University of New Mexico
Using fluorescence microscopy to determine the dynamics and composition of the FcεRI signalosome
Abstract: Complex cellular processes are governed by signal transduction, which in turn is controlled by protein-protein interactions at the plasma membrane and along the signaling cascade. Capturing and quantifying these processes is a long-standing goal in cell biology. We have applied single molecule and super-resolution imaging methods to study the protein interactions and dynamics that regulate FcεRI signaling. FcεRI, is the principal multi-subunit immunoreceptor on the surface of mast cells and basophils. Crosslinking of IgE-FcεRI complexes by multivalent allergen initiates complex signaling pathways, leading to the release of mediators of allergic inflammation. Signal initiation is classically attributed to phosphorylation of FcεRI ITAMs by the Src family kinase (SFC) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. We have we investigate the mechanistic contributions of Lyn, Syk and SHIP1 to the formation of the FcεRI signalosome. Using fluorescence microscopy to quantify protein recruitment across ITAM variants with altered phosphorylation profiles, we were able to dissect the structural requirements of Syk and SHIP1 recruitment to the signaling complex. While Lyn is considered to be the key kinase for FcRI activation, we found that other SFKs can compensate for Lyn, albeit less efficiently. However, even in conditions where ITAM phosphorylation is incomplete, Syk can still be recruited to the crosslinked receptor and propagate signaling. Single molecule imaging was used to directly visualize the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. This approach allows us to determine protein interaction off-rates in living cells. We found that Syk colocalizes transiently to FcRI and that Syk-FcεRI binding dynamics and that Syk activity is tightly correlated with the lifetime of these dynamics. We reveal a novel of SHIP1, where the presence of SHIP1 in the signalosome acts to reduce Syk:receptor binding lifetime and Syk phosphorylation. Super-resolution imaging of FcεRI aggregates induced by antigens of varying valency and size has revealed that the organization of receptors within crosslinked aggregates also regulates the formation of the signalosome. Together, this work highlights the ability of ITAM-based signaling to respond to various ITAM-phosphorylation patterns, Syk binding conformations and the presence/absence of key regulatory molecules. These molecular mechanisms are likely applicable across the family of ITAM-bearing receptors and provide insight in to how disparate immunoreceptors can utilize conserved signaling cascades to tailor their cellular outcomes.
Biosketch: Diane Lidke received her PhD in Biophysical Sciences and Medical Physics from the University of Minnesota in 2002. From 2002 to 2005, she worked as a post-doctoral fellow in the laboratory of Thomas Jovin, located at the Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. There, she worked on the application of fluorescence microscopy techniques to the study of EGFR signaling. In 2005, she joined the Pathology department at the University of New Mexico where she is currently a Professor and Vice Chair for Research. Her research integrates the disciplines of biophysics, bio-imaging and quantitative biology to gain new and fundamental understanding of the components and dynamics of cell signaling pathways, with a focus on signaling in cancer and immune cells.