Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Amy E. Palmer

Second Advisor

Natalie G. Ahn

Third Advisor

Joseph Falke

Fourth Advisor

Hang (Hubert) Yin

Fifth Advisor

Tin Tin Su

Abstract

Zinc (Zn2+) is an important trace element that is found throughout the human body and plays essential roles in proteins to ensure structural integrity and catalytic activity. It is the second most abundant transition metal in the body. Transition metals are required for numerous enzymes, proteins, and cellular processes. It is important for cells to regulate distribution of metals because numerous enzymes and cellular processes depend on transition metals, yet metal imbalance leads to a wide range of diseases. Prostate tissue has the highest Zn2+ concentration in the body compared to other tissues but Zn2+ is significantly depleted upon onset of malignancy. Numerous studies have revealed that cancerous prostate tissue exhibits dramatically reduced levels of Zn2+ and that the levels of Zn2+ appear to correlate with progression from benign to invasive and metastatic cancer. The mechanistic correlation between malignancy and Zn2+ levels is not well understood and in particular, it is not clear whether Zn2+ depletion contributes to or is a consequence of disease progression. Although the majority of Zn2+ found in cells in bound by proteins, enzymes, and cellular ligands, pools of free Zn2+ have been identified in pancreatic islet cells, brain, and prostate. In this current study we used normal and cancerous prostate cells as a model system to map free Zn2+ levels in the nucleus, cytosol, endoplasmic reticulum, and mitochondria to identify the differences in Zn2+ at the subcellular level. This was accomplished by using novel Zn2+ sensors based on fluorescence resonance energy transfer (FRET) targeted to each organelle compartment and carrying out live cell imaging experiments. FRET sensors are powerful tools to monitor Zn2+ dynamics in cells. This type of probes gives us the ability to monitor Zn2+ in multiple cellular comparments or simultaneously track two cellular signals using orthogonal FRET sensors. Such studies allow us to precisely correlate the timing of two interdependent cellular events or to track the movement of ions or molecules from one compartment to another. We discovered that the free Zn2+ pool in the nucleus and cytosol is higher in our prostate cancer cell line models compared to normal cell line. In the ER we observed that the free Zn2+ level in normal prostate cells is about 4 times higher than all three prostate cancer cell lines models used. Similar results were observed in mitochondria, revealing 3-4 times higher Zn2+ in the normal cells compared to all three prostate cancer cell lines. Although all three prostate cancer cell lines used in this study were characterized by over 50% reduction in total cellular Zn2+, surprisingly we discovered this reduction did not translate into across-the-board deficiency in intracellular Zn2+ stores, but rather there was substantial redistribution of Zn2+ between subcellular locations. In an effort to define how and why Zn2+ pools are redistributed in prostate cancer, we measured the changes in expression levels of key Zn2+ regulatory proteins such as transporters (hZIP1, hZIP2, hZIP3, ZnT1, ZnT2, ZnT4, and ZnT7). Our results suggest that there is a dysregulation of the Zn2+ transporters in cancerous prostate cells and there is some heterogeneity between the prostate carcinoma cell lines. To complement existing tools, we developed a suite of sensors using alternately colored FRET pairs using tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE that were used simultaneously with CFP-YFP sensors. Using these combinations of FRET sensors we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600-700 sec. These new green-red sensors provided the starting point for developing vesicle-targeted sensors. In summary, this thesis details efforts to develop new fluorescent sensors for defining free Zn2+ in cells and applies these sensors to quantify free Zn2+ in normal prostate and cancerous cells at the subcellular level. We discovered unprecedented redistribution of Zn2+ stores in all three cancer cell lines and discovered this correlates, at least in part, to alteration of Zn2+ transporters. This work not only provides the first quantitative maps of Zn2+ distribution in cells, it also provides a critical glimpse of how Zn2+ is altered with disease progression.

Included in

Biochemistry Commons

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