Bas Ponsioen

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR–Dll4–Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

Detecting cAMP‐induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP–Epac–YFP fusion construct. When expressed in mammalian cells, CFP–Epac–YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP‐raising agents, whereas it fully recovers after addition of cAMP‐lowering agonists. Thus, by undergoing a cAMP‐induced conformational change, CFP–Epac–YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)‐based sensor, Epac‐based cAMP probes show an extended dynamic range and a better signal‐to‐noise ratio; furthermore, as a single polypeptide, CFP–Epac–YFP does not suffer from the technical problems encountered with multisubunit PKA‐based sensors. These properties make Epac‐based FRET probes the preferred indicators for monitoring cAMP levels in vivo.

A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Next-generation biosensors enable in vivo monitoring of ERK activity and detection of RhoA activity in small cellular extensions. Seeing Signaling in Action Biosensors consist of two fluorophores that produce a light signal when in close proximity and a “sensing module,” which is a protein (or protein fragment) that detects a signaling event, such as the activated state of a guanosine triphosphatase (GTPase) or the activity of a kinase. Producing optimal biosensors to monitor specific signaling events is challenging and time-consuming. Fritz et al. constructed a library of FRET vectors that enabled the rapid generation of highly effective biosensors and created an improved biosensor for RhoA GTPase activity that was used to detect spatial regulation of RhoA activity in filopodia and neuronal growth cones and another that monitors activity of the mitogen-activated protein kinase ERK and was used to detect the activity of this enzyme in living zebrafish. Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal–regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

8-pCPT-2′-O-Me-cAMP-AM: An Improved Epac-Selective cAMP Analogue

Cyclic adenosine monophosphate (cAMP) is a common second messenger involved in the regulation of many different cellular processes through the activation of protein kinase A (PKA), exchange protein directly activated by cAMP (Epac) and cyclicnucleotide-regulated ion channels. Adenylyl cyclases are ACHTUNGTRENNUNGresponsible for catalysing the formation of cAMP from ATP. Levels of cAMP can be raised in cells in response to a large variety of extracellular stimuli, which act via receptors coupled to heterotrimeric G proteins, which stimulate the activity of adenylyl cyclase. In addition, cAMP levels are controlled by phosphodiesterases (PDE), which catalyse the degradation of cAMP to AMP. In cells, cAMP levels can be artificially elevated by forskolin, which activates adenylyl cyclase directly. Furthermore, cAMP levels can be raised by inhibiting PDEs. These approaches are commonly used in tissue culture experiments, but, by generating cAMP, they do not discriminate between the various target proteins that are activated. Alternatively, membrane-permeable cAMP analogues, which selectively interact with particular receptor proteins, can be applied. For example, signalling pathways activated by Epac and PKA can be ACHTUNGTRENNUNGdistinguished by using 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP, respectively. Epac is a guanine nucleotide exchange factor for the small G protein Rap. Rap cycles between a signalling-inactive GDPbound state and a signalling-active GTP-bound state. cAMP-activated Epac catalyses the exchange of Rap-bound GDP for GTP. Epac and Rap function in a number of different cellular processes including insulin secretion, inhibition of cell scattering, neurotransmitter release and cAMP-induced barrier function in endothelial cells. Even though 8-pCPT-2’-O-Me-cAMP has become a widely used tool in Epac-related research, its biological application is limited by its low membrane permeability, caused by the negatively charged phosphate. However, the negatively charged singly bonded oxygen on the phosphate group can be masked by labile esters. Such a precursor is expected to enter the cell efficiently, where the ester is hydrolysed either directly by water or by cellular esterases to liberate the active compound. We therefore synthesised 8-pCPT-2’-O-Me-cAMP-AM from 8pCPT-2’-O-Me-cAMP, whereby acetoxymethyl bromide was used as a donor for the AM group. The product that was obtained had a purity exceeding 97% and consisted of a mixture of the equatorial and the axial isomers of the ester (Figure S1 in the Supporting Information, Scheme 1). Even though the isomers could be resolved by repetitive analytical HPLC runs, efficient separation on a preparative scale was not possible. Orange peel acetylesterase and esterase from porcine liver cleaved the equatorial isomer about five times more efficiently than the axial isomer within minutes (data not shown). The pharmacokinetics of both isomers are thus expected to be similar, justifying the application of a mixture of both isomers to cells. In any case, the isomeric ratio of an individual synthesis can be easily quality controlled by P NMR (Figure S1). To compare the efficiency of 8-pCPT-2’-O-Me-cAMP-AM and 8-pCPT-2’-O-Me-cAMP in activating Epac1 in vivo, an Epac1based fluorescence resonance energy transfer (FRET) probe was used. In this assay, activation of Epac1 by the binding of cAMP to the Epac1-FRET probe is measured as a reduction in the FRET signal. A431 cells transfected with the FRET probe were stimulated with 8-pCPT-2’-O-Me-cAMP-AM or 8-pCPT-2’O-Me-cAMP (Figure 1). Stimulation of cells with 100 mm 8pCPT-2’-O-Me-cAMP resulted in a decrease of the FRET signal that was approximately one order of magnitude slower than the decrease obtained upon stimulation with 1 mm 8-pCPT-2’O-Me-cAMP-AM. Furthermore, activation of Epac1 following stimulation with 100 mm 8-pCPT-2’-O-Me-cAMP could be further enhanced by the addition of forskolin, whereas 1 mm 8pCPT-2’-O-Me-cAMP-AM induced maximal activity of Epac1 under the given conditions. The activation of Epac by 8-pCPT2’-O-Me-cAMP-AM occurs within one minute after application. This is comparable with the kinetics of forskolin-induced Epac activation, and thus 8-pCPT-2’-O-Me-cAMP-AM mimics the “natural” response time of the signalling pathway. The activity of endogenous Epac can be monitored by isolating selectively Rap·GTP from cell lysates. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with different concentrations of 8-pCPT-2’-O-Me-cAMP and 8-pCPT-2’-OMe-cAMP-AM (Figure 2A). Partial activation of Rap was induced by 10 mm 8-pCPT-2’-O-Me-cAMP, and full activation of the G protein was stimulated by 100 mm 8-pCPT-2’-O-Me-cAMP. In contrast, treatment of the cells with just 0.1 mm 8-pCPT-2’-OMe-cAMP-AM was sufficient to induce full Rap activation. [a] M. J. Vliem, W.-J. Pannekoek, Dr. J. Riedl, M. R. H. Kooistra, Prof. Dr. J. L. Bos, Dr. H. Rehmann Department of Physiological Chemistry Centre for Biomedical Genetics and Cancer Genomics Centre University Medical Center Utrecht Universiteitsweg 100, 3584CG Utrecht (The Netherlands) Fax: (+31)88-75-68101 E-mail : j.l.bos@umcutrecht.nl h.rehmann@umcutrecht.nl [b] B. Ponsioen, Dr. K. Jalink Division of Cell Biology, The Netherlands Cancer Institute Amsterdam (The Netherlands) [c] Dr. F. Schwede, Dr. H.-G. Genieser BIOLOG Life Science Institute Flughafendamm 9a, 28071 Bremen (Germany) [] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Direct Spatial Control of Epac1 by Cyclic AMP

ABSTRACT Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

Cell–cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein–coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell–cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) from the plasma membrane. Knockdown of phospholipase Cβ3 (PLCβ3) inhibits PtdIns(4,5)P2 hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P2 depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P2 is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCβ3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P2-hydrolyzing receptors to inhibit cell–cell communication, but not for receptor–PLC coupling. Our results show that PtdIns(4,5)P2 is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCβ3 and Cx43 into a signaling complex to allow regulation of cell–cell communication by localized changes in PtdIns(4,5)P2.

Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

ABSTRACT Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

Spatiotemporal Regulation of Chloride Intracellular Channel Protein CLIC4 by RhoA

Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G(13)-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin, and sphingosine-1-phosphate. CLIC4 recruitment is strictly dependent on Galpha(13)-mediated RhoA activation and F-actin integrity, but not on Rho kinase activity; it is constitutively induced upon enforced RhoA-GTP accumulation. Membrane-targeted CLIC4 does not seem to enter the plasma membrane or modulate transmembrane chloride currents. Mutational analysis reveals that CLIC4 translocation depends on at least six conserved residues, including reactive Cys35, whose equivalents are critical for the enzymatic function of GSTs. We conclude that CLIC4 is regulated by RhoA to be targeted to the plasma membrane, where it may function not as an inducible chloride channel but rather by displaying Cys-dependent transferase activity toward a yet unknown substrate.

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression.