Abstract

This dissertation investigates the role of the translation initiation machinery in the dynamics of stress granules (SGs). Stress granules are cytoplasmic, non-membrane bound organelles composed of mRNA transcripts, RNA-binding proteins, 40S ribosomal subunits, and eukaryotic initiation factors (eIFs). They form in response to environmental stress to promote cell survival and recovery. A major gap in knowledge addressed in this work is the need to measure real- time kinetics of SG assembly and disassembly. Furthermore, the involvement of translation initiation factors in SG assembly remains unclear. My research addresses these gaps by developing a system to monitor SG dynamics using a stable inducible Flp-In™ T-REx™-HeLa Cell Line system, enabling real-time tracking and modeling.Chapter 1 reviews the mechanisms of SG formation, their interplay with the translation initiation machinery, and their regulation under stress conditions. In Chapter 2, I describe the protocols developed to characterize the HeLa Fl-In cell line used throughout this study. Chapter 3 focuses on the role of the RNA Recognition Motif (RRM) of the initiation factor eIF4B in regulating SG assembly. Using single-cell live-imaging, I quantified the kinetics of SG formation and discovered that overexpression of GFP-tagged eIF4B with a point mutation in the RRM domain results in a decreased rate of SG formation, fewer total SGs, and a delayed stress response compared to wild type eIF4B. Furthermore, I demonstrated that eIF4B directly binds to G3BP1, a known SG nucleator, suggesting a mechanism by which eIF4B influences SG assembly. Chapter 4 examines the mechanisms of SG disassembly, a process poorly understood but linked to neurodegenerative disorders due to the inability to disassemble SGs, leaving cells in a constant state of stress. I developed a method to measure the rate of SG disassembly accurately, addressing the challenge of focal drift in time-lapse imaging. Future work will explore overcoming focal drift using microfluidics devices or environmental chambers. Chapter 5 presents preliminary data on the interaction of GFP-tagged eIF4G with SGs. Unexpectedly, modest overexpression of eIF4G in HeLa cells results in hypersensitivity to oxidative stress, even though SGs still form. Proposed future experiments aim to confirm and extend understanding of this phenomenon. Overall, this dissertation provides novel insights into the dynamic interplay between translation initiation machinery and SGs, with potential therapeutic implications for targeting SG dynamics in diseases such as cancer and neurodegeneration.