Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.
Photoreleaseo fc aged compoundsi sa mong the most powerful experimentala pproaches for studying cellularf unctions on fast timescales. However, its full potential has yett ob ee xploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, as traightforward, one-step procedure fore fficiently synthesizing caged compounds was developed.A n in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating variousc aged lipid metabolites, including a numbero fG PCR ligands. By combining the photo-caged ligandsw ith the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells wasd eveloped. Ultimately,t he described synthetic strategy allows rapid generation of photo-caged small moleculesa nd thus greatlyf acilitates the analysis of their biologicalr oles in live cell microscopy assays.
18Every cell produces thousands of distinct lipid species, but methodology for studying the biological 19 roles of individual lipids is insufficient. Using the example of diacylglycerols, prominent second 20 messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular 21 signal specification. We developed novel photo-caged lipid probes, which allow acute manipulation 22 of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with 23 mathematical modelling enabled the determination of binding constants for diacylglycerol-protein 24 interactions and kinetic parameters for diacylglycerol transbilayer movement and turnover in 25 quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of 26 magnitude due to diacylglycerol structural diversity. These differences are sufficient to explain 27 differential recruitment of diacylglycerol binding proteins and thus differing downstream 28 phosphorylation patterns. Our approach represents a generally applicable method for elucidating the 29 biological function of single lipid species on subcellular scales. 30 Intriguingly, a growing body of evidence suggests that changes in the levels of individual lipid species 43 rather than entire lipid classes determine cellular signalling outcome. For instance, early studies 44 reported that activation of individual cell surface receptors leads to the formation of molecularly 45 distinct patterns of diacylglycerol (DAG) species during signal transduction (13-15), suggesting that 46 crucial information could be encoded in the molecular spectrum of signalling lipids generated. 47Supporting this notion, drastically altered levels of distinct lipid species were correlated with cellular 48 processes, e.g. the increase of a phosphatidic acid ether lipid during cytokinesis (16) or the reciprocal 49 regulation of ceramide species during toll-like receptor signaling in innate immunity (17). DAGs 50 appear to be prime targets to study the importance of lipid heterogeneity in cell signalling, as they act 51 as second messengers at the plasma membrane and function in many cellular processes, including 52 insulin signalling, ion channel regulation and neurotransmitter release (18, 19). Many of these 53 processes involve effector proteins such as protein kinase C (PKC) isoforms, which are recruited to 54 cellular membranes by DAG binding to their C1 domains (20). Faithful process initiation thus 55 requires the activation of a subset of DAG effector proteins in the presence of others as observed 56 during the formation of the immunological synapse (21), but the molecular mechanisms of such 57 specific recruitment events are not well understood. Here, specificity could be provided by 58 differential activation of effectors by structurally distinct DAG species which recruit DAG binding 59 proteins due to differences in lipid-protein affinities, local lipid densities and lifetimes. Determining 60 these parameters requires quantitative experim...
Lipids are key components of cellular signaling networks yet studying the role of molecularly distinct lipid species remains challenging due to the complexity of the cellular lipidome and 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 cascade 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 aim to develop a framework that allows to quantitatively determine diacylglycerol-protein affinities and transbilayer movement at the single cell level. A key challenge is that initial uncaging photoreaction yields cannot be measured for single cells and have to be inferred along with the remaining model parameters. We first performed an in silico study on simulated data to understand under which conditions all model parameters are well identifiable. Using profile likelihood analysis, we found that identifiability depends predominantly on the signal-to-noise ratio. The impaired parameter identifiability due to experimental noise can be partially mitigated by increasing the number of single cell trajectories. Using a C1-domain-EGFP fusion protein as a model effector protein in combination with a broad variety of structurally different diacylglycerol species, we acquired multiple sets of single cell signaling trajectories. Using our analytical pipeline, we found that almost all species-specific model parameters are identifiable from experimental data. We find that higher unsaturation degree and longer side chains correlate with faster lipid transbilayer movement and turnover and higher lipid-protein affinities, with the exception of steaoryl-oleoyl glycerol, which noticeably deviated from the general trend. 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 signaling lipid pools.
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