Gregor Reither

The fatty acid composition of diacylglycerols determines local signaling patterns.

Cellular signals are transduced through vast networks of proteins and small molecule metabolites. Rigorous control of the respective signaling molecules is required to ensure a precise and reproducible outcome. On the protein level this is often accomplished by specific reversible chemical modifications such as phosphorylation or by localization of proteins to defined cellular compartments. 2] Much less is known about cellular mechanisms that control small-molecule-mediated signaling events. This is largely due to the intrinsically more difficult observation of small-molecule turnover and localization in living cells. These difficulties are potentiated when lipid signaling is investigated. The variety of known lipid backbones is fairly comprehensive but the diversity and combinations of fatty acids attached to these backbones provides many thousand possibilities and lipidomics shows that a large portion of this diversity is available in cells. This overwhelming and generally not addressable complexity has led to a situation where lipid signaling events are treated as head-group signaling events and the existing chemical differences between individual species of the same lipid class are widely ignored although a number of in vitro studies suggest significant differences in potency. Along the same lines, the influence of subcellular concentration gradients of defined lipid species on intracellular signaling has not been studied thoroughly so far. We hypothesized that both lipid species diversity and subcellular concentration gradients of distinct lipid species might serve as molecular mechanisms to drive specific lipid-mediated signaling events. Experimentally, both fatty acid diversity and locally elevated levels of a given species may be generated by using photoactivatable lipids in intact cells. We chose to analyze diacylglycerol (DAG) signaling due to its important role in several cellular signaling pathways that include G-protein coupled receptors as well as growth factor triggered and calcium-based signaling networks. Recent lipidomics analyses demonstrated the co-existence of 30–50 DAG species with different fatty acid compositions in mammalian cells. While DAGs are best known to activate various protein kinase C (PKC) isoforms by binding to their C1 domains and recruiting them to cellular membranes, DAG-induced translocation and activation of proteins such as RasGRPs, Munc13, and DGKg have also been described. In addition, DAGs have been shown to directly activate human transient receptor potential C3 (TRPC3) and TRPC6 channels. So far, locally elevated DAG levels have either been experimentally achieved by liberation of a photoactivatable T-cell receptor agonist which causes downstream DAG production or by local uncaging of a nonphysiological DAG analogue. 23] While these approaches have led to important insights into the mechanism of T-cell receptor mediated signaling and microtubule-organizing center (MTOC) polarization in T-cells, they cannot be utilized

Protein Kinase C: The “Masters” of Calcium and Lipid

The coordinated and physiological behavior of living cells in an organism critically depends on their ability to interact with surrounding cells and with the extracellular space. For this, cells have to interpret incoming stimuli, correctly process the signals, and produce meaningful responses. A major part of such signaling mechanisms is the translation of incoming stimuli into intracellularly understandable signals, usually represented by second messengers or second-messenger systems. Two key second messengers, namely the calcium ion and signaling lipids, albeit extremely different in nature, play an important and often synergistic role in such signaling cascades. In this report, we will shed some light on an entire family of protein kinases, the protein kinases C, that are perfectly designed to exactly decode these two second messengers in all of their properties and convey the signaling content to downstream processes within the cell.

Fluorescence and bioluminescence procedures for functional proteomics

This review aims to provide an overview of current optical procedures used in functional proteomics, investigating protein localization, protein–protein interaction, intracellular signaling events, and second messenger generation in living cells. Reporter assays using proteins tagged with fluorescent or bioluminescent moieties are discussed. Recently, intracellular biosensor assays, flow cytometry‐based techniques (fluorescent cell barcoding), as well as transfected cell microarray assays involving RNA interference coupled with automated imaging were introduced and have been adopted as screening platforms for annotating small molecules, investigating signaling events, or in phenotype analysis. These novel methodological advances include improved image acquisition and processing techniques and help linking in vitro observations to in vivo processes. In addition, the acquired data are increasingly quantitative in nature and will therefore pave the way for modeling of signaling cascades and other complex cellular events, an important step toward systems biology.

Contribution of fluorophores to protein kinase C FRET probe performance.

Intracellular signaling is frequently monitored by using fluorescent molecules, especially when high temporal and spatial resolution is required. The various designs include simple fusion proteins that translocate from the membrane to the ACHTUNGTRENNUNGcytosol or vice versa, sensors that change in fluorescence ACHTUNGTRENNUNGintensity after binding to a ligand, or probes with two fluorophores that exhibit Fçrster resonance energy transfer (FRET). 7, 8] The last kind can be used for ratiometric imaging, which has the advantage of being fairly independent of probe concentration. Many FRET probes consist of a construct with two genetically encoded fluorescent proteins, most often ECFP and EYFP. All naturally occurring fluorescent proteins have the tendency to oligomerize, with varying affinities and distinct stoichiometry. Concern has been raised that this characteristic could negatively influence probe performance. Fluorescent proteins from the Aequorea family, including CFP and YFP, have been widely used in probe design, because of their relatively low tendency to dimerize. Introduction of a single point mutation, A206K, renders these fluorescent proteins monomeric while retaining the same photophysical properties, thus making them ideal for use in many biological applications. Alternatively, extensive modifications of the originally tetrameric Discosoma sp. red fluorescent protein DsRed have generated a number of monomeric proteins fluorescing in the red area of the optical spectrum. Recently, we developed two FRET probes, KCP-1 and KCP-2, for monitoring protein kinase C activities in living cells. Both are based on a pleckstrin fragment sandwiched between two fluorescent proteins, GFP and EYFP. The probes comprise a N-terminal pleckstrin homology domain (PH1) linked to a DEP domain by a PKC-sensitive substrate loop and a C-terminal linker region (KCP-1: aa 1-239; KCP-2: aa 1-221; Figure 1A). In in vivo measurements, the EYFP/GFP emission ratio is followed over time. The longer construct, KCP-1, responds to phosphorylation by protein kinase C with an increase in emission ratio of about 10–15% which is fully reversible by adding the PKC inhibitor Gç6983 (Figure 1B). Mutants in which all PKC-sensitive phosphorylation sites Ser, Thr, and Ser were replaced by alanine residues were nonresponsive. The shorter version, KCP-2, lacking the C-terminal linker region, displays a decrease in emission ratio of 15% upon PKC activation. The dimerization constant of the two fluorophores in solution is expected to be around 110 mm. Intramolecularly, the effective concentration of fluorophores is much higher and dimerization is more relevant. It was previously shown that the use of monomeric fluorescent proteins resulted in improved FRET probe performance, for example for monitoring mechanotransduction with a Src sensor. However, in other cases, the dimerization of fluorophores might be beneficial, as was shown for the protease sensor CLY9. Therefore in this work, we explored the effect of dimerization on PKC probe performance. We first equipped both constructs, KCP-1 and KCP-2, with nondimerizing fluorophores by introducing an A206K mutation. The fluorescent proteins carrying the mutation were named mGFP or mYFP, respectively. Surprisingly, mutants carrying A206K mutations in both or only one fluorescent ACHTUNGTRENNUNGprotein were close to nonresponsive. As depicted in Figure 1B, the original KCP-1 probe showed an increase of emission ratio whereas the original KCP-2 probe responded with a decrease in emission ratio upon PKC stimulation by a phorbol ester (PMA). Nondimerizing KCP-1, however, resembled KCP-2 in its response pattern, albeit with a smaller amplitude. Monomeric sensors based on KCP-2 did not change the direction of the signal, but gave a much weaker signal than the original KCP-2 probe (Figure 1B). These observations were independent of which of the fluorescent proteins (or both) was monomeric. In addition, when the monomeric fluorescent proteins mOrange and mCherry were used, no significant FRET change was ACHTUNGTRENNUNGobserved in either the KCP-1 or KCP-2 constructs (Figure 1C). These results suggest that fluorophore dimerization significantly contributes to probe performance of KCP-1 and KCP-2. The mOrange/mCherry FRET pairs exhibited low starting FRET (Figure S1C), which probably further prevents significant ratio changes by this FRET pair. By performing acceptor photobleaching experiments in living cells, we confirmed that KCP-1 has higher FRET efficiency after phosphorylation (27%). Before this event, FRET is reduced to the level of nondimerizing fluorophores (17%; Figure 2). Original KCP-2 gave higher starting FRET efficiencies than after phosphorylation or in its monomeric versions, likely due to initial dimerization of the fluorophores. The original dimerizing KCP-2 has a FRET efficiency of 28%, which decreases to 17% after phosphorylation. Monomeric constructs of KCP-2 exhibited FRET levels of 19–20% and no significant change following phosphorylation. This corresponds well with the idea of a sensor molecule in which fluorophore dimerization serves as a clamp pulling both ends of the molecule together. Phosphorylation breaks the pulling force and the fluorophores separate from each other leading to a decrease in FRET (Figure 3C). In KCP-1, however, the fluorophores are initially unable to dimerize in a fashion favorable for FRET (see below). Only after phos[a] Dr. C. A. Jost, Dr. G. Reither, Priv. Doz. Dr. C. Schultz EMBL Heidelberg Meyerhofstrasse 1, 69117 Heidelberg (Germany) Fax: (+49)6221-387-206 E-mail : schultz@embl.de [b] Dr. C. Hoffmann Universit6t W8rzburg, Institut f8r Pharmakologie Versbacher Strasse 9, 97078 W8rzburg (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Chemical Activators of Protein Phosphatase-1 Induce Calcium Release inside Intact Cells

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase that is involved in numerous cellular processes. PP1-disrupting peptides (PDPs) are selective chemical tools used to study PP1. They generate catalytically active PP1 inside cells but do not bind to the closely related PP2A. Here, we show that PDPs also do not act directly on PP2B, thus demonstrating the selectivity of PDPs toward PP1. We present PDPs with different properties, enabling reversible versus permanent activation of PP1. We also show that Ca(2+) spiking is an acute effect caused by PDP-induced activation of PP1. The Ca(2+) is released from internal stores. Our data show that PDPs can be used as selective chemical genetics tools to study acute and long-term effects of PP1 activation in intact cells, and PDPs will therefore be valuable tools to study PP1 biology.