Optimization of fluorescence lifetime imaging microscopy (FLIM) for studying the activity of enzymes in live cancer cells
This dissertation describes the process of optimizing a Fluorescence Lifetime Imaging Microscopy (FLIM) system in order to observe the dynamics of enzymes in live cancer cells. The enzyme studied throughout this research is Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) which is a membrane-bound protein principally responsible for degrading extra-cellular matrix (ECM) proteins in the local environment of a migrating cell. However, MT1-MMP has an intricate role in the regulation of the cell’s migration separate from its simple proteolytic functions. In addition, the increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. In spite of the importance of MT1-MMP in understanding a cancer cell’s decision making as it leaves a tumor, very few reports have quantitatively studied the activity of this enzyme in live cells. Even fewer reports have examined the spatiotemporal activity of MT1-MMP in live cells cultured in 3-dimensional settings such as matrices of ECM proteins. These 3-dimensional settings can parallel the environment encountered by metastasizing cells in tissues. Studying live cells in 3-dimensional matrices is crucial for biologically relevant investigations. A cell’s morphology and migratory behavior can vary significantly when comparisons are made between cells cultured on two dimensional substrates and those cultured in 3-dimensional matrices. The purpose of this project was to understand the coordinated functions of MT1-MMP as live cancer cells interact with and move through a 3-dimensional matrix of ECM proteins. Specifically, we are ultimately interested in the spatiotemporal activation patterns of MT1-MMP in live cancer cells in order to build a quantitative (systems-level) model describing MT1-MMP’s role in the cell’s decision making as it is leaves a tumor site.
Fluorescence Lifetime Imaging Micorscopy (FLIM), Cancer, Fluorescent Proteins, Metastasis, Enzymatic Activity
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign