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.

The continuous detection of enzyme activities and their application in medical diagnostics is one of the challenges in the translational sciences. Proteinases represent one of the largest groups of enzymes in the human genome and many diseases are based on malfunctions of proteolytic activity. Fluorescent sensors may shed light on regular and irregular proteinase activity in vitro and in vivo and provide a deeper insight into the function of these enzymes and their role in pathophysiological processes. The focus of this review is on Förster resonance energy transfer (FRET)-based proteinase sensors and reporters because these probes are most likely to provide quantitative data. The medical relevance of proteinases are discussed using lung diseases as a prominent example. Probe design and probe targeting are described and fluorescent probe development for disease-relevant proteinases, including matrix-metalloproteinases, cathepsins, caspases, and other selected proteinases, is reviewed.

Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gαs and Gαq, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gαs and Gαq resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gαs-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca(2+) signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.

Proteases such as neutrophil elastase (NE) and matrix metalloprotease 12 (MMP-12) are key factors in inflammatory processes and contribute the gradual destruction of extracellular lung matrix in chronic inflammation. We now hypothesize that activity levels of inflammation-relevant proteases may be useful indicators for the onset and progression of obstructive lung diseases such as cystic fibrosis or COPD. With the recently published small molecule FRET protease reporters NEmo and LaRee, for detection of NE and MMP-12 activity, respectively, it is possible to monitor protease activity at the single cell level.

Photoactivatable or “caged” derivatives of signaling molecules are important tools for manipulating intracellular events with spatial and temporal resolution. In their Communication on page 6330 ff., C. Schultz and co-workers demonstrate that regionally limited photoactivation simultaneously leads to a local protein kinase C and a global calcium response. Surprisingly, the orchestration of these effects seems to depend on the fatty acid composition of the released lipid.

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 Ca2+ spiking is an acute effect caused by PDP-induced activation of PP1. The Ca2+ 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.

Photoaktivierbare Derivate von Signalmolekülen sind wichtige Hilfsmittel für die räumlich und zeitlich aufgelöste Manipulation intrazellulärer Ereignisse. C. Schultz und Mitarbeiter demonstrieren in ihrer Zuschrift auf S. 6455 ff., dass eine regional beschränkte Photoaktivierung Proteinkinase C lokal aktiviert, während simultan der Calciumspiegel global ansteigt. Überraschenderweise scheinen diese Effekte von der Fettsäurezusammensetzung des freigesetzten Lipids abzuhängen.

Protein Kinase C delta (PKCδ), a member of the novel PKC family, is ubiquitously expressed and involved in many intracellular signal pathways. Compared to conventional PKCs, the translocation behavior of the nPKCs is unclear, although they also contain C1 and C2 domains. We found that, when co-expressing fluorescent protein fusion proteins of PKCα and PKCδ in the same cell, following "physiological" stimulation of the cells with ATP (100μM), PKCα translocated to the plasma membrane and PKCδ to intracellular membrane structures that we identified as the ER. In order to investigate whether C1 or C2 domains of the PKC were responsible for such a different re-distribution patterns, we designed several chimera PKCs by exchanging C1 and C2 domains between PKCα and PKCδ and analysing subcellular translocation with various stimuli. Chimera II and III with their α-C2 domain not only displayed rapid and Ca2+ dependent plasma membrane translocation but also retained the slower ER translocation of wt-PKCδ. In contrast, chimeras I and IV did not display ATP or Ca2+ dependent translocation while their PMA-induced behavior was still intact. These data demonstrated the independent translocation capabilities of C1 and C2 domains in PKCs. But we were still puzzled to why the C1 domains of PKCs cause different recruitment schemes. To further enlighten this, we designed constructs in which we swapped C1a and C1b domains of PKCα and PKCδ. Expression and translocation experiments unequivocally showed that the C1b rather than C1a domain of PKCδ determined ER targeting, which was further confirmed by single amino acid mutation in C1b. To elucidate the signalling cascade contributing to PKCδ translocation, several specific chemical compounds and genetic inhibitors were applied. The results strongly indicated that the Gαs-cAMP-EPAC signal pathway played an essential role in the ER targeting PKCδ translocation.

exceeded. Application of the gap-junction blocker carbenoxolone stopped pacing activity in the coupledmyocyte and/orHEK293/SCN5acell;washout of carbenoxolone restored activity. Hence, the currents from a IfþIK1-injected cell delivered to a cardiac myocyte (or another cell type) via gap junctions can generate spontaneous APs allowing the cell pair to function as a pacemaker unit. Pacing activity also was investigated artificially by electrically connecting two separate single cells via a dual-cell dynamic clamp, permitting varying of coupling conductancewhilemodulating If and/or IK1withineitherorbothof the twocoupledcells. These results demonstrate that the dynamic clamp can be used to study the determinants of pacemaker activity. Supported by HL28958, GM88180, GM088181.

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.

In this study we investigated putative correlations between key proteins of the Gq-coupled pathway and atrial arrhythmia (AA) in humans. For this purpose we used quantitative real time PCR to investigate transcription levels of various genes including: protein kinase C (PKC), phospholipase C and inositol-1,4,5-trisphosphate receptor. The mRNA was isolated from human auricles obtained from patients undergoing heart surgery that we divided into four groups: 1. patients with AA without cardio-specific medication 2. patients with AA receiving ACE- and/or beta-blocker 3. patients with sinus rhythm receiving ACE- and/or beta-blocker 4. patients with sinus rhythm without cardio-specific medication Initial analysis of the mRNA appeared to suggest significant alterations of gene transcription with respect to the groups (1-4). Gene transcripts that were up-regulated during disease were found to be down-regulated in patients receiving cardio-specific medication. During atrial fibrillation structural remodeling occurs. Thus we investigated whether such remodeling was also reflected in the transcriptional activity of tissue specific marker genes. We found that e.g. the transcriptional activity of the PKCα gene strongly correlated with markers for endothelial cells and fibroblasts but did not show any correlation with myocyte specific markers. This finding suggested that in human atria PKCα is not expressed in the myocyte. Instead, transcriptional signals of this gene product most likely solely originate from non-muscle cells. Results such as the one described strongly suggest that qPCR analysis of entire cardiac tissue ought to be interpreted in light of possible changes of the tissue composition that usually accompanies cardiac diseases. This work was supported by: German Science Foundation (DFG), SFB530 and KliFor196

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.

Streptococcus pneumoniae ist eine häufige Ursache der ambulant erworbenen Pneumonie. Lungenepithelzellen stellen eine wichtige Barriere gegen Atemwegskeime dar. Wir haben in der vorliegenden Arbeit untersucht, ob die Proteinkinase C (PKC) an der Regulation der Immunantwort gegen S. pneumoniae teilnimmt.

Pairing probes: Recently, we prepared several probes that monitor protein kinase C activities in living cells based on a pleckstrin fragment sandwiched between two fluorescent proteins, GFP2 and EYFP. Herein, we replaced the fluorescent proteins (FPs) with monomeric variants which resulted in nonfunctional probes. This suggested that fluorophore dimerization actively participated in probe performance.

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.

Calcium signals are ubiquitous in all living cells and require the readout of their information by proper detector proteins such as calmodulin. Another important readout of cellular calcium signals are the subfamily of conventional protein kinase Cs (cPKCs) which are activated by calcium and diazylglyzerol (DAG) produced following the activation of e.g. Gq-coupled membrane receptors. One important requirement of such readout mechanisms is their ability to detect the entire cellular calcium signalling toolkit, from global calcium waves to elementary calcium signals, such as sparks and puffs. This has, up to now, not been shown for any of those readout elements. We have previously shown that the cPKCα can read out all calcium signals generated in living cells, from calcium waves, spatially restricted calcium signals to elementary calcium signals.