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

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.

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size.

We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of 13 C-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an 127 I-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long-carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a single cell level.

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

Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level.

Chemical Imaging and Analysis of Single Nerve Cells by Secondary Ion Mass Spectrometry Imaging and Cellular Electrochemistry.

A nerve cell is a unit of neuronal communication in the nervous system and is a heterogeneous molecular structure, which is highly mediated to accommodate cellular functions. Understanding the complex regulatory mechanisms of neural communication at the single cell level requires analytical techniques with high sensitivity, specificity, and spatial resolution. Challenging technologies for chemical imaging and analysis of nerve cells will be described in this review. Secondary ion mass spectrometry (SIMS) allows for non-targeted and targeted molecular imaging of nerve cells and synapses at subcellular resolution. Cellular electrochemistry is well-suited for quantifying the amount of reactive chemicals released from living nerve cells. These techniques will also be discussed regarding multimodal imaging approaches that have recently been shown to be advantageous for the understanding of structural and functional relationships in the nervous system. This review aims to provide an insight into the strengths, limitations, and potentials of these technologies for synaptic and neuronal analyses.