Focus Issue: A Sharp Focus on Cell Signaling

Abstract

Among the techniques used to study signal transduction, imaging and microscopy have the undeniable appeal of allowing the experimentalist to see cellular structures and--with the advent of new techniques in live-cell light microscopy--to visualize dynamic interactions among molecules in cell signaling pathways. This week's Science and Science 's STKE collate material on imaging techniques in cell biology to bring this dynamic area into a sharp focus. Two Reviews in Science provide fundamental information on live-cell imaging. In an overview of live-cell imaging techniques, Stephens and Allan provide advice for researchers considering fluorescence microscopy and discuss such approaches as total internal reflection fluorescence microscopy (TIRFM), fluorescence resonance energy transfer (FRET), and photobleaching and photoactivation. In a complementary Review, Lippincott-Schwartz and Patterson trace the development and applications of green fluorescent protein (GFP) and its variants, which can be used to create chimeric proteins that can be expressed in cells and used to monitor protein localization and protein-protein interactions. In a Science Review, Weijer puts the focus squarely on signal transduction, emphasizing the spatial and temporal relationships among signaling molecules. Weijer discusses fluorescent approaches to visualizing the dynamic changes in intracellular messengers, such as calcium (Ca2+), nicotinamide adenine dinucleotide phosphate, and adenosine 3′, 5′ monophosphate (cAMP), as well as the translocation of signaling proteins, and cytoskeletal dynamics. The STKE Perspective by Bers emphasizes the highly localized changes in second messenger dyanamics, which are critical for regulating cellular functions. Bers highlights the insights gained from the use of small organic fluorescent Ca2+ indicators to study excitation-contraction coupling in cardiac myocytes and discusses this approach and complementary techniques, including FRET, electrophysiological measurements of Ca2+-sensitive proteins, and TIRFM, for measuring localized changes in Ca2+ and other second messengers. In a recent STKE Protocol, Fiala and Spall describe the application of FRET, using a calmodulin-GFP-based calcium sensor protein cameleon that was selectively expressed in Drosophila olfactory neurons, to measure in vivo brain activity. In Perspectives in the STKE Archive, Gaits and Hahn compare several fluorescence-based approaches to studying live-cell signaling dynamics, focusing on FRET biosensors, whereas Zacharias discusses possible problems arising from oligomerization of fluorescent proteins and how this can impact FRET measurements. Several other recent Protocols describe applications of GFP variants to live-cell imaging. In the current issue of STKE, Bunnell et al . discuss using GFP chimeras to monitor dynamic changes in signaling complexes in live T lymphocytes activated by antibodies fixed to a planar surface, as well as a fixed cell Protocol that employs GFP-labeled proteins in combination with immunofluorescence. This Protocol includes movies tracking fluorescently tagged proteins in activated cells. In a Protocol from the STKE archives, Tengholm et al . describe the simultaneous measurement of phosphatidylinositol 3-kinase activity and glucose transporter insertion into the plasma membrane using dual color TIRFM in single adipocytes and the application of this technique to study insulin signaling. For STKE protocols using GFP published a year or more ago, see the Editorial Guide by Gough from the last STKE Focus Issue on imaging in signal transduction. Powerful as these approaches may be for visualizing dynamic relationships among signaling molecules, live-cell light microscopy is not the only imaging technique that can provide valuable information on signaling mechanisms. A recent STKE Protocol by Zheng and Zagotta describes Patch-Clamp Fluorometry, a technique in which ion channels are selectively labeled on cysteines with sulfhydryl-reactive fluorophores so that localized changes in structure can be monitored by fluorescence microscopy, simultaneously with electrophysiological measurements of channel function. In an STKE Protocol in this issue, Prior et al . bring signaling into still sharper focus in an STKE Protocol using immunogold electron microscopy to visualize signaling domains on the cell surface. With this technique, the authors have observed cellular Ras signaling domains and determined the size and distribution of lipid rafts. In contrast to the preceding articles, which have tended to focus on cell signaling in individual cells--or subcellular regions--the Perspective by Ramm and Thomas discusses imaged-based approaches, generally based on fluorescence or luminescence assays, for high-throughput screening of pharmacological agents that impact signaling pathways. How is all of the signaling information garnered through such imaging techniques to be analyzed and correlated? Readers who have been inspired to set up various imaging techniques may want to take a detour on the way to the lab to consider the Viewpoint by Swedlow et al ., which discusses the Open Microscopy Environment, an approach to imaging bioinformatics encompassing the storage and analysis of optical microscope image data. Featured in This Focus Issue Related Resources at STKE