Kinases are known to be integral to all cellular signal transduction, such as relaying receptor activation from the plasma membrane and modulating gene transcription. Despite the central role of kinases in cellular physiology, it has so far been very difficult to measure their activity in single, living cells. We propose to develop a method that will measure the activation of these essential enzymes in subcellular regions of a single cells, the oocyte of the frog Xenopus laevis. Xenopus oocytes and eggs are widely used throughout the biomedical sciences as a model to study ion channels, Ca/2+ waves, the cell cycle, fertilization, and many other aspects of cell function. Not only will the technique described in this proposal make measures in a single oocyte, but it will have the sensitivity and spatial resolution to detect subcellular activation of kinases in a defined region of the oocyte. This methodology draws upon principles from several different disciplines to develop a completely new paradigm in single cell measurement. Once developed the technique can be applied to other important model systems used in the study of cellular biology, such as the giant neurons of Aplysia or the eggs of the sea urchin. It addition it should be possible to measure many different kinases not only individually, but simultaneously in the same cell. The strategy will be to use fluorescently-labeled peptides that not specific kinases substrates as detectors of kinase activity. An oocyte containing the peptide will be sampled in a defined region with a capillary. The phosphorylated and non-phosphorylated peptide will be separated by electrophoresis through the capillary, and quantitated by their fluorescence. The ratio of the two peptide species is a direct measure of the activity of the kinase. The laboratory has made considerable progress in demonstrating the feasibility of this approach. Previous work demonstrates the ability to perform rapid, subcellular sampling followed by electrophoresis and detection of peptide in that sample. Peptide phosphorylated in vivo can be measured in a cytoplasmic sample obtained from a small region of an intact, living oocyte.
The specific aims of the work described in the following proposal are specifically directed at method development. They are the specification of the spatial and temporal resolution, determination of the detection limit, validation of the measurement of kinase activity, and measurement of the relevant kinetic properties of the archetypical kinases for the fluorescently-labeled peptide substrates.
