A new methodology for quantitative and spatiotemporal sensing of key signaling lipids.

摘要

Elucidating the spatiotemporal dynamics of signaling lipids in-vivo is of great importance in understanding a large array of signaling pathways. One lipid of particular importance is the ubiquitous second messenger, diacylglycerol (DAG). Originally, DAG was known to have only one protein receptor, Protein Kinase C (PKC). However, more recently several other families of downstream effectors of DAG have been found, namely RasGRP's, Mucnc13's, Chimaerins and Diacylglycerol Kinases. With so many receptors, and with each potentially playing multiple roles in signaling, the question arises---how is DAG able to regulate so many signaling pathways with an apparent selectivity? The only way to successfully answer this question is to understand, quantitatively, the spatiotemporal dynamics of DAG, upon stimulation of the cell.;To achieve this goal a new methodology for quantitatively sensing the spatiotemporal dynamics of DAG has been established. A high-affinity DAG-specific binding domain has been successfully labeled with an environmentally sensitive fluorophore. The labeled domain binds DAG in a 1:1 ratio and penetrates the membrane, exposing the fluorophore to the hydrophobic acyl chains of the bulk lipids. This in turn changes the local environment of the fluorophore, providing a dramatic blue shift and intensity increase in fluorescence signal. From this change in signal, a ratiometric analysis of the lipid-sensor can provide quantitative information about the formation of DAG in the membrane. Also, the concentration of this sensor can be easily controlled, making it susceptible to Fluorescence Correlation Spectroscopy (FCS) analysis.;In designing an effective and practical lipid-sensor, the greatest challenge was in introducing the fully functional lipid-sensor to the intracellular space of living cells. This challenge was overcome by lowering the positive charge density of a large cationic patch, found on the surface of the lipid-binding domain, by means of site-directed mutagenesis. This ultimately lowered non-specific interactions between the lipid-sensor and negatively charged delivery systems. The general approach of this method may be applied to other lipid-binding domains, which also tend to have a cationic patch, paving the way for the design of several other functional sensors for a variety of important signaling lipids.