Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling.
The allergic response is initiated on the plasma membrane of mast cells by phosphorylation of the receptor for immunoglobulin E (IgE), FcεRI, by Lyn kinase after IgE-FcεRI complexes are cross-linked by multivalent antigen. Signal transduction requires reorganization of receptors and membrane signaling proteins, but this spatial regulation is not well defined. We used fluorescence localization microscopy (FLM) and pair-correlation analysis to measure the codistribution of IgE-FcεRI and Lyn on the plasma membrane of fixed cells with 20- to 25-nm resolution. We directly visualized Lyn recruitment to IgE-FcεRI within 1 min of antigen stimulation. Parallel FLM experiments captured stimulation-induced FcεRI phosphorylation and colocalization of a saturated lipid-anchor probe derived from Lyn's membrane anchorage. We used cytochalasin and latrunculin to investigate participation of the actin cytoskeleton in regulating functional interactions of FcεRI. Inhibition of actin polymerization by these agents enhanced colocalization of IgE-FcεRI with Lyn and its saturated lipid anchor at early stimulation times, accompanied by augmented phosphorylation within FcεRI clusters. Ising model simulations provide a simplified model consistent with our results. These findings extend previous evidence that IgE-FcεRI signaling is initiated by colocalization with Lyn in ordered lipid regions and that the actin cytoskeleton regulates this functional interaction by influencing the organization of membrane lipids.
Department of Chemistry and Chemical Biology and Field of Biophysics, Cornell University, Ithaca, NY 14853.
Sarah A
SA
Shelby
Department of Biophysics, University of Michigan, Ann Arbor, MI 48109.
Sarah L
SL
Veatch
Department of Chemistry and Chemical Biology and Field of Biophysics, Cornell University, Ithaca, NY 14853.
David A
DA
Holowka
Department of Chemistry and Chemical Biology and Field of Biophysics, Cornell University, Ithaca, NY 14853 bab13@cornell.edu.
Barbara A
BA
Baird
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D003198:Computer Simulation; D007073:Immunoglobulin E / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D017455:Receptors, IgE / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism*
D017455:Receptors, IgE; D007073:Immunoglobulin E; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases
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