Manipulating and visualizing the energy metabolism in single cells using chemogenetic tools and genetically encoded biosensors

Çokluk, Zeynep (2021) Manipulating and visualizing the energy metabolism in single cells using chemogenetic tools and genetically encoded biosensors. [Thesis]

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Mitochondria directly or indirectly affect cell fate. These organelles are known as the powerhouse of the cell and are responsible for generating ATP. As a consequence of energy production, highly reactive byproducts such as superoxide anion (O2 - ), hydroxyl radical (OH- ), hydrogen peroxide (H2O2), and peroxynitrite (ONOO- ), all collectively known as reactive oxygen species (ROS) or reactive nitrogen species are produced. In recent years it has been established that lower ROS levels drive important cellular signaling pathways, particularly H2O2. In a recent study, we developed an H2O2 generating chemogenetic tool referred to as D-amino acid oxidase (DAAO) that permits the generation of subcellular H2O2 levels with high spatial and temporal resolution. This study exploits multiparametric imaging approaches by combining chemogenetic tools with genetically encoded biosensors HyPer7 for H2O2, MaLionR for ATP, R-GECO for Calcium, and FLII12Pglu-700μδ6 for Glucose, and also chemical sensors such as tetramethylrhodamine methyl ester perchlorate (TMRM) for multispectral live-cell imaging of the membrane potential in cancer cells. These approaches allowed us to simultaneously generate ROS and visualize the downstream effects of H2O2 levels on various signaling pathways (subcellular calcium signaling) and the energy metabolism (mitochondrial membrane potential, glucose uptake, and ATP generation) in cancer cells. We observed that chemogenetically produced H2O2 did not affect Ca2+ and glucose levels while ATP levels were significantly affected. We documented that robust increase in mito-H2O2 levels disrupted the mitochondrial membrane potential. We also documented that chronic H2O2 production in HeLa cells reversed mitochondrial ATP production upon glucose deprivation. Our results demonstrate that utilizing chemogenetic tools paired with genetically encoded biosensors may serve as an informative approach to investigate the implications of oxidative stress in cancer energy metabolism.
Item Type: Thesis
Uncontrolled Keywords: Mitochondria. -- ROS signaling. -- hydrogen peroxide (H2O2). -- DAAO. -- cancer metabolism. -- genetically encoded biosensors. -- ATP. -- Glucose. -- Mitokondri. -- ROS sinyali. -- hidrojen peroksitç -- kanser metabolizması. -- genetik olarak kodlanmış biyosensörler. -- Glikoz
Subjects: T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Depositing User: Dila Günay
Date Deposited: 20 Jun 2022 16:32
Last Modified: 20 Jun 2022 16:32

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