Biomedical Sciences ETDs

Publication Date

5-1-2011

Abstract

The high affinity IgE receptor (FcεRI) plays a primary role in the pathogenesis of allergic disease and shares significant similarities with the two other multichain immune recognition receptor family members, the B-cell receptor and T-cell receptor. A wealth of information exists in all three of these receptor systems with regard to the signaling cascades occurring subsequent to receptor activation. It is also known that all three require binding of multivalent antigen to initiate signaling. However, very little is known about the precise mechanism by which multivalent antigen binding initializes downstream signaling. It has long been known that, in response to antigen binding, FcεRI reorganizes into large aggregates on the cell surface and that the receptor transitions from freely diffusing to highly immobile. The extent of aggregation and immobilization appears to correlate strongly with the extent of cellular activation, as measured by release of pre-formed mediators of allergic inflammation from intracellular granules. These observations have fueled speculation that immobilization of FcεRI may be the primary driver behind signal initiation. However, technical limitations related to the challenges of imaging highly dynamic, nanometer scale phenomena in living cells has precluded detailed examination of these processes. Here, we describe the development of novel live cell imaging techniques and quantum dot (QD) based probes to address the role played by receptor dynamics in FcεRI signaling. Using multi-color single QD tracking, we rigorously quantified the diffusion of FcεRI in the absence of multivalent antigen and discovered a novel role for the actin cytoskeleton in modulating the diffusion of transmembrane proteins on micron length scales. We developed a real-time assay to monitor the kinetics of antigen-induced immobilization of FcεRI and report that this process is influenced by the actin cytoskeleton and heavily dependent on multivalent antigen concentration. We describe the relationship between immobilization, clustering and signal intiation and demonstrate that immobilization is not required for robust signaling. We also show that antigen-induced aggregation and internalization of FcεRI is not dependent on downstream signaling. From these data, we propose that the size of receptor clusters alone dictates the mobility, signaling competence, and internalization of FcεRI.

Keywords

IgE, high affinity IgE receptor, cytoskeleton, actin, quantum dots, single particle tracking

Sponsors

University of New Mexico School of Medicine MD/PhD Program, The National Institutes of Health, The National Science Foundation

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Advisor

Lidke, Diane

First Committee Member (Chair)

Oliver, Janet

Second Committee Member

Wilson, Bridget

Third Committee Member

Shuttleworth, C. William

Fourth Committee Member

Boyle, Timothy

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