In-cell NMR and beyond: high resolution metabolic profiling in Caenorhabditis elegans by in vivo NMR spectroscopy

BRUNO HERNÁNDEZ CRAVERO; DIEGO DE MENDOZA; GASTÓN PREZ; ANDRÉS BINOLFI; VERÓNICA A. LOMBARDO

Rosario

Congreso; Second Latin American Worm Meeting; 2020

The Latin American Worm Society

To understand the functional properties of a biomolecule, such a small metabolite, a protein or nucleic acids, we ought to detect them with high resolution in their native environment. Recent efforts have aimed at the development of in vivo techniques for metabolomics and structural biology studies. NMR spectroscopy, a biophysical method for investigations of biomolecules at the atomic level, allows for the direct observation of NMR-active nuclei in complex, undefined environments and can thus be employed to investigate labeled molecules inside cells. We call this methodology In-cell NMR spectroscopy and denotes the application of high-resolution solution-state NMR methods to study biomolecules directly in live cells. This allows to evaluate the properties and/or the composition of a molecule or a group of molecules in a physiological environment and to resolve their functional characteristics in a cellular context. These newly developed methods can be used on intact bacteria, yeasts or cultured mammalian cells. However these cells are clonally grown at high densities in artificial media, lacking the complex tissue context present in higher organisms and all its associated biological activities. Here, we extend In-cell NMR applications to in vivo conditions using C. elegans as a model organism. By isotopically enriching live C. elegans with 13C atoms we detect 1H-13C NMR correlations that allow delineating metabolic compositions in wt and mutant worm strains with impaired unsaturated fatty acids (UFAs) biosynthesis. Perturbations in UFAs metabolism are associated with the onset of severe cardiovascular and neurodegenerative disorders, including cardiac infarction and Parkinson?s disease. Therefore, a detailed knowledge of its fluctuations in physiological and pathological conditions in vivo is essential for advancing our knowledge of these life-threatening diseases and for the design of novel targets for therapeutic intervention. Overall, our strategy represents a high-resolution, multi-dimensional NMR study in a live multicellular organism and constitutes a methodological advancement with regard to genuine in vivo NMR applications.