Design and implementation of high-content imaging platforms: lessons learned from end user-developer collaboration.

Automated high-content screening and analysis (HCS/HCA) technology solutions have become indispensable in expediting the pace of drug discovery. Because of the complexity involved in designing, building, and validating HCS/HCA platforms, it is important to design, build, and validate a HCS/HCA platform before it is actually needed. Managed properly, collaboration between technology providers and end users in research is essential in accelerating development of the hardware and software of new HCS/HCA platforms before they become commercially available.

High-content screening of known G protein-coupled receptors by arrestin translocation.

G protein-coupled receptors (GPCRs) have proven to be one of the most successful target classes for drug discovery. Accordingly, many assays are available to screen GPCRs, including radioactive-binding assays, second messenger signaling assays, and downstream reporter assays. One of the more novel approaches is the Transfluor technology, a cell-based assay that uses a detectable tag on a cytosolic protein, called arrestin, that is involved in the desensitization or inactivation of GPCRs.

The ligand-independent translocation assay: an enabling technology for screening orphan G protein-coupled receptors by arrestin recruitment.

Finding natural and/or synthetic ligands that activate orphan G protein-coupled receptors (oGPCRs) is a major focus in current drug discovery efforts. Transfluor is a cell-based GPCR screening platform that utilizes an arrestin-green fluorescent protein conjugate (arrestin-GFP) to detect ligand interactions with GPCRs. The assay is ideally suited for oGPCRs because binding of arrestin-GFP to activated receptors is independent of the interacting G protein.

A large T cell invagination with CD2 enrichment resets receptor engagement in the immunological synapse.

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation.

Interface accumulation of receptor/ligand couples in lymphocyte activation: methods, mechanisms, and significance.

Cellular interaction is vital to the activation of most lymphocytes. At the interface between the lymphocyte and the cell that activates it, multiple receptor/ligand pairs accumulate in distinct patterns. This accumulation is intriguing, as it is likely to shape the quality of receptor signaling and thereby lymphocyte behavior.

Costimulation and endogenous MHC ligands contribute to T cell recognition.

To initiate an immune response, key receptor-ligand pairs must cluster in "immune synapses" at the T cell-antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-E(k), at a T cell-B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation.