Date of Award
Fall 2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical Engineering (ENAS)
First Advisor
Levchenko, Andre
Abstract
Proper cell functions are dependent on the precise regulation of signal transduction pathways to relay specific external environmental cues into the cell for processing and yielding a specific response. Dysregulation of signaling function or localization is a hallmark of numerous diseases including neurodegenerative, autoimmune, and immunological disorders and most human cancers. Given that signaling pathways are large networks of nodes capable of interacting in complex manners, a notable question that arises for many studying the intricacies of signaling regulation is: “How is signaling tightly controlled in different subcellular compartments?” Here we examine and elucidate mechanisms that begin to answer this fundamental question in the context of EGF signaling. Conventional techniques have been successful in investigating the functions of individual components and in mapping interactions between signaling components. However, discrete time snapshots of activity in a population of cells or the analysis of single components of signaling pathways outside of their natural biological context leave much information about spatial regulation, cell-cell variability, and time-resolved dynamical information unrevealed. Addressing these limitations, FRET-based biosensors provide a powerful means for dissecting the complexities of signaling pathway components in real-time with single cell resolution in their native live biological context. Utilizing engineered and optimized ERK Activity Reporters (EKARs), we examine ERK activity dynamics in the context of epidermal growth factor (EGF) signaling in PC12 cells, a cell line historical to questions related to signal specificity. We reveal distinct ERK activity dynamics in different subcellular compartments, the presence of switch-like activation of plasma membrane (PM) ERK, and elucidate mechanisms involved in controlling the switch-like activation of PM ERK. Next, we examine how the signaling network topology we uncovered at the PM is coupled (or uncoupled) to cytoplasmic ERK activity. Lastly, we show distinct functional roles for the different pools of ERK activity.
Recommended Citation
Hamidzadeh, Archer, "Elucidating Extracellular Signal-Regulated Kinase (ERK) Dynamics With FRET-based Biosensors" (2022). Yale Graduate School of Arts and Sciences Dissertations. 717.
https://elischolar.library.yale.edu/gsas_dissertations/717
