Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes.

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C12, the flipper dye FliptR, as well as the dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C16, lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic dyes, molecular rotors, or flipper in a wide range of biophysics experiments. Compared to other methods, fluorescence lifetime imaging is a minimally invasive and nondestructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis, and the probe's intracellular distribution, as well as potential side effects induced by labeling and imaging.

Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.