Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Chemotaxis is one of the best characterised signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterised the properties of the chemosensory response of Rhodobacter sphaeroides, an -proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber’s law. Mathematical modelling also shows that, like E. coli, R. sphaeroides is capable of showing Fold-Change Detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber’s law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Key point summary • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia. • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice. • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’ du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit, α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell (GC) layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone-Purkinje cell(IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1Rmediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants; effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

CalDAG-GEFI is at the nexus of calcium-dependent platelet activation.

The importance of the second messengers calcium (Ca(2+)) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca(2+) sensor(s). We here identify Ca(2+) and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca(2+)-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A(2) (TxA(2)) release. CalDAG-GEFI-dependent TxA(2) generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca(2+)-induced integrin activation, TxA(2) generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI- and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.

Nutrient sensing and signalling in plants: Potassium and phosphorus

Potassium and phosphorus are important macronutrients for crops but are often deficient in the field. Very little is known about how plants sense fluctuations in K and P and how information about K and P availability is integrated at the whole plant level into physiological and metabolic adaptations. This chapter reviews recent advances in discovering molecular responses of plants to K and P deficiency by microarray experiments. These studies provide us not only with a comprehensive picture of adaptive mechanisms, but also with a large number of transcriptional markers that can be used to identify upstream components of K and P signalling pathways. On the basis of the available information we discuss putative receptors and signals involved in the sensing and integration of K and P status both at the cellular and at the whole plant level. These involve membrane potential, voltage-dependent ion channels, intracellular Ca and pH, and transcription factors, as well as hormones and metabolites for systemic signalling. Genetic screens of reporter lines for transcriptional markers and metabolome analysis of K- and P-deficient plants are likely to further advance our knowledge in this area in the near future.

Variation in the shoot calcium content of angiosperms

This study describes the variation in the mean relative shoot Ca content within the angiosperms at the ordinal level. Data were derived from studies in the literature in which the shoot Ca content of two or more species had been compared, and from a hydroponic experiment in which plants were selected to represent the relative number of species within each angiosperm order. Across all angiosperms, most of the variation in shoot Ca content occurred at and above the level of the order. Relative shoot Ca contents and variances correlated between literature and experimental data. In general, orders of commelinoid monocots had lower Ca contents than other monocot or eudicot orders. These results are used to illustrate how physiological and ecological hypotheses can be formulated using literature data.

Shoot calcium and magnesium concentrations differ between subtaxa, are highly heritable, and associate with potentially pleiotropic loci in Brassica oleracea

Calcium (Ca) and magnesium (Mg) are the most abundant group II elements in both plants and animals. Genetic variation in shoot Ca and shoot Mg concentration (shoot Ca and Mg) in plants can be exploited to biofortify food crops and thereby increase dietary Ca and Mg intake for humans and livestock. We present a comprehensive analysis of within-species genetic variation for shoot Ca and Mg, demonstrating that shoot mineral concentration differs significantly between subtaxa (varietas). We established a structured diversity foundation set of 376 accessions to capture a high proportion of species-wide allelic diversity within domesticated Brassica oleracea, including representation of wild relatives (C genome, 1n = 9) from natural populations. These accessions and 74 modern F-1 hybrid cultivars were grown in glasshouse and field environments. Shoot Ca and Mg varied 2- and 2.3-fold, respectively, and was typically not inversely correlated with shoot biomass, within most subtaxa. The closely related capitata (cabbage) and sabauda (Savoy cabbage) subtaxa consistently had the highest mean shoot Ca and Mg. Shoot Ca and Mg in glasshouse-grown plants was highly correlated with data from the field. To understand and dissect the genetic basis of variation in shoot Ca and Mg, we studied homozygous lines from a segregating B. oleracea mapping population. Shoot Ca and Mg was highly heritable (up to 40). Quantitative trait loci (QTL) for shoot Ca and Mg were detected on chromosomes C2, C6, C7, C8, and, in particular, C9, where QTL accounted for 14 to 55 of the total genetic variance. The presence of QTL on C9 was substantiated by scoring recurrent backcross substitution lines, derived from the same parents. This also greatly increased the map resolution, with strong evidence that a 4-cM region on C9 influences shoot Ca. This region corresponds to a 0.41-Mb region on Arabidopsis (Arabidopsis thaliana) chromosome 5 that includes 106 genes. There is also evidence that pleiotropic loci on C8 and C9 affect shoot Ca and Mg. Map-based cloning of these loci will reveal how shoot-level phenotypes relate to Ca 21 and Mg 21 uptake and homeostasis at the molecular level.

Distribution of calcium (Ca) and magnesium (Mg) in the leaves of Brassica rapa under varying exogenous Ca and Mg supply

Background and Aims Leafy vegetable Brassica crops are an important source of dietary calcium (Ca) and magnesium (Mg) and represent potential targets for increasing leaf Ca and Mg concentrations through agronomy or breeding. Although the internal distribution of Ca and Mg within leaves affects the accumulation of these elements, such data are not available for Brassica. The aim of this study was to characterize the internal distribution of Ca and Mg in the leaves of a vegetable Brassica and to determine the effects of altered exogenous Ca and Mg supply on this distribution. Methods Brassica rapa ssp. trilocularis ‘R-o-18’ was grown at four different Ca:Mg treatments for 21 d in a controlled environment. Concentrations of Ca and Mg were determined in fully expanded leaves using inductively coupled plasma-mass spectrometry (ICP-MS). Internal distributions of Ca and Mg were determined in transverse leaf sections at the base and apex of leaves using energy-dispersive X-ray spectroscopy (EDS) with cryo-scanning electron microscopy (cryo-SEM). Key Results Leaf Ca and Mg concentrations were greatest in palisade and spongy mesophyll cells, respectively, although this was dependent on exogenous supply. Calcium accumulation in palisade mesophyll cells was enhanced slightly under high Mg supply; in contrast, Mg accumulation in spongy mesophyll cells was not affected by Ca supply. Conclusions The results are consistent with Arabidopsis thaliana and other Brassicaceae, providing phenotypic evidence that conserved mechanisms regulate leaf Ca and Mg distribution at a cellular scale. The future study of Arabidopsis gene orthologues in mutants of this reference B. rapa genotype will improve our understanding of Ca and Mg homeostasis in plants and may provide a model-to-crop translation pathway for targeted breeding.

Antithrombotic actions of statins involve PECAM-1 signalling

Statins are widely prescribed cholesterol-lowering drugs that are a first-line treatment for coronary artery disease and atherosclerosis, reducing the incidence of thrombotic events such as myocardial infarction and stroke. Statins have been shown to reduce platelet activation, although the mechanism(s) through which this occurs is unclear. Since several of the characteristic effects of statins on platelets are shared with those elicited by the inhibitory platelet adhesion receptor PECAM-1, we investigated a potential connection between the influence of statins on platelet function and PECAM-1 signalling. Statins were found to inhibit a range of platelet functional responses and thrombus formation in vitro and in vivo. Notably, these effects of statins on platelet function in vitro and in vivo were diminished in PECAM-1-/- platelets. Activation of PECAM-1 signalling results in its tyrosine phosphorylation, the recruitment and activation of tyrosine phosphatase SHP-2, the subsequent binding of phosphoinositol 3-kinase (PI3-K) and diminished PI3-K signalling. Statins resulted in the stimulation of these events, leading to the inhibition of Akt activation. Together, these data provides evidence for a fundamental role of PECAM-1 in the inhibitory effects of statins on platelet activation, which may explain some of the pleiotropic actions of these drugs.

Enhancement in corneal permeability of riboflavin using calcium sequestering compounds

Ethylenediaminetetraacetic acid, ethylenediamine-N,N′-disuccinic acid and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid are polyaminocarboxylic acids that are able to sequester metal ions. Calcium is implicated in maintenance of intercellular matrix, zonula occludens (tight junctions) and zonula adherens of epithelium and endothelium cells. Corneal epithelium is impervious to many aqueous formulations due to it being lipophilic, whereby transcellular drug transit is resisted, whilst tight junctions restrict access via the paracellular route. Research has shown that integrity of tight junctions breaks down through loss of Ca2+ for endothelial and epithelial cells. This study investigates different Ca2+ sequestering compounds and their effect on corneal permeability of riboflavin at physiological pH. Riboflavin is a topically administered ocular drug applied during UV-induced corneal cross-linking for the treatment of keratoconus.

Use of mutants to dissect the role of ethylene signalling in organ senescence and the regulation of yield in Arabidopsis thaliana

The role of ethylene in regulating organ senescence in Arabidopsis has been investigated by studying the development of mutants that have an attenu- ated capacity to perceive the gas. The onset of leaf senescence and floral organ abscission was delayed in the ethylene-insensitive mutant etr1. The photosynthetic life span of rosette leaves was similarly extended in the gain- of-function mutant ers2, and this mutant also exhibited a delay in the timing of pod dehiscence primarily as a con- sequence of an extension in the final stages of senescence. A detailed analysis of yield revealed that whilst thousand grain weight was increased, by as much as 20 %, in etr1, ein4, and the loss-of-function mutant etr2, only the latter showed a significant increase in total weight of seeds produced per plant. The other studied mutants exhibited a reduction in total seed yield of almost 40 %. These observations are discussed in the context of the possible role of ethylene in regulating organ senescence and their significance in the breeding of crop plants with enhanced phenotypic characteristics.

Environmental controls on the production of calcium carbonate by earthworms

Lumbricus terrestris earthworms produce calcium carbonate (CaCO3) granules with unknown physiological function. To investigate carbon sequestration potential, the influence of temperature and CO2 concentration ([CO2]) on CaCO3 production was investigated using three soils, five temperatures(3-20 C) and four atmospheric [CO2] (439-3793 ppm). Granule production rates differed between soils, but could not be related to any soil characteristics measured. Production rates increased with temperature, probably because of higher metabolic rate, and with soil CO2 concentration. Implications for carbon sequestration are discussed. CaCO3 production in earthworms is probably related to pH regulation of blood and tissue fluid in the high CO2 environment of the soil.

EphB2 regulates contact-dependent and independent signalling to control platelet function

The Eph kinases, EphA4 and EphB1 and their ligand, ephrinB1 have been previously reported to be present in platelets where they contribute to thrombus stability. While thrombus formation allows for Eph-ephrin engagement and bidirectional signalling, the importance specifically of Eph kinase or ephrin signalling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signalling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signalling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together these data demonstrate that EphB2 signalling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Pharmacological actions of nobiletin in the modulation of platelet function

Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. The fibrinogen binding, α-granule secretion and calcium mobilisation assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore nobiletin may represent a potential antithrombotic agent of dietary origins.