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Abstract:
Background: The molecular analysis of intracellular signal transduction requires technologies that quantitatively address the activation of signaling proteins and formation of molecular complexes without disrupting the cellular integrity.
Methods: Cells expressing the epidermal growth factor receptor (EGFR) or a GFP fusion protein thereof were incubated with 1 mm 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 recovering the microsphere-associated fluorescence as well as the fluorescence in the local environment.
Results: EGF-functionalized microspheres were internalized in an activation-dependent similar to that of the soluble growth factor. The correlation of receptor activation and recruitment of a signaling protein was quantitatively analyzed by isolating immunofluorescence signals from the microspheres as well as from the immediate environment of the microspheres.
Conclusions: The microsphere-based approach enables the quantitative analysis of cellular signal transduction with subcellular resolution under conditions maintaining cellular integrity. The analysis of signaling-induced colocalization of proteins about a microsphere complements other technologies directly probing for molecular interactions such as fluorescence resonance energy transfer.
