Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.

Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells.

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).

Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

Cell cycle dependence of protein subcellular location inferred from static, asynchronous images.

Protein subcellular location is one of the most important determinants of protein function during cellular processes. Changes in protein behavior during the cell cycle are expected to be involved in cellular reprogramming during disease and development, and there is therefore a critical need to understand cell-cycle dependent variation in protein localization which may be related to aberrant pathway activity. With this goal, it would be useful to have an automated method that can be applied on a proteomic scale to identify candidate proteins showing cell-cycle dependent variation of location. Fluorescence microscopy, and especially automated, high-throughput microscopy, can provide images for tens of thousands of fluorescently-tagged proteins for this purpose. Previous work on analysis of cell cycle variation has traditionally relied on obtaining time-series images over an entire cell cycle; these methods are not applicable to the single time point images that are much easier to obtain on a large scale. Hence a method that can infer cell cycle-dependence of proteins from asynchronous, static cell images would be preferable. In this work, we demonstrate such a method that can associate protein pattern variation in static images with cell cycle progression. We additionally show that a one-dimensional parameterization of cell cycle progression and protein feature pattern is sufficient to infer association between localization and cell cycle.

Developmental Expression of Echinonectin, an Endogenous Lectin of the Sea Urchin Embryo.

Echinonectin (EN) is a galactose-binding lectin present in eggs and embryos of the sea urchin Lytechinus variegatus. Recent studies have suggested that EN is a hyaline layer protein that may function as a substrate adhesion molecule (SAM) during development. We have used monoclonal and affinity-purified polyclonal antibodies that specifically recognize this protein to determine its spatial and temporal expression during embryogenesis. EN is stored in granules or vesicles in the unfertilized egg. After fertilization, these granules are rapidly redistributed to the apical cytoplasm of the zygote. Our results show that at subsequent stages of development the lectin is expressed by cells of all three germ layers, including cells of the developing gut, coelomic pouches, and ectoderm, and by both primary and secondary mesenchyme cells. In contrast to previous observations based solely upon light level immunofluorescent staining, immunoelectron microscopy demonstrates that EN is localized in intracellular, membrane-bounded vesicles. In epithelial cell types these vesicles have a highly polarized distribution and are found in the apical cortical cytoplasm. In mesenchyme cells the distribution of EN-containing vesicles is not obviously polarized. Steady-state levels of EN protein in the embryo remain almost constant from fertilization to the pluteus larva stage, Metabolic labeling studies show that synthesis of EN in L. variegatus begins immediately after fertilization and continues throughout embryogenesis. Monospecific antibodies raised against L. variegatus EN have also been used to determine whether this lectin is expressed in other echinoid species.