Developing Red Fluorescent Zn2+ Sensors and Characterizing Zn2+ Homeostasis in Mucolipidosis Type IV Disease

<p>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.</p> <p>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.</p>

Name thesis:degree_name

M.S.

Level thesis:degree_level

Masters Thesis

Year dc:date.available

2023

Author dc:creator

• Dischler, Anna M.

Contributors dc:contributor

• Yan Qin
• Martin Margittai
• Scott Barbee
• Dan Linseman

• Fluorescence
• Lysosomes
• Mucolipidosis
• Neurodegenerative
• Zinc
• Biochemistry, Biophysics, and Structural Biology
• Cell and Developmental Biology
• Cell Biology
• Life Sciences
• Molecular Biology

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2220

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3208