Nanoscience and Microsystems ETDs

Publication Date



Healthy immune cell behavior requires sensitive and robust control over the processes that regulate signal transduction. In this work we employ single molecule fluorescence imaging techniques to quantify adapter protein recruitment, lateral mobility, receptor aggregation, and cytoskeletal organization to create a better understanding of many key processes in immune cell regulation. We focus on understanding the initiating events in FceRI signaling in mast cells. Mast cell signaling encompassing a wide array of cellular outcomes including calcium flux, release of pre-formed inflammatory mediators and the production of cytokines. Careful control over appropriate reactions to external antigens is necessary for mast cells to properly function within the context of an immune response. The antigen binding and signaling domains of FceRI are carried on different subunits. Because no studies to date have thoroughly investigated the integrity of the FceRI complex, it remains unclear how antigen binding regulates the dynamics and spatio-temporal distribution of the intracellular signaling ITAM domains. We address this by quantifying the dynamics and localization of the of the FceRI-gamma subunit relative to the FceRI-alpha subunit. We utilize a Fluorogen Activating Protein (FAP) tag to fluorescently label the FceRI-gamma subunit, and demonstrate its versatility as a novel probe for single particle tracking. Direct labeling of the FceRI-gamma also allows us to address long standing questions regarding the influence of IgE binding on receptor behavior. Given our understanding for how antigen addition regulates FceRI signaling domains, we next explore how FceRI aggregation regulates its association with the intracellular tyrosine kinase Syk. Proper initiation of tyrosine kinase activity is absolutely necessary for all receptor dependent signaling, yet it is not completely understood how Syk function is regulated. In this work we use single molecule imaging to follow individual Syk molecules at the plasma membrane and investigate how FceRI distribution influences its spatial and temporal behavior. We combine these findings with biochemical readouts to correlate the behavior of Syk with functional consequences.\\\\\\\\ In addition to investigating FceRI signaling, we also demonstrate how localization based super-resolution imaging can provide new insights into immune cell biology. First, we report on the development of FAP as a novel probe for super-resolution imaging and live cell super-resolution imaging of intracellular structures (specifically the actin cytoskeleton). Second, we use super-resolution imaging to quantify the nanoscale aggregation of FcgRI and assess the influence of cytokine stimulation on FcgRI distribution. Third, we use super-resolution imaging to explore the nanoscale architecture of the podosome mechano-sensory assembly in dendritic cells.


IgE Signaling, Single Molecule Flourescence Microscopy, Super-resolution Fluorescence Microscopy, Mast cell Signaling

Document Type




Degree Name

Nanoscience and Microsystems

Level of Degree


Department Name

Nanoscience and Microsystems

First Advisor

Lidke, Diane

First Committee Member (Chair)

Wilson, Bridget

Second Committee Member

Wandinger-Ness, Angela

Third Committee Member

Lidke, Keith

Available for download on Wednesday, May 16, 2018