Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Xuedong Liu

Second Advisor

Amy Palmer

Third Advisor

William Old

Fourth Advisor

Sabrina Spencer

Fifth Advisor

Natalie Ahn

Abstract

A multitude of cellular functions are controlled by the activity of the membrane-bound protease TACE, including immune response and development. Through cleavage of its membrane anchored substrates, TACE can activate a myriad of signaling pathways in an auto- or paracrine manner, transferring signals across a tissues and systems. Despite its importance, it is highly debated how TACE is regulated. I helped generate TSen, a fluorescence-based sensor of TACE activity, a critical tool that proved necessary to interrogate the underlying signaling that leads to TACE activation. TSen enabled us to uncover a wide variation between cell lines and their underlying signaling in regards to TACE which may account for much of the confusion about its regulation. Furthermore, we used this sensor to find that TACE is intimately tied actin cytoskeletal dynamics, which appears to be a novel mechanism of TACE activation. I then used TSen to monitor TACE activity in single cells in conjunction with an ERK reporter to measure both activities simultaneously for the first time. I was able to confirm past results showing the propagation of ERK pulses from cell to cell, as well as identify a new type of activity pulse: spikes in ligand shedding. These pulses of TACE activity occur in neighbor cells prior to an ERK pulse, which is followed by another spike in ligand shedding, validating the model that ERK pulses propagate between cells through ligand shedding. I then probed cell junctions for their role in TACE signaling. Because the aggressiveness of an epithelial-derived cancer is highly correlated with loss of cell junctions, I generated an α-catenin knockdown in HaCaT cells, recreating a phenotype of mesenchymal morphology and hyper proliferation. I found that silencing α-catenin in this system generates a large increase in ligand shedding and subsequent proliferation and that the hyper proliferative phenotype does not occur in the absence of TACE. My findings contribute to the overall understanding of TACE regulation, as well as the downstream effects of this signaling molecule in ERK pulses and proliferation.

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