Regulatory Roles of Cellular Diacylglycerol
thesisposted on 22.10.2017, 00:00 by Svetlana P. Kurilova
Kinetics of diacylglycerol and C1 domain interaction Lipids in biological membranes play significant roles in cellular signaling. Lipids vary not only in head groups but also in acyl chains. It has been reported that the acyl chain of lipids plays an important role in membrane curvature and signal transduction. In this work we investigated the importance of the degree of unsaturation in the acyl chain of diacylglycerol (DAG) in its interaction with the C1 domain. Fluorescence quenching assay showed that DAG with a highly unsaturated acyl chain binds more tightly to the C1 domain than DAG with saturated acyl chains. Moreover, our single molecule tracking studies using supported lipid bilayers as a model system and NIH 3T3 cells explain the kinetic aspect of that binding. Although more unsaturated DAG binds C1 domain tighter it dissociates from the membrane faster than in case of saturated DAG. This finding can explain why unsaturated lipids participate in cell signaling when saturated lipids mainly play a role of bulk lipids. This work provides the new inside about the dynamics of protein-lipid interaction. Extracellular function of diacylglycerol The heterogeneous distribution of lipid molecules in biological membranes depends on a specific region and situation. Selective distribution between bilayer leaflets can be achieved through diffusion (flip-flop) across membranes. DAG is one these lipids which is able to flip-flop rapidly because of a small, uncharged head group. Since the precise cellular localization of DAG determines its function and activity, it is important to understand its distribution and dynamics within a membrane leaflet and between inner and outer parts of cellular bilayers. We developed DAG sensor using mutagenesis and chemical labeling, which allowed accurate quantification of the lipid. Using this sensor we were able to detect very low concentration of DAG at the plasma membrane. It was found that ATP activation in NIH 3T3 cells elevates DAG in plasma membrane 5-7 times. Preliminary data suggest a significant basal level of DAG. It is known that there are two major sources of DAG: from phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylcholine (PC) and both can be initiated by ATP. Physiological roles of exofacial DAG remain unknown. It requires further investigation to understand the flip-flop process and its initiation. We discuss the novel four-color imaging with application of ratiometric analysis that can resolve these questions.