Functional Promiscuity of Gene Regulation by Serpentine Receptors in Dictyostelium discoideum

Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1’s mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.Recent years have seen the discovery of critical roles in animal development for serpentine receptors, which are usually coupled to heterotrimeric G proteins. The insect sigaling peptides hedgehog and wingless and their mammalian counterparts sonic hedgehog, desert hedgehog, and indian hedgehog and the wnt factors control a multitude of inductive events during all stages of embryogenesis. The hedgehog signal is detected by two different serpentine receptors, smoothened (1, 40) and patched (21, 38), whereas the wingless or wnt signal is detected by the serpentine receptor D-frizzled-2 (3). In the social amoeba Dictyostelium discoideum, serpentine cyclic AMP (cAMP) receptors (cARs) control induction of cell differentiation during the entire course of development. Starving cells secrete cAMP pulses that induce chemotaxis and expression of genes required for the aggregation process. Cells aggregate to form mounds, which ultimately transform into fruiting structures that consist of a globular spore mass supported by a column of stalk cells. cAMP induces entry into the spore differentiation pathway as well as synthesis of a lipophilic factor, differentiation-inducing factor (DIF), which induces entry into the stalk differentiation pathway (see reference 5). At an early stage of development cAMP synergizes with DIF to induce prestalk genes, but later it becomes an inhibitor of stalk gene expression (2). cARs were shown previously to mediate induction of aggregative genes by cAMP pulses (20) as well as cAMP induction of prespore genes and repression of prestalk genes (31, 37). Remarkably, the target for the latter critical step in cell fate determination is glycogen synthase kinase 3 (GSK-3), a zeste white-3 homolog, which is the target for the effects of wingless and wnt in insects and vertebrates, respectively (7, 34).Four cARs, showing 54 to 69% amino acid identity, are expressed in a stage- and cell-type-specific manner. cAR1 is predominantly expressed before and during aggregation (18). cAR3 is expressed at late aggregation, and expression is later restricted to the prespore cell population (13, 44). cAR2 and cAR4 are both expressed exclusively in the prestalk cell population after aggregation (19, 30). cAR knockout cell lines were generated to examine the role of the individual cARs in Dictyostelium development. car1 null cells neither aggregate nor express developmental genes but can be triggered to express aggregative and postaggregative genes by stimulation with cAMP (37, 39). car3 null cells aggregate and develop normally (13). car1 car3 double gene disruptants do not aggregate, and developmental gene expression cannot be restored with cAMP, indicating that cAR1 or cAR3 shows functional redundancy and that either one or the other has to be present for gene induction to occur (10, 36). car2 null cells are blocked in the mound stage, while car4 null cells show abnormal slug morphogenesis and culmination. Both lines show reduced expression of prestalk genes and enhanced expression of prespore genes (19, 29).To understand the function of the four cARs, it is essential to know whether each receptor is coupled to a specific signal transduction pathway that controls a specific cell differentiation event or whether each receptor can activate multiple cell differentiation pathways. In the latter case, it is not the presence of a specific receptor that determines whether a response occurs but the availability of the downstream signaling pathway. To determine whether individual receptors have unique functions in developmental gene expression, we examined gene regulation in cell lines that display about equal levels of cAR1, cAR2, and cAR3 in a car1 car3 mutant background. Our results show that with two exceptions, all three receptors can transduce both the excitation and adaptation components of the different cAMP-regulated gene induction events with almost equal levels of efficiency.     

Dry mass changes in germinating spores of Diplodia maydis

Summary The dry mass of two-celled Diplodia maydis spores was measured both before and after germination by quantitative interference microscopy. The dry mass of spores declined approximately 50% during germination. However, the dry mass of germinating spores plus the dry mass of their germ tubes was greater than the dry mass of spores before germination. We conclude that the germinating spores absorbed nutrients released from non-germinating spores.The dry mass of fungal spores can be estimated by weighing large numbers of spores and determining the mean from sample spore counts. Mumford and Pappelis(4) determined the total dry mass of individual spores of Fusarium roseum and the contained lipid bodies before and after spores germinated using quantitative interference microscopy. The mean spore dry mass before germination was 57 pg. Lipid bodies accounted for about 61% of that mass and decreased as spores germinated. The total dry mass of the spore and germ tube 24 hr later greatly exceeded that of the spore before germination. Quantitative interference microscopy has been used to measure the dry mass of various types of cells. Kulfinski and Pappelis (3) recently reviewed how this technique has been applied to plant cells. Technical aspects of interference microscopy have been described by Ross (6).The purpose of this study was to examine the dry mass changes in Diplodia maydis (Berk.) Sacc. with and without germ tubes through the use of interference microscopy.     

Cellular Source and Mechanisms of High Transcriptome Complexity in the Mammalian Testis

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Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.     

Life history tactics shape amphibians’ demographic responses to the North Atlantic Oscillation

Over the last three decades, climate abnormalities have been reported to be involved in biodiversity decline by affecting population dynamics. A growing number of studies have shown that the North Atlantic Oscillation (NAO) influences the demographic parameters of a wide range of plant and animal taxa in different ways. Life history theory could help to understand these different demographic responses to the NAO. Indeed, theory states that the impact of weather variation on a species’ demographic traits should depend on its position along the fast–slow continuum. In particular, it is expected that NAO would have a higher impact on recruitment than on adult survival in slow species, while the opposite pattern is expected occur in fast species. To test these predictions, we used long‐term capture–recapture datasets (more than 15,000 individuals marked from 1965 to 2015) on different surveyed populations of three amphibian species in Western Europe: Triturus cristatus, Bombina variegata, and Salamandra salamandra. Despite substantial intraspecific variation, our study revealed that these three species differ in their position on a slow–fast gradient of pace of life. Our results also suggest that the differences in life history tactics influence amphibian responses to NAO fluctuations: Adult survival was most affected by the NAO in the species with the fastest pace of life (T. cristatus), whereas recruitment was most impacted in species with a slower pace of life (B. variegata and S. salamandra). In the context of climate change, our findings suggest that the capacity of organisms to deal with future changes in NAO values could be closely linked to their position on the fast–slow continuum.     

Uropathogenic E. coli induces DNA damage in the bladder

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.     

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

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Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the α2 → 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with ∼22% tri-antennary and most were core fucosylated (68%). About 13% contained α-galactose. In the lung, MR exhibited more terminal sialic acids in the α2 → 3- rather than in the α2 → 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11–q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.