Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging

Authors:

Stefania Rabasco, Alicia A Lork, Emmanuel Berlin, Tho D K Nguyen, Carl Ernst, Nicolas Locker, Andrew G Ewing, Nhu T N Phan

Keywords:

stress granules; NanoSIMS; protein turnover; neuronal progenitor cells; mass spectrometry imaging

Abstract:

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (¹⁵N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the ¹⁵N/¹⁴N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

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