Synthetic Notch Activation Patterns in a Proliferating Tissue.

Cell-cell communication through direct contact is essential during fundamental biological processes such as tissue repair and morphogenesis. Synthetic forms of contact-mediated cell-cell communication can generate custom gene expression outputs, making them valuable for tissue engineering and regenerative medicine. To precisely control the location and timing of synthetic signal outputs in growing tissues, it is necessary to understand the mechanisms underlying its spatiotemporal patterns. Towards this goal, we combine theory and experiments to study patterns of synthetic Notch (synNotch) activation - a custom synthetic gene circuit that we implement within Drosophila wing imaginal discs. We show that output synthesis and degradation rates together with cell division are the key minimal parameters that predict the heterogeneous spatiotemporal patterns of synNotch activation. Notably, synNotch output forms a graded exponential spatial profile that extends several cell diameters from the signal source, establishing evidence for signal propagation without diffusion or long range cell-cell communication. Furthermore, we discover that the shape of the interface between ligand and receptor cells is important in determining the synNotch output. Overall, we elucidate key biophysical principles that underlie complex emergent spatiotemporal patterns of synNotch output in a growing tissue.

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins.

The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. In the absence of ligand, a negative regulatory region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site;1,2 ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a "pulling force" on Notch that is essential to expose the cleavage site in the NRR.4-6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a "leucine plug" that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.

Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.

Changing directions in the study of chemotaxis.

Chemotaxis--the guided movement of cells in chemical gradients--probably first emerged in our single-celled ancestors and even today is recognizably similar in neutrophils and amoebae. Chemotaxis enables immune cells to reach sites of infection, allows wounds to heal and is crucial for forming embryonic patterns. Furthermore, the manipulation of chemotaxis may help to alleviate disease states, including the metastasis of cancer cells. This review discusses recent results concerning how cells orientate in chemotactic gradients and the role of phosphatidylinositol-3,4,5-trisphosphate, what produces the force for projecting pseudopodia and a new role for the endocytic cycle in movement.

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization.

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.

The pattern of intravenous drug administration during the transfer of critically ill children by a specialist transport team.

BACKGROUND: There are few published data on the patterns of intravenous drug administration by specialist pediatric intensive care unit (PICU) transport teams during the transfer of critically ill children between hospitals. METHODS: A retrospective review of retrieval documentation was undertaken for all patients transported by the Royal Manchester Children's Hospital PICU transport team over a period of 1 year. RESULTS: A total of 257 patients were transported during the study period, 82 patients (32%) were excluded owing to incomplete or absent documentation, leaving a sample of 175 available for analysis. Intravenous drugs were administered to 168 of these patients (96%). In total, 38 different drugs were administered. The four most commonly administered drugs were midazolam (130 patients), morphine (129 patients), atracurium (108 patients), and heparin (53 patients). Ten drugs accounted for 90% of all prescription episodes (total number of infusions and bolus doses administered), whilst 16 drugs were prescribed only once. The mean number of drugs administered per patient was 3.25 with a mean of 1.96 drug infusions and 1.29 bolus drugs administered per patient. CONCLUSIONS: A relatively small number of drugs are used frequently during the retrieval of critically ill children, but the total range of drugs that are used is large. This has implications for the rational carriage of drugs by PICU transport teams, the potential for drug errors and also for the development of advanced nurse practitioners whose prescribing-like activities may depend on the development of Patient Group Directions.

Blebbing of Dictyostelium cells in response to chemoattractant.

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.

Inducible gene silencing in podocytes: a new tool for studying glomerular function.

Glomerular filtration is one of the primary functions of the kidney. Podocytes, a highly specialized cell type found in glomeruli, are believed to play a critical role in that function. Null mutations of genes expressed in podocytes like WT1, nephrin, and NEPH1 result in an embryo and perinatal lethal phenotype and therefore do not allow the functional analysis of these genes in the adult kidney. Here is describes the generation of a model that will allow such studies. We have engineered transgenic mice in which the disruption of targeted genes can be induced in a temporally controlled fashion in podocytes. For this, a transgene encoding the mutated estrogen receptor-Cre recombinase fusion protein was introduced into the mouse genome. Animals were crossed with Z/AP reporter mice to test for efficient and inducible recombination. We found that, after injection of inducer drug tamoxifen, Cre fusion protein translocates to the nuclei of podocytes, where it becomes active and mediates recombination of DNA carrying loxP target sequences. These animals provide for the first time a tool for silencing genes selectively in podocytes of adult animals.

Reduction of chronic sorrow: a health promotion role for children's community nurses?

Families who have a child with a chronic illness face losses in their lives and react in a variety of ways. The theory of constuctivism is used to examine these losses. The ongoing nature of these losses can lead to ongoing grief rather than acceptance. This is discussed within the themes of time-limited grief and chronic sorrow. The role of children's community nurses in assisting families with resolving this sorrow is discussed, the main suggestions being the provision of an empathetic presence, time and the provision of accurate and specific information regarding the illness and the ways of managing it within the familys life.