Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: Application to the EGF receptor

Background: The molecular analysis of intracellular signal transduction requires technologies that address quantitatively the activation of signaling proteins and formation of molecular complexes without disrupting cellular integrity. Methods: Cells expressing the epidermal growth factor receptor (EGFR) in its endogenous form or fused to green fluorescent protein were incubated with 1-mum microspheres covalently functionalized with EGF. The disposition of the plasma membrane about the microspheres was analyzed by high-resolution confocal microscopy in combination with computational resolution enhancement and optimized fixation procedures. Receptor activation and translocation of signaling proteins to the microspheres was quantitated by image processing protocols for recovering the microsphere-associated fluorescence and the fluorescence in the local environment. Results: EGF-functionalized microspheres were internalized in an activation-dependent manner similar to that of the soluble growth factor. The correlation of receptor activation and recruitment of a signaling protein was analyzed quantitatively by isolating immunofluorescence signals from the microspheres and from their immediate environment. Conclusions: The microsphere-based approach provides a quantitative analysis of cellular signal transduction with subcellular resolution under conditions maintaining cellular integrity. The analysis of signaling-induced (co)localization of proteins around a microsphere complements other technologies directly probing for molecular interactions such as fluorescence resonance energy transfer. (C) 2003 Wiley-Liss, Inc.