Probing the subcellular molecular architecture and turnover of neural cell models using correlative mass spectrometry imaging

Abstract

Neurons are fascinating cellular units of the nervous system and are responsible for the propagation of signals that let us move and think. Understanding cellular mechanisms that maintain neurons and enable their communication is crucial for a deeper understanding of the nervous system, including diseases that affect it. Elucidating the intricacies of biological processes is a core purpose of analytical methods. However, often the use of one technique does not provide sufficient information to answer scientific questions. Correlative chemical imaging, the combination of two or more imaging modalities, is a useful analytical tool to obtain comprehensive knowledge of a sample that could not be obtained otherwise. In the work included in this thesis, correlative chemical imaging was used to investigate exocytosis, the process by which neurotransmitter is released from cellular vesicles to the extracellular space to communicate with other cells. The predominantly partial release of neurotransmitters and concurrent chemical transport into vesicles was visualized with correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging. Additionally, it was found that the process of partial release is independent of vesicle size. Furthermore, protein turnover, an important mechanism in cells to maintain protein homeostasis was investigated in human stem cell-derived neural progenitor cells (NPCs) and their further differentiation to neurons. Utilizing correlative TEM and NanoSIMS imaging, protein turnover could be tracked at a single organelle level by incubating cells with isotopically labeled amino acids. It was found that protein turnover is heterogeneous across the cell and that different amino acids result in different spatial turnover patterns. In the differentiation from NPCs to neurons it was found that protein turnover overall slowed down and that the protein lifetime of different organelles in different stages of differentiation was highly distinguished which could potentially be used to assess activities of these organelles and their involvement in the regulation of specific cell states. Additionally, it was found with correlative fluorescence microscopy and NanoSIMS imaging that NPCs recovering from stress have reduced protein turnover and that stress granules, organelles that are formed when cells undergo stress, are displaying similar turnover than that in the cytoplasm. Taken together, this thesis provides insights into the biological mechanisms of neural cell models via correlative mass spectrometry imaging.