Andrew G. Ewing

An overall emphasis of our research efforts is the development and application of bioanalytical chemistry techniques to small-volume and single cell neurochemistry, biology, and biophysics. The importance of this work to the scientific community lies in that it defines the limits for measurements in small biological environments and it provides a means to examine molecular mechanisms of neuronal function and dysfunction (illness) at a level difficult except with molecular biology techniques and with fluorescent chelating agents. Although these techniques are certainly very powerful and highly useful, the molecular methods we have been developing permit a much wider range of messengers to be examined. Small-scale electrochemical methods have been used in our laboratories to monitor catecholamine, ascorbic acid, glucose, and oxygen dynamics at the single cell level. A major emphasis has been qualitative and quantitative analysis of single exocytosis (release) events and development of an understanding of the molecular intricacies of these events. We are particularly interested in understanding the membrane mechanics driving vesicle opening, release of transmitter through the fusion pore, and the source of lipid allowing vesicles to contract and expand when treated pharmacologically.

Small-scale separations are under development for cellular and subcellular analysis. Capillary electrophoresis with electrochemical, fluorescence, and mass spectrometric detection has been used to monitor catecholamines, amino acids, peptides, and proteins in single cells. In addition, we have developed dynamic electrophoresis techniques that permit continuous separations from ultrasmall biological environments with a large effort in understanding the function of biogenic amines in the brain of the fruit fly Drosophila melanogaster in collaboration with Professor Kyung-An Han in the Penn State Biology Department.

Collaborating with the group of Nick Winograd in Chemistry, imaging with time-of-flight secondary ion mass spectrometry promises measurements of phospholipids in specific regions of neuronal membranes and peptides in single cross fractured vesicles in cells. Here we are particularly interested in understanding the structure and function of membrane segments of high curvature (vesicles, fusion pores, etc.).

The significance of this work to neuroscience and to human health lies in a better molecular, physiological and pharmacological understanding of neurotransmission, transport of neurotransmitter across membranes, and messengers regulating the neuroimmune axis. The potential to image membrane phospholipids with submicron spatial resolution provides the means to define the role of specific phospholipids in defining membrane structure during exocytosis and vesicle recycling. An artificial cell model using liposomes and lipid nanotubes has been developed, in collaboration with Owe Orwar's group at Chalmers in Sweden, and is being used to examine the biophysics and chemistry of exocytosis events and release via the fusion pore. Overall, analytical methods under development can be used for in work ranging from fundamental neuroscience to molecular mechanisms of disease.