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. The section is comprised of Drs. Steven Vogel (Chief), Srinagesh Koushik (Research Fellow), Christopher Thaler (Postdoctoral IRTA), and Jose Fernando Covian-Nares (Visiting Fellow). The sections initial efforts have been in building and testing a laser scanning microscope specifically designed for studying protein-protein interactions in living cells. The microscope is a fully functional laser scanning two-photon microscope, with the additional capabilities of measuring florescent emission spectra (spectral imaging), fluorescent lifetime decays (FLIM), and fluorescent anisotropy lifetime decays (rFLIM). These added capabilities make it specifically useful for monitoring Forster?s Resonance Energy Transfer (FRET) between either dissimilar (Hetero-FRET) or similar (Homo-FRET) fluorophores. We have used this microscope to develop a new, photon efficient method for measuring FRET in living cells based on spectral imaging. This study has been accepted for publication in the Biophysical Journal (due out in October 2005) and will be highlighted on the journals cover. A second manuscript, evaluating how quantitative spectral imaging is for discriminating proteins labeled with either blue (CFP) or yellow (YFP) variants of green fluorescent protein (GFP) has been submitted and is currently under review. We are currently working on 5 projects in the lab. Three of them are methodological, and the last two utilize our microscopes unique capabilities to answer biological questions: 1. We are in the final steps of data collection for a study that describes the generation and characterization of a set of FRET standards to aid in the interpretation of FRET analysis. In the course of this study we have identified two important potential artifacts that can complicate the proper interpretation of FRET analysis. 2. We are also in the final data collection stage for a study describing the generation of Homo-FRET standards that will be used in interpreting anisotropy decay experiments. This relatively new method has the potential for monitoring how proteins form multimers, and their stoichiometry in living cells. The generation of fluorescent protein anisotropy standards required the generation of a mutant variant of GFP that has the same fundamental structure as GFP but is not capable of participating in FRET reactions. 3. The third methodological project investigates how FRET efficiencies change when multiple acceptors are present. This type of problem is important, because many proteins form multi-meric complexes, and the assembly and disassembly of these complexes might play an important role in regulating cell functions. The accepted mathematical formalism for dealing with this class of FRET problems assumes that the rates of energy transfer (to single acceptors) sum linearly when multiple acceptors are present. Our preliminary data suggests that this treatment underestimates the true FRET efficiency. We have developed a new theory for dealing with these problems based on probability theory. We are currently testing this new formalism. 4. The fourth project, in collaboration with Dr. Dave Lovinger?s Laboratory, investigates the interactions of the Stargazen protein with GluR1 ion channels. GluR1 has been shown to play a decisive role in synaptic efficacy, as activity causes GluR1 to migrate to dendritic spines in hippocampal CA1 neurons. Stargazin, a member of the transmembrane AMPA receptor regulatory protein family has also been shown to affect the trafficking of GluR1 as well as its kinetics. We are using spectral analysis, FLIM analysis, and anisotropy analysis to investigate the interactions of stargazing with GluR1, as well as their multi-meric structure. 5. Compensatory endocytosis is a distinct form of endocytosis that compensates for exocytotic activity. It plays an essential role in the maintenance of cell surface homeostasis. In our fifth project we have developed a new imaging based assay for testing the effects of either over expression of exogenous proteins, or down regulation of endogenous proteins, on compensatory endocytosis in developing sea urchin embryos. Using this assay we find that over expression of Src kinase, an oncogene, inhibits compensatory endocytosis. Inhibitors of tyrosine phosphatase had a similar effect, suggesting that the balance of tyrosine kinase and phosphatase activity plays a key role in how cells regulate their cell surface area. Cell proliferation, as observed in tumors, requires a constant increase in cell surface area to support multiple rounds of cell division. Our finding suggests that the increase in Src kinase activity observed in many tumors might disrupt cell surface homeostasis by inhibiting compensatory endocytosis.