One of the most significant endeavors of modern cell biology is to understand mechanisms that mediate signalling between and within cells. Notable examples presently under intensive study in our laboratories include: 1) the differentiation of ion channels and neurotransmitter receptors in embryonic neurons; 2) the interactions of immune cells with antigens and with their target cells; 3) the development of synaptic contacts in neural systems; 4) the regulation of cell-cell junctional coupling; 5) the packaging and directed transport of intracellular components; and 6) the assignment of cell phenotype during neural development. A group of faculty members representing these areas of interest will combine their expertise to develop a facility based on a laser-scanning confocal microscope and video imaging. Part of the recent revolution in cell biology is due to the development of fluorescent indicator dyes that are visualized using computer-enhanced microscopy techniques. These dyes permit quantitative measurement of intracellular ions, messenger molecules, membrane potential, and specific labeled membrane and intracellular constituents of living cells with temporal and spatial resolutions capable of faithfully monitoring the sites and time courses of intracellular events. We propose to develop the next generation of microscope-based video acquisition and analysis system with which we will investigate the three-dimensional distributions of second messenger molecules in cells in situ, and the kinetics of rapidly changing transduction events. This new system will incorporate improved optics, three-dimensional imaging capabilities, and a detector system capable of acquiring, analyzing, and storing processed images in real time. We will incorporate patch-clamp techniques, enabling resolution of conformational changes in single ion-channel proteins while measuring the three-dimensional distributions of intracellular messenger molecules. Photolysis of caged compounds will allow for rapid changes in the concentration of active second messengers. This facility will greatly enhance the existing capabilities and further the investigation of cell- signalling mechanisms.
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