediated interaction between activated receptors in live cells.

Keywords: Diffusion; Epidermal growth factor receptor; Live cell; Plasma membrane; Single-molecule trajectory.

Fig. 1

Schematic diagram of epidermal growth factor receptors (EGFRs) in the environment of plasma membrane. EGF-Qdot585 is a fluorescent epidermal growth factor (EGF) synthesized by conjugating EGF with a quantum dot Qdot585. Here cholesterol molecules are shown in yellow, the pink lipids represent the raft lipid species, and the dark green lipids are nonraft lipids

Fig. 2

An EMCCD image of single-molecule EGFRs in a living cell taken with an exposure time of 25 ms; Three typical trajectories of EGFRs diffusing in a confined region were extracted from measured trajectories using the confinement quantification procedure [30]

Fig. 3

Two-dimensional contour plot on the $V\left(\overline{{R_{\tau }}^2}\right)$-$\overline{{R_{\tau }}^2}$ plane for Qdot585-Ab-CD59 diffusing in the plasma membrane of (a) native HeLa cells and (b) M βCD-pretreated HeLa cells

Fig. 4

a, b, c Histograms of mean-square displacements (MSD) and (d, e, f) $V\left(\overline{{R_{\tau }}^2}\right)$-$\overline{{R_{\tau }}^2}$ plots of unliganded EGFR (Qdot585-Ab-EGFR) diffusing in the plasma membrane of (a, d) native cells, (b, e) nystatin-pretreated cells, and (c, f) M βCD-pretreated cells. Trajectories were sampled with a frame period of τ=25 ms. Data are shown in red for HeLa cells, green for A431 cells, and blue for MCF12A cells

Fig. 5

a, b, c Histograms of MSDs and (d, e, f) $V\left(\overline{{R_{\tau }}^2}\right)$-$\overline{{R_{\tau }}^2}$ plots of liganded EGFR (Qdot585-EGF-EGFR) diffusing in the plasma membrane of (a, d) native cells, (b, e) nystatin-pretreated cells, and (c, f) M βCD-pretreated cells. Trajectories were sampled with a frame period of τ=25 ms. Data are shown in red for HeLa cells, green for A431 cells, and blue for MCF12A cells

Fig. 6

a, b, c Histograms of MSDs and (d, e, f) $V\left(\overline{{R_{\tau }}^2}\right)$-$\overline{{R_{\tau }}^2}$ plots of correlated Qdot585-EGF-EGFRs diffusing in the plasma membrane of (a, d) native cells, (b, e) nystatin-pretreated cells, and (c, f) M βCD-pretreated cells. Trajectory segments with a degree of correlation exceeding 0.8 were analyzed. Trajectories were sampled with a frame period of τ=25 ms. Data are shown in red for HeLa cells, green for A431 cells, and blue for MCF12A cells

Fig. 7

Schematic representation of correlated EGFRs (green) in a cholesterol (yellow) enriched lipid domain (center) and in a cholesterol-depleted nonraft lipid domain (right), drawn to illustrate the conclusion of this study. The red object on EGFR denotes the EGF ligand. Cholesterol molecules increase receptor-receptor interaction and thus promote the stability of correlatively moving EGFRs