Biology subjects

Kuhn, S. M.

Publications and source records attributed to Kuhn, S. M..

4 recordsLinked to original sources

bioRxiv2025

GEQO biosensors for absolute analyte quantification in single cells

Genetically encoded fluorescent biosensors are widely used to monitor small molecule and ion levels in living cells. Quantitative FRET and FLIM sensors and highly sensitive intensiometric sensors have been developed for many analytes. Notwithstanding notable advances over the last years, a universal high-performance sensor design for absolute quantification that does not require specialized equipment has yet to be developed. We here report the GEQO platform of quantitative biosensors featuring calcium, ATP, cAMP, and organelle-specific variants. We used GEQO sensors to follow calcium and cAMP transients in immortalised cells, human pancreatic progenitor cells, and zebrafish embryos. We show that GEQO-based absolute quantification allows to account for analyte buffering and retains information in time trace data lost during relative quantification. GEQO biosensors will enable quantitative analyte measurements across a wide range of imaging platforms, a key prerequisite for diagnostic applications and quantitative approaches in basic cell biology.

cell biology↗

bioRxiv2025

Disruption of Plasma Membrane Lipid Asymmetry Alters Cellular Energetics

Biological membranes often feature an unequal and actively maintained concentration gradients of lipids between bilayer leaflets, termed lipid asymmetry.1-3 Lipid asymmetry has been linked to many cellular processes4,5, but the mechanisms that connect lipid trans-bilayer concentration gradients to cellular functions are poorly understood. Here we systematically map the cellular processes affected by dysregulation of lipid asymmetry by knocking out flippases, floppases, and scramblases in mammalian cells. We identify broad alterations to core metabolic pathways including nutrient uptake, neutral lipid turnover, pentose phosphate pathway and glycolysis. Lipidomics, respirometry, lipid and metabolic imaging, and live-cell calorimetry revealed elevated neutral lipid and ATP consumption rates which are coupled with increased heat loss per unit of biomass synthesized despite slower growth. This suggests that compensatory maintenance of lipid asymmetry strains the cellular energy budget, inducing a shift from a growth-promoting anabolic to a more energy-using catabolic state. Our data indicate that lipid asymmetry and its active maintenance is key for proper cell energetics, highlighting its putative role as a cellular store of potential energy akin to proton and ion transmembrane gradients.

cell biology↗

bioRxiv2025

Quantification of lipid sorting during clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question if lipids are also sorted by this process. To address this question, we combined super-resolution STED imaging of bifunctional lipid probes with mathematical modeling. Quantification of 10 different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. We find that the lipid asymmetry in the plasma membrane is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not significantly contribute to lipid sorting compared to non-vesicular trafficking. More broadly, we believe that lipid super-resolution imaging will be a powerful approach to quantify lipid partitioning into membrane structures in cells.

cell biology↗

bioRxiv2023

Quantifying single cell lipid signaling kinetics after photo-stimulation

Studying the role of molecularly distinct lipid species in cell signaling remains challenging due to a scarcity of methods for performing quantitative lipid biochemistry in living cells. We have recently used lipid uncaging to quantify lipid-protein affinities and rates of lipid transbilayer movement and turnover in the diacylglycerol signaling pathway using population average time series data. So far, this approach does not allow to account for the cell-to-cell variability of cellular signaling responses. We here report a framework that allows to uniquely identify model parameters such diacylglycerol-protein affinities and transbilayer movement rates at the single cell level for a broad variety of structurally different diacylglycerol species. We find that lipid unsaturation degree and longer side chains generally correlate with faster lipid transbilayer movement and turnover and higher lipid-protein affinities. In summary, our work demonstrates how rate parameters and lipid-protein affinities can be quantified from single cell signaling trajectories with sufficient sensitivity to resolve the subtle kinetic differences caused by the chemical diversity of cellular signaling lipid pools.

cell biology↗