Cholesterol Dictates the Freedom of EGF Receptors and HER2 in the Plane of the Membrane

Abstract

The flow of information through the epidermal growth factor receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, human epidermal growth factor receptor 2 (HER2), in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its depolymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin depolymerization partially restored receptor mobility in cholesterol-depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possibly by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.

Submicrometer and Nanoscale Inorganic Particles Exploit the Actin Machinery To Be Propelled along Microvilli-like Structures into Alveolar Cells

“…The single-molecule fluorescence technique was described earlier in detail. 43 Briefly, three criteria were used to identify individual fluorophores. (1) The size of the fluorescent spot should www.acsnano.org be at the diffraction limit, which is about half of the excitation wavelength, or 3ϫ3 pixels (300 nm 2 ), under our experimental conditions.…”

The impact of high density receptor clusters on VEGF signaling

“…Experimental and theoretical investigation of EGF binding [3,9,17] emphasized the importance of spatial distribution of receptors. Ample experimental evidence indicates that EGF receptors can have a highly inhomogeneous distribution characterized by small areas of high density [30], and exhibit anomalous diffusion [20]. There are other examples of receptors that exhibit clustering and anomalous diffusion [1].…”

“…The current picture [28] is more structured, with microdomains of lipids and proteins [6,13,24]. Modern microscopy techniques [22,29] provide direct evidence of the effect of these structures on membrane receptor localization and movement [1,18,20], revealing receptor clusters in static images, and intervals of confinement in small areas separated by jumps or "hops" in single particle tracking.…”

Single-Molecule Study of Lateral Mobility of Epidermal Growth Factor Receptor 2/HER2 on Activation

“…Many single molecule studies on signaling receptors have offered new insights into their localization, assembly, and activation during the singal transduction process. [18][19][20][21][22][23][24][25][26] In these studies, a decrease in receptor lateral mobility after receptor activation is usually observed. For example, single-molecule imaging of small G protein Ras revealed that Ras diffusion slowed down on activation.…”