Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dr. Sashko Damjanovski

Abstract

During development the extracellular matrix is cleaved and remodeled to facilitate the large-scale cell rearrangements that are necessary for processes like gastrulation, neurulation, angiogenesis and organogenesis. ECM remodeling occurs primarily through secreted enzymes called matrix metalloproteinases (MMPs). Regulation of MMP activity is achieved through the tissue inhibitor of metalloproteinases (TIMPs), a small family of secreted proteins that bind MMPs in a 1:1 manner to inhibit their activity. Although TIMPs were originally characterized based on their MMP-inhibitory activities, in vitro studies have revealed that TIMPs are multifunctional proteins, with structurally and functionally distinct N- and C-terminal domains. TIMP N-terminal domains bind to and inhibit MMPs, whereas their C-terminal domains have cell signaling activity in apoptosis, cell migration and cell proliferation pathways. Using Xenopus laevis as a model organism, this study examined the unique roles of the TIMP N- and C-terminal domains during development to investigate the balance between TIMP MMP-inhibitory activity and signaling activity in vivo. Microinjection was used to overexpress the three X. laevis TIMPs or their individual domains in early stage embryos. This research demonstrated that the balance between MMPs and inhibitors in the ECM is very important, and that the ECM functions as an intricate network. Here I showed that the TIMP-1 and -2 C-terminal domains downregulated MMP expression/activity within this network independent of MMP-inhibition. Additionally the TIMP-1 and -2 C-domains altered signaling markers including caspase-3 and RECK, which was not observed with N-domain constructs, and resulted in severe developmental defects. In contrast, the TIMP-3 C-terminal domain performed no independent role in development. This research is the first comprehensive comparison of TIMP domain function in vivo, and demonstrates for the first time a role for TIMP signaling during development.

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