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Updated: Jul 6, 2026
A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
Published on: March 22, 2012
Real-time cross-correlation image analysis of early events in IgE receptor signaling
Raibatak Das1, Stephanie Hammond, David Holowka
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Insights
Researchers developed an automated imaging method to track early signaling events in mast cells. This technique quantifies protein interactions with IgE receptors after antigen stimulation, revealing Syk
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Mast cells and basophils mediate allergic responses via IgE receptors (FcepsilonRI).
- Receptor crosslinking by antigens triggers degranulation and mediator release.
- Understanding early signaling events is crucial for allergic disease research.
Purpose of the Study:
- To develop and validate an automated image analysis method for quantifying early signaling events in mast cells.
- To investigate the spatio-temporal dynamics of signaling protein recruitment to IgE receptors upon stimulation.
- To assess protein interactions with crosslinked IgE receptors using multicolor fluorescence confocal microscopy.
Main Methods:
- Utilized multicolor fluorescence confocal microscopy to monitor GFP-/CFP-labeled signaling proteins in RBL-2H3 mast cells.
- Employed a fluorescently tagged antigen and cholera toxin B for visualizing plasma membrane and receptor localization.
- Developed an automated image analysis scheme for quantifying protein recruitment and colocalization with crosslinked receptors via cross-correlation analysis.
Main Results:
- The automated method successfully quantified the recruitment of signaling proteins to the plasma membrane.
- Demonstrated time-dependent colocalization of signaling proteins with crosslinked IgE receptors.
- Observed rapid translocation of Syk-CFP to the plasma membrane but limited colocalization with aggregated receptors.
Conclusions:
- The developed automated method provides a quantitative and efficient way to monitor protein interactions during cell signaling.
- This technique is valuable for studying the early molecular events in IgE receptor-mediated signaling pathways.
- The findings offer insights into the dynamic behavior of signaling proteins like Syk in response to receptor activation.
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.
