Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc

<p>Zinc (Zn²⁺) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn²⁺ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn²⁺, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn²⁺ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn²⁺ sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn²⁺ sensors by introducing the first generation of red-shifted cpFP Zn²⁺ sensors. We demonstrate that not only can these Zn²⁺ sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn²⁺ concentration. We also show that red-shifted cpFP Zn²⁺ sensors display high sensitivity for detecting labile Zn²⁺, similar to the green-shifted cpFP Zn²⁺ sensors. These discoveries add to the current knowledge of labile Zn²⁺ within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn²⁺ in cells that was previously unavailable.</p>

Name thesis:degree_name

M.S.

Level thesis:degree_level

Masters Thesis

Year dc:date.available

2021

Author dc:creator

• Maslar, Drew

Contributors dc:contributor

• Yan Qin
• Michelle Knowles
• Daniel Linseman
• Dinah Loerke

• Endoplasmic reticulum
• Fluorescent probe
• Zinc
• Biochemistry, Biophysics, and Structural Biology
• 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/1959

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-2949