STRESS GRANULES: SIGNAL FOR FORMATION UNDER ARSENIC AND OXIDATIVE STRESS Item Type Electronic Thesis; text Authors Wu, Emma Citation Wu, Emma. (2020). STRESS GRANULES: SIGNAL FOR FORMATION UNDER ARSENIC AND OXIDATIVE STRESS (Bachelor's thesis, University of Arizona, Tucson, USA). Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/09/2021 19:23:30 Item License http://rightsstatements.org/vocab/InC/1.0/ Link to Item http://hdl.handle.net/10150/651400 STRESS GRANULES: SIGNAL FOR FORMATION UNDER ARSENIC AND OXIDATIVE STRESS By EMMA DEAP WU ____________________ A Thesis Submitted to The Honors College In Partial Fulfillment of the Bachelor of Science Degree With Honors in Molecular and Cellular Biology THE UNIVERSITY OF ARIZONA M A Y 2 0 2 0 Approved by: ______________________ Dr. Qin Chen Department of Pharmacology Stress Granules: Signal for Formation Under Arsenic and Oxidative Stress Emma D. Wu, Jennifer N. Daw, Qin M. Chen ______________________________________________________________________________ Abstract Aerobic metabolism generates Reactive Oxygen Species (ROS) as by-products. ROS production increases during xenobiotic stress and under various pathological conditions. Generally, ROS are considered detrimental since they are capable of attacking cellular macromolecules, resulting in DNA damage, protein oxidation, and lipid peroxidation. Previous research indicates that a signaling function of ROS, with the cascade leading up to and regulating transcriptional and post-transcriptional series, causes cell death or even cell survival and adaptation. One out of the many cellular defense mechanisms in which ROS induces is the development of stress granules (SGs). Stress granules are dense aggregations composed of RNA and proteins that appear when the cell is under stressed conditions in the cytosol. The stalled translational framework, along with messenger (m)RNA, aggregates into molecularly dynamic and microscopically observable stress granules. We discovered that stress granules were formed under oxidative stress with hydrogen peroxide (H2O2) and arsenic stress with sodium arsenite (NaAsO2). T-Cell Intracytoplasmic Antigen-1 (TIA-1), G3BP1, and HuR were all effective stress granule markers while La/SSB and FBP1 were not so much. Understanding how RNA aggregation is controlled by healthy cells will be a significant objective for studies in the future and the advanced technology is promising for this field to progress forward. Because of incomplete mitochondrial electron transport, aerobic metabolism generates ROS as by-products. Superoxide that is formed during impeded mitochondrial respiration is transformed by superoxide dismutase to hydrogen peroxide. In the presence of iron, hydrogen peroxide produces hydroxyl radicals, a highly reactive species that will attack a cell's DNA, lipids, and proteins. ROS will induce multiple signaling events to control various cellular endpoints. Many of these events were initially determined as an abrupt reaction to receptor binding of growth or endocrine factors. ______________________________________________________________________________ Introduction Various cellular defense mechanisms have evolved to cope with pathogenic and environmental stresses. Interrupting protein synthesis usually serves to conserve energy, yet avoid creating wrong sequences or protein conformations. Before termination of protein translation, ribonucleoprotein complexes composed of 40S ribosomes quickly gather and assemble stress granules. This protective mechanism is to shelter the translation framework and mRNA species. Stress Granules (SGs) are non-membrane bound RNA-protein complexes that condensate in the cytoplasm of cells during several stress responses. This stalled translation complex is believed to affect mRNA function, localization, and signaling pathways. After arsenic and oxidative stress, translation initiation in the cell has been limited and blocked. Ribosomes continue translation elongation, thus releasing over hundreds of polysome-free messenger (m)RNAs in which are unable to initiate further rounds of translation. These halted translation initiation complexes include translation initiation factors, RNA-binding proteins, and eukaryotic initiation factors that combine together to develop stress granules. Sequencing analyses of the stress granule transcriptomes from mammalian cells and yeast have shown that more than 99% of mRNA species translocate into stress granules. The formation of stress granules indicates a self-defense mechanism and a stage for cell survival under arsenic and oxidative stress conditions. There have been a plethora of studies that have analyzed the dynamics of proteins during the process of assembling stress granules. ______________________________________________________________________________ Research Methods Cell culture, H2O2 treatment, and NaAsO2 treatment Under tissue culture conditions, HeLa cells were treated with a weekly subculture in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) along with supplementation of 10% Fetal Bovine Serum (FBS) and 100 U/mL of each streptomycin and penicillin. Cells had been seeded at 6.5x10E4 cells per well onto 24-well plates. Cells had reached 80% confluency before they were treated with hydrogen peroxide (H2O2) and sodium arsenite (NaAsO2). Immunocytochemistry (ICC) Immunocytochemistry (ICC) is a staining technique that is used in the laboratory to anatomically detect and visualize the localization of a particular antigen or protein in cells by utilizing a specific primary antibody that will bind to it. Immunocytochemical markers are antibodies used to identify specific proteins in tissue sections. Markers used in this study included TIA-1, G3BP1, HuR, La/SSB, and FBP1. The primary antibody binds to the protein being assessed and a fluorescent secondary antibody label with a green fluorescent protein (GFP). This is a method to identify if the protein of interest is present. The proteins of interest that we labeled indicate the formation of stress granules. Immunocytochemical Staining for Different Stress Granule Proteins Protocol Before seeding cells, the number of cells was estimated using a hemocytometry. Cells were seeded onto individual cover glasses in a 24-well plate. This is done by placing 12 mm circular cover glasses to each individual well of 24 wells in which 6.5x10E4 cells were seeded. We pressed down the cover glass using a pipette to ensure that there is no air between the cover glass and the bottom of the well. About 2-3 days after seeding when cells are nearly 80% confluent, the 10% medium was changed to 0.5% medium and serum-starved between 16-20 hours. Serum-starve is unnecessary for the cells, but cells were then fixed. About 0.5 mL of PBS was added to each well. Cells were then treated with hydrogen peroxide (H2O2) and sodium arsenite for 2 hours. Following each conditional treatment, we removed the culture medium and washed the cells once with phosphate-buffered saline (PBS). Cells were fixed with 4% paraformaldehyde in PBS for 5 minutes with occasional shaking. The solution was removed and 1 mL of 4% paraformaldehyde was added to incubate with shaking for 30 minutes. After incubation, the 4% paraformaldehyde was removed, and the 24-well plate was stored in a wash of PBS in 4 degrees Celsius. When prepared for immunocytochemical staining, the cover glasses were removed to a new well containing PBS. A fine needle was used to find the edge of the cover glass that was then lifted up and then moved to a new well with fine forceps where the surface containing the cells facing up. After two washes of 0.5 mL of PBS for each well preceding three washes of 0.5 mL of PBS containing 0.25% of Triton X-100 for 5 minutes each, 200 μL of PBS containing 1% Bovine Serum Albumin (BSA) and 0.1% Tween 20 were added to the cells. This was incubated for 40 minutes with shaking and then later incubated the cells with primary antibodies (1:100 dilution in PBS with 0.5% BSA) in 200 μL volume for 60 minutes at room temperature or overnight in 4 degrees Celsius. Overnight was more efficient. The antibodies that were incubated with the cells included anti-TIA-1 (sc-1751), anti-TIA-1 (sc- 166247), anti-G3BP-1 (sc-81940), anti-HuR (sc-5261), anti-La/SSB (sc-80656), and anti FBP1 (sc-136137) (Santa Cruz Biotechnology, CA) After removal of primary antibodies, five washes of 200 μL of PBS containing 1% BSA and 0.1% Tween 20 immediately after one another were performed. Again, we incubated the cells, this time with 200 μL Alexa Fluor 488 conjugated secondary antibody (1:600 dilution) for 60 minutes in the dark. The incubation was performed in the dark by wrapping the plate with aluminum foil to prevent bleaching of the fluorescence. After incubation and removal of unbound antibodies, cells were washed five times with 0.5 mL of PBS for five minutes each. The cover glasses were each mounted onto a microscope slide with 8 μL of Vectashield Antifade Mounting Medium with DAPI and allowed to harden overnight in 4C. The negative control slide contained no primary antibody. Stress Granule Counting Cells and stress granules that formed were chosen for counting at random. In order to measure the percentage of cells that contained the formed