Date of Award

2023

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natural Science and Mathematics, Biological Sciences

First Advisor

Yan Qin

Second Advisor

Martin Margittai

Third Advisor

Scott Barbee

Fourth Advisor

Dan Linseman

Keywords

Fluorescence, Lysosomes, Mucolipidosis, Neurodegenerative, Zinc

Abstract

Zinc (Zn2+) is a divalent, redox-inert metal that plays a vital role in many cellular processes either by acting as a catalytic cofactor or a labile signaling molecule in the cytoplasm. Cytosolic Zn2+ levels are tightly regulated to stay within a narrow window of concentrations, but fluctuations in Zn2+ signals have been detected in a variety of cells. We developed a genetically encoded, single red fluorescent protein (RFP) based Zn2+ sensor, RZnP1, that can be used alongside green-wavelength sensors for the simultaneous detection of different signaling molecules within the same cells or among different subcellular compartments. The excitation wavelength of many RFPs is less phototoxic than that of GFPs which extends imaging time. We demonstrate live cell multi-compartmental imaging of cytosolic and mitochondrial Zn2+ dynamics using RZnP1 and mitochondria targeted GZnP2 and discovered that high concentrations of cytosolic Zn2+ are not sequestered into mitochondria in healthy neurons.

My second project aims to study Zn2+ homeostasis in Mucolipidosis type IV (MLIV). MLIV is an inherited neurodevelopmental and neurodegenerative disorder, which has severe developmental delay, psychomotor deficits, and vision loss. It is caused by loss-function mutations in the lysosomal channel TRPML1. We assess the Zn2+ and Ca2+ permeability and localization of 10 different TRPML1 patient mutants. Furthermore, we quantify Zn2+ concentrations in the cytosol, lysosomes, and mitochondria in MLIV patient fibroblasts. We found that mitochondrial Zn2+ concentrations are significantly higher in MLIV cells. Next, we demonstrate that high lysosomal Zn2+ can be imported into the mitochondria. Lastly, we assessed whether higher mitochondrial Zn2+ in MLIV cells can affect mitochondrial morphology.

Publication Statement

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

Rights Holder

Anna M. Dischler

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

82 pgs

Discipline

Cellular biology, Molecular biology

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Available for download on Friday, August 01, 2025



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