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

Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging

“…This could be due to free 15 N-phenylalanine being oxidized generating m-tyrosine, which can be incorporated into proteins and result in cellular stress. 46 Cellular stress in turn could inhibit the overall protein turnover, 16 resulting in a atter curve of the turnover of 15 N-phenylalanine across all the cell compartments. NPCs incubated with 15 N-phenylalanine show high enrichment in mitochondria aer 6 h chase time.…”

“…Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a valuable tool in studying the subcellular distribution of analytes in mammalian cells. [10][11][12][13][14][15] NanoSIMS has been used to study protein turnover in cells and tissues at a subcellular level for example in stress granules, 16 stereocilia, 17 synapses, 10 and lysosomes. 18 In NanoSIMS, a primary ion beam erodes a sample surface pixel by pixel, generating atomic and diatomic secondary ions from the sample surface.…”

“…NanoSIMS has been used with rare isotopic labelling to track protein turnover in cells and tissues. 16,20 NanoSIMS is oen combined with other imaging techniques such as uorescence and/or electron microscopy to identify detailed morphological structures and obtain complementary information of sample properties. 10,[21][22][23] We have previously demonstrated correlative TEM and NanoSIMS imaging to assess the neurotransmitter content at the nanometer vesicles in cells.…”