Membrane protein targeting and regulation by exocytosis-

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The Section on Cellular Biophotonics, of the Laboratory of Molecular Physiology, NIAAA, was established in April 2003. The principle aim of this section is to use imaging techniques to study how protein complexes, with special emphasis on complexes comprised of integral membrane proteins, are formed and maintained in living cells. Membrane proteins, such as receptors, channels, and their regulatory partners, are typically synthesized by ribosome?s associated with the endoplasmic reticulum, but ultimately function at the cell surface. Thus, the logistics of their production, assembly, transport, and exocytotic insertion into the cell surface is key to understanding both normal physiological function, and abnormal physiological states associated with human maladies. Furthermore, the delivery of membrane protein complexes to cell surface micro-domains must involve the coordination of exocytotic and endocytotic reactions. Lesions in exocytosis have been tied to some forms of muscular dystrophy, and may also be involved in alcohol induced myopathy. Drs. Srinagesh Koushik , and Christopher Thaler, joined the section in May 2003 as a Research Fellow, and as a postdoctoral IRTA respectively. Over the past six months we have primarily been involved in assembling and testing a new microscope capable of multi-photon laser scanning imaging, spectral imaging, and time-resolved fluorescence imaging. Of these three imaging modes, the first two are currently operational, and we expect the time-resolved fluorescence to be operational within the next two months. During this period, we have also initiated the following research projects in the section: 1. The generation of integral membrane protein constructs, labeled with green fluorescent protein variants on extracellular segments. These constructs will be used as markers for cell surface micro-domains, and the quenching of their intrinsic fluorescence by extracellular quenchers, will be used to monitor the dynamics of their delivery to the cell surface. 2. The evaluation of Zeiss 510 META spectral un-mixing algorithms in multi-photon and confocal modes for CFP-YFP fluorescence energy transfer (FRET) applications using purified GFP, CFP, YFP, and a CFP-YFP construct. 3. Determining the role of tyrosine phosphatase in regulating the uncoupling of constitutive exocytosis and its compensatory endocytosis during early sea urchin development. Additionally, we have also been involved in two collaborations that are associated with the primary aims of the section. In collaboration with Dr. Stephen Ikeda?s section, we have been involved in developing and evaluating genetically engineered reagents to monitor G-protein subunit localization and activation in living cells. With Dr. Margaret Davis (in Dr. Dave Lovinger?s Laboratory) we have been involved in developing methods for two-photon uncaging of photactivatible GFP constructs in living cells. This method will be used to study the dynamics of tyrosine-kinase activation and regulation of ion channels on the neuronal cell surface.