Date of Award

2020

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natual Science and Mathematics, Biological Sciences

First Advisor

Yan Qin

Second Advisor

Schuyler Van Engelenburg

Third Advisor

Michelle K. Knowles

Fourth Advisor

Balsingham Murugaverl

Fifth Advisor

Joseph Angleson

Keywords

Cellular trafficking, Microtubule trafficking, Mitochondria, Polyamines, Zinc

Abstract

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors we found that exposure to high labile zinc only overloaded the cytosol and the mitochondrial IMS with zinc while the mitochondrial matrix did not sequester excess labile zinc. We highlighted the importance of mitochondrial labile zinc dynamics and concentrations across different mitochondrial locations and distinguished the mitochondria as not being a storage site for zinc.

In this work we revealed, for the first time, a novel vital role zinc plays in the regulation of cellular trafficking. High load of zinc can arrest the motility of mitochondria, lysosomes and vesicles in both HeLa cells and neurons. The zinc-microtubule interaction was found to prevent motor proteins from landing on microtubules obstructing microtubule-based motility. Zinc was identified as interacting with two histidine residues on the α-tubulin monomer of microtubules. This finding resolved the long lasting controversy regarding the signaling roles of labile zinc and provided solid, direct and strong evidence confirming that zinc act as a significant intracellular signaling molecule. In addition to zinc, polyamines were investigated as a potential specific regulator of mitochondrial microtubule-based trafficking.

We found a correlation between mitochondrial microtubule-based trafficking and abnormal polyamine concentrations resulting from Alzheimer’s disease (AD) in a Down syndrome (DS) cell model. Decreased mitochondrial motility was found in primary cultured rat hippocampal neurons as well as in the DS mouse hippocampal neuron model with increased polyamines. Polyamine production can be blocked using difuoromethylornithine (DFMO) a drug that inhibits ornithine decarboxylase (ODC) preventing the production of polyamines. DFMO restored mitochondrial motility in the DS mouse model. Mitochondrial motility in relation to polyamines has never been connected prior to these results indicating a novel pathway for the pathology of AD in DS brains. Two new regulatory mechanisms for axonal motility and exposure of previously unknown zinc mitochondrial relationships were discovered which has magnified our understanding of how the cell functions.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Dylan Hunt Fudge

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

264 p.

Discipline

Biophysics, Biology, Chemistry

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