.

Aim: Cellular protein turnover has often been studied with mass spectrometry bulk analysis, however this method does not account for spatial differences of the turnover at single cell and subcellular level (e.g., organelle specificity). In this study, correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging were employed to investigate subcellular protein turnover in neuronal progenitor cells (NPCs). This integrative tool is a useful approach for many other investigations of biological mechanisms such as protein metabolism and cellular differentiation.
Methods: NPCs were incubated with different isotopically labeled (¹⁵N) amino acids for 48 h followed by an incubation with regular cell medium for pre-determined periods (chase time), from 0 h to 96 h. The cells collected after each chase time were then chemically fixed, embedded in resin and cut into thin sections. First, cells were imaged using TEM and the locations of various organelles within the cells were determined. Subsequently, the same cells were imaged with NanoSIMS providing information about the isotopic enrichment at the organelle level. By superimposing TEM and NanoSIMS images of the same cells, the ¹⁵N enrichment of individual organelles at the different chase times could be obtained.
Results and discussion: A heterogenous pattern of protein turnover across different cellular organelles was obtained, which could be related to previous data from Yousefi et al. about a protein’s localization influencing its turnover (Yousefi et al. 2021). This highlights a possible relationship between protein turnover and subcellular function. Additionally, significant differences in the subcellular distribution of ¹⁵N-enrichment from different ¹⁵N-amino acids were observed. These findings highlight the roles of different protein precursors in cellular protein turnover, as they might undergo distinctive metabolic pathways and thus are incorporated into specific proteins at specific cellular regions.

References: Yousefi, R.; Jevdokimenko,K.; Kluever, V.; Pacheu-Grau, D.;Fornasiero, E.F. 2021 Influence of Subcellular Localization and Functional State on Protein Turnover. Cells, 10, 1747

The presented study focused on two polymers poly(tert-butyl methacrylate) (PtBMA, Tg=117 °C) and poly(n-butyl methacrylate) (PnBMA, Tg=20 °C) and their interactions with biological materials.

In the first step, the impact of PtBMA stereoregularity on its interactions with peptides, proteins, and bacteria strains was studied for three stereoregular forms: isotactic (iso), atactic (at), and syndiotactic (syn). Principal component analysis (PCA) of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) data recorded for thin polymer films indicated a different orientation of ester groups, which in the case of iso-PtBMA are exposed away from the surface whereas for at-PtBMA and syn-PtBMA these are located deeper within the film. This arrangement of chemical groups modified the interactions of iso-PtBMA with biomolecules when compared to at-PtBMA and syn-PtBMA. For peptides, the affected interactions were explained by preferential hydrogen bonding and electrostatic interaction between the exposed polar ester groups of iso-PtBMA and positively charged peptides. In turn, for protein adsorption no impact on the amount of adsorbed proteins was observed. However, the polymer stereoregularity influenced the orientation of immunoglobulin G (IgG) and induced conformational changes in bovine serum albumin (BSA) structure. Moreover, the impact of polymer stereoregularity occurred equally for their interactions with Gram-positive bacteria (S. aureus), which absorbed preferentially onto iso-PtBMA films as compared to two other stereoregularities.

In the next step, the effect of temperature on the protein adsorption onto the PnBMA grafted brush coatings was studied. Due to glass temperature around 20 °C PnBMA is extremely interesting material for biomedical applications since its properties can be modified within the range of physiological temperatures. The combination of proteins molecules structure analysis with PCA of ToF-SIMS data revealed a temperature-dependent orientation of BSA as well as IgG molecules. BSA adsorbed to PnBMA coatings below Tg adopts such an orientation that Albumin 1 and Albumin 2 are exposed out of the surface, whereas for temperatures above transition, Albumin 3 is exposed. Similarly to BSA, the PCA of the ToF-SIMS data obtained for IgG immobilized at different temperatures onto PnBMA coatings clearly separates the IgG adsorbed onto the surface PnBMA below and above Tg. The analysis revealed a dominant head-on orientation for IgG adsorbed above Tg compared to a dominant end-on orientation for molecules adsorbed below the temperature of transition, which has a strong impact on their biological activity.

Biological molecules have been shown to exhibit specific cellular localizations, which relate closely to their functions. To understand the molecular mechanism of a cellular process, it is essential to obtain the spatial information of biomolecules and organelles in the cells, and their turnover dynamics and activity. Secondary ion mass spectrometry, with high a sensitivity, versatility, and spatial resolution, has been increasingly applied in biological research and neuroscience allowing the visualization of molecular localizations and turnover at the single cell and subcellular resolution. However, applications of SIMS alone is difficult for the biological interpretation due to its limitation in identifying specific cellular structures and organelles. Besides, information of large molecules, such as proteins, is completely lost due to the intense fragmentation, especially in NanoSIMS. Here, we resolved these problems using a correlative imaging approach using NanoSIMS and super resolution stimulate emission depletion microscopy (STED) to characterize the protein turnover dynamics of human neural progenitor cells (NPCs) at the organelle level. Alternatively, we developed dual labelling probes capable of binding to specific cellular proteins which can be visualized by both fluorescence microscopy and NanoSIMS imaging.

Correlating STED and NanoSIMS, we identified individual stress granules (SGs) in NPCs by STED microscopy and determined their protein turnover by NanoSIMS under the conditions of cellular ER stress (using ER stressor Thapsigargin) and stress recovery. SGs are membraneless organelles formed during cell stress as a cellular defensive mechanism to protect important cellular translational and signalling materials. However, the molecular mechanism of SG assembly and disassembly, and how they affect the cell recovery is not fully known. We found that SGs assemble by recruiting the proteins from the cytoplasm that exist before stress. In addition, the ER stress causes significant protein turnover impairment which could remain longer than commonly expected stress recovery period.

We have developed labelling probes, each containing a fluorophore and an element that is easily ionized by SIMS, particularly fluorine, boron, or iodine. These probes were demonstrated to label specific cellular proteins via click chemistry or immunostaining, and to visualize their localizations by fluorescence microscopy and NanoSIMS. We also generated a probe containing gold nanoparticles conjugated to nanobodies for specific protein imaging with NanoSIMS at subcellular resolution.

The correlative imaging approach and all the developed labelling probes offer a possibility of specific molecular imaging in SIMS extending to high mass biomolecules above metabolites and lipids.