Metabolism in microbial colonies responds to competing species, rapidly evolving genetic makeup, and sometimes dramatic environmental changes. Conventional characterization of the existing and emerging microbial strains and their interactions with antimicrobial agents, e.g., the Kirby-Bauer susceptibility test, relies on time consuming methods with limited ability to discern the molecular mechanism. Assessing the metabolic adaptation of microbial colonies requires their non-targeted molecular imaging in a native environment. Laser ablation electrospray ionization (LAESI) is an ambient ionization technique that in combination with mass spectrometry (MS) enables the rapid analysis and molecular imaging of over 400 metabolites and lipids. In the first part of this presentation, we report on the application of LAESI-MS imaging to gain a deeper molecular insight into microbe-antibiotic interactions, and enhance the speed and quantitative nature of antibiotic susceptibility testing.
Mitosis in eukaryotic cells is divided into four successive stages, i.e., prophase, metaphase, anaphase, and telophase, based on the distribution of chromosomes and the morphologies of the mitotic spindles. Consumption of ATP during the two stages of anaphase, are thought to be different, i.e., during the movement of the chromosomes towards the two spindle poles (anaphase A) does not require ATP, whereas the elongation of the spindles (anaphase B) proceeds with ATP consumption. However, the intracellular energy states and metabolic composition during the other mitotic stages, e.g., metaphase, and the following cytokinesis have not been characterized. Recent advances in capillary microsampling mass spectrometry enable the analysis of the metabolic makeup, energy and redox states, and lipid composition of individual cells. Here, we show the changes of energy states, such as adenylate energy charge (AEC), in single cells at distinct mitotic stages. Cellular AEC at metaphase showed the highest level and narrowest distribution. Reduced AEC levels were observed in the anaphase, telophase, and cytokinesis. These findings might be linked to the consumption of ATP in spindle elongation during late anaphase and telophase. The [GTP]/[GDP] ratio exhibited higher values in metaphase and decreased in the anaphase and telophase, and rebound during cytokinesis. Our results inform on the dynamic changes of cellular physiological states and the corresponding metabolic noise during the movement of chromosomes and elongation of spindles in mitosis.
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