Gonzales, David T.Schuhmacher, Milena MariaLennartz, H. MathildaIglesias-Artola, Juan M.Kuhn, Sascha M.Barahtjan, PavelZechner, ChristophNadler, Andre2024-02-212024-02-212024-02-212023-12-1910.1016/j.bpj.2023.11.013https://infoscience.epfl.ch/handle/20.500.14299/205104WOS:001145665200001Studying 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 -pro-tein affinities and rates of lipid trans-bilayer movement and turnover in the diacylglycerol signaling pathway. This approach is based on acquiring live-cell dose-response curves requiring light dose titrations and experimental determination of uncaging photoreaction efficiency. We here aimed to develop a methodological approach that allows us to retrieve quantitative kinetic data from uncaging experiments that 1) require only typically available datasets without the need for specialized additional con-straints and 2) should in principle be applicable to other types of photoactivation experiments. Our new analysis framework al-lows us to identify model parameters such as diacylglycerol-protein affinities and trans-bilayer movement rates, together with initial uncaged diacylglycerol levels, using noisy single-cell data for a broad variety of structurally different diacylglycerol species. We find that lipid unsaturation degree and side-chain length generally correlate with faster lipid trans-bilayer movement and turn-over and also affect 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 the chemical of cellularLife Sciences & BiomedicineActivationtext::journal::journal article::research article