Membrane-Protein Dynamics: New Perspectives in FcepsilonRI Signaling through Single Particle Tracking

Author

Patrick Cutler

Publication Date

12-1-2013

Abstract

Under physiological conditions IgE binds to its high affinity receptor, FcεRI. The aggregation of IgE-FcεRI by crosslinker initiates intracellular signaling resulting in the release of histamines and other cellular mediators that are the basis for allergic disease. In the field of IgE-FcεRI, receptor immobilization was thought to trigger signaling. Recent single particle tracking (SPT) experiments presented evidence supporting the hypothesis that small mobile aggregates are signaling competent. SPT is an important method for investigating spatio-temporal relationships at the subcellular level. New SPT tools are necessary to adequately investigate this hypothesis. The studies presented here both develop and apply tools for investigating IgE-FcεRI. A Bayesian based approach for the estimation of diffusion coefficients for SPT is presented. The diffusion estimator accounts for several experimental realities including finite camera exposure time, trajectory intermittency, and variable localization uncertainty. Derivation of the method and applications to both real and simulated data are presented. This method provides the capacity to accurately estimate a diffusion coefficient with a credible interval for small sets of data. In order to better interrogate the aggregation of IgE-FcεRI a high speed hyperspectral line-scanning microscope was developed to perform multi-color SPT (mcSPT) experiments at ~30 fps with up to 8 spectrally distinct colors of quantum dots (QDs). A full description the microscope and software necessary for mcSPT are included in this work. Additionally, several applications of the hyperspectral microscope are presented to highlight its capabilities. The dynamics of IgE-FcεRI aggregates are interrogated using the hyperspectral microscope by perform mc SPT with 5- colors of QD probes. Surprisingly, many transient aggregates of QD-IgE are observed. Corroborating this observation is the observation that transient interactions of a QD crosslinker analog (DNP-QD) are also observed. Multi-color single molecule imaging of 4-colors of DNP-QD with 3-colors of Alexa Fluor IgE are used to characterize the valency of DNP-QD. Finally, signaling competency of a crosslinked aggregate is observed in SPT experiments with DNP-QD and Syk-GFP. This set of experiments provides significant evidence supporting the previous hypothesis and extends it to include the signaling competency of transient interactions.

Keywords

IgE, FcepsilonRI, membrane protein dynamics, single particle tracking, fluorescence microscopy, hyperspectral microscopy

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Lidke, Keith

Second Committee Member

Wilson, Bridget

Third Committee Member

Haaland, David

Recommended Citation

Cutler, Patrick. "Membrane-Protein Dynamics: New Perspectives in FcepsilonRI Signaling through Single Particle Tracking." (2013). https://digitalrepository.unm.edu/biom_etds/76