Combining coherent Raman scattering with fluorescence microscopy to investigate sugar uptake in living cells

• Applicant: Dr. Evelyn Plötz
• Subject Area: Biophysics ; Analytical Chemistry
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 426814631

Sugars are essential molecules in life. Being actively transported over the cell membrane by specialized membrane transporters, they represent a major source of energy and carbon in glycan biosynthesis. Glycosylation, as ubiquitous post-translational modification has been known for a long time. All cells are covered with a dense and complex layer of glycans. This so-called glycocalyx is involved in various processes including signalling, cell-adhesion and immunity based on glycan-driven pattern recognition. Despite its importance, only little is known about the composition, structure and precise function of the glycocalyx due to the extraordinary variability of glycan structures as well as dynamic nature of glycosylation. It is unanswered, how sugar uptake influences the chemical composition of the cellular glycocalyx. Combined structural approaches have identified a growing catalogue of monosaccharides and glycan structures however they are restricted to ex vivo analysis. Metabolic oligosaccharide engineering allowed for fluorescence imaging of sugar moieties in vivo. Nevertheless, labelling of sugars remains challenging. Fluorescent labels can interfere with the sugar’s biological function. Sugars species are present at intracellular concentrations ranging from nM to mM range under physiological conditions. Single-molecule imaging is not suited to simultaneously visualize low and high concentrations of different sugar species, or the involved membrane transporter and substrate at the same time. Despite its immense biomedical importance, we lack a high-speed, multiplexed imaging technology that allows for studying natural sugars during uptake, metabolism and storage as glycans in situ. We propose to establish stimulated Raman microscopy (SRS), which I pioneered during my PhD, as tool for chemical mapping of sugars. I will design a cutting-edge imaging platform, that combines single-molecule live cell imaging with broadband stimulated Raman microscopy. It is dedicated for multiplexed in vivo imaging with chemical specificity at high concentrations and simultaneous single-molecule sensitivity. I will establish the developed methodology to investigate uptake and storage of sugar within the cell. The combined approach serves for the quantification of sugar uptake mediated by GLUT transporters with cellular resolution. Moreover, we will study whether inserted Raman-active moieties in MOE affect the affinity and uptake and if unnatural sugars are taken up actively or via passive diffusion. By investigating the cellular storage of sugars, we ultimately aim to establish this approach as a chemical characterization method for the cellular glycocalyx.

• DFG Programme: Research Grants