The Composition of Stress Granules Impact its Dynamics and Fluidity

Sarkar, Mainak (2025) The Composition of Stress Granules Impact its Dynamics and Fluidity. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Mainak Sarkar (20MS037)) 20MS007_Thesis_file.pdf Restricted to Repository staff only Download (4MB)

Abstract

Stress granules (SGs) are dynamic biomolecular condensates that form in response to cellular stress and are increasingly implicated in the pathogenesis of neurodegenerative diseases. Emerging evidence suggests that persistent SGs, along with altered SG dynamics and material properties, contribute to disease progression. Motivated by this, I investigated whether targeted perturbations in SG composition could modulate their biophysical characteristics. Using structure-guided single-point mutations within the NTF2-like (NTF2L) domain of Ras GTPase-activating protein-binding protein 1 (G3BP1)—a key SG scaffold protein—we selectively disrupted specific protein–protein interactions within the SG network. Live-cell imaging revealed mutation-specific alterations in SG formation and disassembly dynamics, particularly under sodium arsenite-induced oxidative stress and one hour of heat stress. Fluorescence recovery after photobleaching (FRAP) analysis further demonstrated a trend of increased SG fluidity in G3BP1 mutants relative to wild-type because of higher mobile fraction for the G3BP1 mutants, although differences across mutants did not reach statistical significance. Control experiments confirmed that the mutations did not alter the intrinsic properties or expression stability of mEmerald- G3BP1 (mEme-G3BP1), validating that the observed effects on SG behavior are attributable to disrupted interactions. Extending our approach, I identified a single-point mutation (F511A) in the intrinsically disordered protein NUFIP2 that abolished its binding to G3BP1, thereby exploring and demonstrating the mutational disruption of G3BP1 interactors impacting SG composition. Together, these findings underscore the importance of SG composition in regulating their dynamics and material state, and suggest that compositional perturbations may underlie SG persistence in disease contexts. Given our limited understanding of the cellular functionality of biomolecular condensate interaction networks, studying SG as a prototypical condensate can significantly enhance our comprehension of the underlying principles of biomolecular condensates. This principle may also be applied to delve deeper into understanding the cellular functionality of the interaction network of the other biomolecular condensates. i

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