Publication Date

2015-08-07

Availability

Embargoed

Embargo Period

2017-08-06

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2015-06-09

First Committee Member

Akira Chiba

Second Committee Member

Vance Lemmon

Third Committee Member

Michael D. Kim

Fourth Committee Member

Gerhard P. Dahl

Fifth Committee Member

Hans P. Larson

Sixth Committee Member

Rodney Murphey

Abstract

Obtaining high-resolution information from a complex network while maintaining its natural context is a key challenge in biology. This is especially true of molecular networks, largely governed by protein-protein interactions, which underlie life’s dynamic processes. Despite the invaluable information they have provided, genetic interaction screens and biochemical assays lack either evidence of direct protein association or an intact endogenous environment, respectively. As a result, the static interaction maps we currently possess do not reveal when and where interactions take place within a living system, limiting our ability to recreate or control processes at the cellular level. Using optically transparent Drosophila embryos and Fluorescence Lifetime Imaging Microscopy (FLIM), this project visualizes protein interactions directly within their natural context in order to understand some of the molecular events responsible for neuron morphogenesis. I demonstrate that a ubiquitously present and developmentally essential molecular switch, Cdc42, physically associates with its signaling partners, WASp and Par-6, within the nervous system. Although broadly co-localized, Cdc42 interacts with each partner only at distinct times and places. Within single neurons, both interactions corresponded to the time and place of dendrite morphogenesis, where they exhibited both overlap and complementarity. Taken together with experiments that assessed these proteins’ morphological contributions, these studies indicate that divergent signaling pathways coordinate to execute morphological change during neurodevelopment. Looking forward, a dynamic in situ interactome can help clarify how molecular networks underscore normal and abnormal cellular physiology.

Keywords

neurodevelopment; FLIM-FRET; Cdc42; Rac1; protein-protein interactions; neuron morphology

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