In neurons, enzymes, such as kinases, phosphatases, and proteases are key molecules that regulate and connect signaling pathways. These enzymes are known to be involved in almost every aspect of neuronal function from differentiation to synaptic plasticity, aging, and response to injury. The overarching goal of the proposed research is to gain quantitative measures of the activities of intra-neuronal enzymes to test current theories of neuronal signaling and function and to guide future theory and understanding. This has become possible with the advent of a new combination of instrument and assay referred to as the laser-micropipet system (LMS). Use of the LMS begins with the intracellular introduction of fluorescently-labeled peptide substrates that are reporters of the activities of specific enzymes. After reporter molecule loading and any other experimental conditions, such as electrical stimulation, the LMS is used to lyse the neuron and terminate the cellular chemical reactions on a sub-second timescale. The LMS also collects the contents of the single neuron, separates the contents by capillary electrophoresis and detects (with very high sensitivity) the fluorescent substrate and product molecules, by laser-induced fluorescence. The substrates and products are identified by their electrophoretic mobilities and a quantitative measure of enzyme activity comes from the ratio of their fluorescence peak areas. The first hypothesis to be tested is whether Ca2+/calmodulin dependent kinase II (CamKII) acts, in vivo, as a Ca2+ signal frequency decoder within neurons when all of the other intracellular signaling mechanisms remain intact.
| Meredith, Gavin D; Wu, Hayley Y; Allbritton, Nancy L (2004) Targeted protein functionalization using His-tags. Bioconjug Chem 15:969-82 |
