Day One:

Sunday, November 14, 2004

4-6pm    Early Registration

Monday, November 15

7:00am    Registration and Morning Coffee

8:30    Chairperson's Remarks
Dr. Andrew M. Bradbury, Staff Scientist, Los Alamos National Laboratory

8:40    Improving Spectral Properties of Engineered Fluorescent Proteins
Dr. Mark Prescott, Lecturer and Research Fellow, Department of Biochemistry and Molecular Biology, Monash University 
Detailed investigations of the GFP (green fluorescent protein)-like family of proteins have revealed novel biochemistry that underpin their intriguing and biotechnologically useful spectral properties. Photoconversion of particular proteins is promoted through light-induced polypeptide cleavage or decarboxylation reactions. Our studies using X-ray crystal structural analysis indicate that chromophores within the GFP-like family, in particular those with red-shifted spectral properties, adopt alternative conformations despite having the same chromophore sequence. We will discuss our most recent findings that suggest the conformation of the chromophore appears to influence the fluorescent properties the protein. This fundamental knowledge will allow fluorescent proteins with improved spectral properties to be engineered, adding to their biotechnological value.

9:10    GFP to Track GPCR Activation
Dr. Carson R. Loomis, Senior Vice President, Research, Norak Biosciences 
Norak Bioscience's Transfluor® technology provides a cell-based assay for high throughput screening of G protein-coupled receptors (GPCRs). Transfluor measures the redistribution of GFP-labeled ß-arrestin to the plasma membrane upon agonist activation and subsequent desensitization of the receptor. This redistribution has been shown to accurately detect agonists, partial agonists and antagonists of GPCRs in live or fixed cells. Transfluor has the advantage that it is nearly a universal assay for all GPCRs. Highly accurate concentration dependent curves are easily obtained with many of the cell-based image analyzers currently on the market. The presentation will show that GFP from different sources were found to provide surprising results specific to this technology. 

9:40    Using Flow Cytometric FRET Measurement to Study Signal Transduction of TNF Receptors 
Dr. Francis Ka-Ming Chan, Assistant Professor, Department of Pathology, University of Massachusetts Medical School 
Tumor necrosis factor (TNF) and its receptors are crucial regulators of diverse immune functions such as the induction of inflammation and cell death. These biological effects of TNF receptor signaling are mediated by multiple signal transduction components. Using flow cytometric measurements of FRET, we have examined the molecular interactions of TNF receptor signal transduction pathway. Our results revealed novel properties about the molecular requirements for TNF receptor signaling and demonstrate that flow cytometric FRET analysis is an effective tool to examine complex receptor signal transduction pathways.

10:10    Coffee Break and Poster Viewing

10:45    Novel Drug Screens using GFP/Small-Molecule FRET
Dr. George Hanson, R&D Scientist, Applied Chem, Invitrogen
Engineered mutants of GFP can serve as an excellent partner in fluorescent resonance energy transfer (FRET) with a variety of small-molecule fluorescent probes, including organelle-specific organic dyes and time-resolved lanthanide chelates. Several applications of GFP-FRET technology related to drug discovery will be presented: intramolecular labeling of GFP fusions using His tag-specific dyes, membrane localization assays using GFP fusions and lipophilic dye combinations, and a drug screen for a protein aggregation disease using a GFP fusion protein with terbium-linked antibody probes. The performance of these applications in plate-based formats, as well as the breadth of potential applications for this technology will be discussed.

11:15    Use of Fluorescent Proteins in Assay Development for High Content Image Based Screening
Dr. O. Joseph Trask, Jr., Senior Biochemist, Eli Lilly and Company 

11:45    Fluorescent Proteins in Automated Sub-Cellular Imaging for Drug Discovery
Dr. Stefan Prechtl, Group Leader, Imaging, SCHERING AG 
Industrial screening applications based on subcellular imaging prove to be useful for direct screening of drug effects on targets at the cellular level. Advances in fluorescence imaging methods and microscope systems offer the opportunity to investigate kinetic properties of proteins in living cells and to analyze the statistical distribution of this events within mixed cell populations. An increasing list of fluorescent dyes and target specific antibodies as well as autofluorescent proteins like green fluorescent protein (GFP) and its derivatives act as versatile reporters to monitor spatial and temporal phenomena in living cells. Their use in automated subcellular imaging technology facilitates the study of protein-protein interactions, intracellular signalling and gene expression at the single cell level and furthermore enables the quantification of their abundance. It remains to be seen, if this will mainly be done for target identification and validation or for high throughput screening (HTS) and subsequent compound profiling processes. All together, this provides an outstanding stimulus to investigate the impact of novel pharmacologic relevant substances in living cells which hopefully in turn will result in more effective and cost reduced drug screening. 

12:15    Luncheon Technology Workshop	Sponsored by
Cellular Imaging and High Content Screening Dr. Jeffery Cook, Scientist, GE Healthcare Cellular imaging technology is advancing rapidly and is quickly becoming a vital tool in both basic research and high throughput screening. GE Healthcare will present an overview of the IN Cell Analysis system of instruments, software ,reagents and professional services as well as some of the latest tools for studying cell cycle, protein translocation and morphology including: New Developer software- Versatile and flexible image analysis toolbox Functional screening of the cell cycle, apoptosis and micronuclei analysis

1:30    Chairperson's Remark

1:40    Visualization of Protein Interactions and Modifications in Living Cells Using Biomolecular Fluorescence Complementation Analysis
Dr. Tom K. Kerppola, Associate Professor, Department of Biological Chemistry, University of Michigan Medical School and Associate Investigator, Howard Hughes Medical Institute 
Protein interactions and modifications serve to integrate signals from different extracellular stimuli and developmental programs. We have developed a new approach, designated bimolecular fluorescence complementation (BiFC) analysis for the visualization of protein interactions and modifications in living cells. The BiFC approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments. This approach enables visualization of the subcellular locations of protein interactions and modified proteins in the normal cellular environment. We have used BiFC analysis to visualize interactions and modifications of a variety of structural classes of proteins. 

2:10    Towards a Global Pharmacology of the Cell 
Dr. Stephen Michnick, Associate Professor, Département de Biochimie, Université de Montreal 
The modular architecture and dynamics of biochemical networks provide a framework for determining the actions of environment and drugs on specific cellular processes. Using a fluorescence-based protein fragment complementation assay (PCA), we have observed spatially and temporally distinct effects of drugs within a class with activities beyond the intended target of the molecules. Visualizing drug effects in time and space reveals otherwise hidden mechanisms, potentially beneficial or harmful consequences of a molecule can be investigated, thus enhancing the efficiency of therapeutic discovery. 

2:40    The SNAP-tag: A Self Labeling Protein in Intracellular Studies
Dr. Andreas Brecht, Chief Technology Officer, Covalys Biosciences AG 
The SNAP-tag is a protein tag, which couples itself covalently and with high specificity to a well defined molecular structure. Substrates are guanine derivates substituted in the O6-position, from which the O6-alkylsubstituent is transferred in an SN2-reaction to a reactive Cysteine residue in the SNAP-tag. This results in the formation of a stable thioether bond and allows to label SNAP-tag fusion proteins under mild conditions, with defined stoichiometry, and high specificity with a wide range of SNAP-tag substrates. In intracellular applications cell membrane permeable fluorophore conjugates are used. Two important differences to autofluorescent proteins are that the time and the extent of labeling can be freely chosen and that sequential labeling with different fluorophors is possible, allowing to study dynamic processes within cells.

3:10    Refreshment Break and Poster Viewing in the Exhibit Hall

3:50     The HaloTag Fusion Reporter for Cellular Analysis and High-Content Screening
Dr. Keith V. Wood, Research Fellow, Promega Corporation
High-content screening can be augmented by the simultaneous analysis of multiple cellular markers, differentiable through a range of fluorescent colors. The HaloTag(tm) Fusion Marker provides a new option for highlighting subcellular structures designed to complement existing methods. HaloTag works in a manner similar to GFP except that the fluorophore can be interchanged among a variety of standard dyes. This allows living cells to be imaged at different wavelengths without requiringchanges to the underlying genetic constructs, and the colors can be rapidly switched to allow temporal analysis of protein fate. Because the dyes are covalently attached to the HaloTag, they retain their fluorescence evenunder denaturing conditions. This can be important for applications using chemically fixed cells, where the fluorescence of many IFP's such as dsRED can be quickly destroyed. The dyes can also be exchanged with other functional molecules, such as biotin to serve as an affinity handle. The interchangeability allows visualization of subcellular localization before these alternative groups are attached to the HaloTag.

4:20    Technology Workshop

Sponsored By:

Cell-based Applications of BD Living Colors™ Fluorescent Proteins, Including Novel Monomers Dr. Francine Yu Fang,BD Biosciences Clontech BD Biosciences Clontech will present recent developments of BD Living Colors™ Fluorescent Proteins in cell-based applications. The Novel Fluorescent Proteins include proteins isolated from both reef corals (RCFPs) and the jellyfish Aequorea coerulescens (AcGFP1). They represent the widest collection of fluorescent proteins currently available, with colors ranging from cyan to far red, and emission maxima from 488 nm to 618 nm. Topics presented will include new novel monomeric fluorescent proteins, stable cell lines including the ZsGreen Proteasome Sensor, and the detection of multiple fluorescent proteins using both confocal microscopic imaging and flow cytometry. Applications include: • Novel monomer fluorescent protein expression with fusion tags and subcellular targeting sequences • Use of fluorescent proteins for shRNA target sequence screening and validation • Use of fluorescent proteins as transcription reporters for gene expression • Monitoring proteasome activity in live cells

5:00    Selected Poster Presentation

5:25    Poster Session in the Exhibit Hall

6:15    Close of Day One