Date of Award

2021

Document Type

Thesis

Degree Name

M.S.

Department

Biological Sciences

First Advisor

Yan Qin

Second Advisor

Michelle Knowles

Third Advisor

Daniel Linseman

Fourth Advisor

Dinah Loerke

Keywords

Endoplasmic reticulum, Fluorescent probe, Zinc

Abstract

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.

Publication Statement

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

Provenance

Received from ProQuest

Rights holder

Drew Maslar

File size

65 pgs

File format

application/pdf

Language

en

Discipline

Molecular biology, Cellular biology, Biology

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