PMID:18326662

Citation	Das, R, Hammond, S, Holowka, D and Baird, B (2008) Real-time cross-correlation image analysis of early events in IgE receptor signaling. Biophys. J. 94:4996-5008
Abstract	Signaling in mast cells and basophils is mediated through IgE and its high affinity cell surface receptor, FcepsilonRI. Crosslinking of the receptors by a cognate multivalent antigen leads to degranulation and release of mediators of the allergic immune response. Using multicolor fluorescence confocal microscopy, we probed the spatio-temporal dynamics of early events in the IgE receptor signal cascade. We monitored the recruitment of GFP-/CFP-labeled signaling proteins by acquiring sequential images with time resolution of 3 s during stimulation of RBL-2H3 mast cells with multivalent antigen. A fluorescent tag on the antigen allowed us to visualize the plasma membrane localization of crosslinked receptors, and fluorescent cholera toxin B served as a plasma membrane marker. We developed an automated image analysis scheme to quantify the recruitment of fluorescent intracellular proteins to the plasma membrane and to assess the time-dependent colocalization of these and other membrane-associated proteins with crosslinked receptors as measured by cross-correlation between the plasma membrane distributions of the two fluorophores. This automated method permits analysis of thousands of individual images from multiple experiments for each cross-correlation pair. We systematically applied this analysis to characterize stimulated interactions of IgE receptors with several signaling proteins, including the tyrosine kinases Lyn and Syk, and the adaptor protein LAT. Notably, for Syk-CFP we observed a rapid stimulated translocation to the plasma membrane but very little colocalization with aggregated receptors. Our results demonstrate the utility of this simple, automated method to monitor protein interactions quantitatively during cell signaling.
Links	PubMed PMC2397348 Online version:10.1529/biophysj.107.105502
Keywords	Animals; Cell Line; Computer Systems; Mast Cells/metabolism; Microscopy, Fluorescence/methods; Protein Interaction Mapping/methods; Rats; Receptors, IgE/metabolism; Regression Analysis; Signal Transduction/physiology; Statistics as Topic
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RAT:LYN	part_of	GO:0045121: membrane raft	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete

References

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