Isoform-specific induction of nuclear free calcium oscillations by platelet-derived growth factor.

Confocal laser scanning microscopy was used to analyze alterations in nuclear free calcium (Ca2+n) levels induced by platelet-derived growth factor (PDGF) isoforms in BALB/c3T3 fibroblasts loaded with the calcium-sensitive fluorescent indicator Fluo-3. Both AA-PDGF and BB-PDGF caused a transient increase in Ca2+n. Analysis of PDGF-induced Ca2+n alterations as a function of time revealed that BB-PDGF stimulation resulted in the generation of Ca2+n oscillations that diminished over time. The frequency of BB-PDGF-stimulated oscillations was modulated by extracellular Ca2+ and could not be mimicked by increasing intracellular inositol 1,4,5-trisphosphate levels in the absence of growth factor stimulation. Caffeine alone had no effect on Ca2+n levels, but exposure of cells to caffeine after BB-PDGF stimulation augmented Ca2+n oscillations, either by increasing the frequency or reinitiating preexisting oscillations. The genesis of these oscillations in Ca2+n appears to be in the region just outside of the nucleus, as perinuclear cytoplasmic free calcium (Ca2+i) increased just prior to Ca2+n. In contrast, AA-PDGF stimulation resulted in the generation of one or two irregular, transient Ca2+n spikes. Caffeine pretreatment followed by AA-PDGF stimulation resulted in Ca2+n oscillations very similar to those produced by BB-PDGF alone. Additionally, the AA-PDGF and BB-PDGF isoforms appeared to modulate distinct pools of cellular Ca2+, as BB-PDGF was still capable of inducing Ca2+n oscillations subsequent to prior induction of oscillations by AA-PDGF/caffeine. These PDGF isoform-specific changes in nuclear free Ca2+ could serve as a mechanism by which isoform-specific cellular signaling pathways may be manifested by the growth factors

Plant calcium dynamics: signalling the way to sustainable food production

In this work we explore the calcium signalling pathway mediating symbiotic associations between legumes and Nitrogen-fixing bacteria. This symbiotic process has been identified as a possible 'biological' alternative to using environmentally damaging synthetic fertilisers in crop production. Therefore, motivated by the challenge of ensuring worldwide food security whilst utilising increasingly degraded soils, we look to advance the knowledge of the signalling pathway that facilitates nodulation in order to progress towards the transfer of this favourable trait from legumes to cereal crops. A mathematical model representing the flux and diffusion of calcium in the plant cell nucleus was derived for up to 3-dimensions in both spherical and prolate spheroidal coordinates. Our final model is the first 3-dimensional representation of nuclear plant calcium signalling. It is also the first to reproduce the exact spiking dynamics reported in the literature, without the need for any additional inputs such as refractory periods or buffers. In a novel and exciting finding, we show that nuclear calcium oscillations can be generated autonomously provided that the channels which facilitate them form clusters. The resulting calcium signatures are presented for clustered and distributed channels and we examine the microdomains over which these patterns occur. It is also found that the formation of these microdomains is necessary for calcium concentrations to exceed the binding threshold of downstream sensory protein CCaMK. This provides new insight into the possible mechanism of generation of the nuclear calcium signature, suggesting a potential multi-functional role for cluster formation. The mathematical method through which this was achieved involved solving the heat equation semi-analytically in order to obtain a series of Green's functions. This provides us with a useful 3-dimensional framework which allows us to perform simulations significantly faster than could be achieved through traditional finite-element methods. An experimental methodology, using plant lines expressing GCAMP suspended in a FlowCell device, was developed in order to successfully image calcium signalling over longer times, with easier stimulus application. This was used to compare the calcium response in \textit{Arabidopsis thaliana}, hereby Arabidopsis, with \textit{Nicotiana benthamiana} highlighting the difference in calcium signalling between species with and without nodulation abilities. Original data on the geometry of the nucleus was collected for parameterisation of our model. Analysis of this data revealed statistically significant differences in nuclear morphology between cortical and root hair cells and also revealed a correlation between the nuclear morphology of cells in the elongation zone and their distance from the root apex

Plant calcium dynamics: signalling the way to sustainable food production

In this work we explore the calcium signalling pathway mediating symbiotic associations between legumes and Nitrogen-fixing bacteria. This symbiotic process has been identified as a possible 'biological' alternative to using environmentally damaging synthetic fertilisers in crop production. Therefore, motivated by the challenge of ensuring worldwide food security whilst utilising increasingly degraded soils, we look to advance the knowledge of the signalling pathway that facilitates nodulation in order to progress towards the transfer of this favourable trait from legumes to cereal crops. A mathematical model representing the flux and diffusion of calcium in the plant cell nucleus was derived for up to 3-dimensions in both spherical and prolate spheroidal coordinates. Our final model is the first 3-dimensional representation of nuclear plant calcium signalling. It is also the first to reproduce the exact spiking dynamics reported in the literature, without the need for any additional inputs such as refractory periods or buffers. In a novel and exciting finding, we show that nuclear calcium oscillations can be generated autonomously provided that the channels which facilitate them form clusters. The resulting calcium signatures are presented for clustered and distributed channels and we examine the microdomains over which these patterns occur. It is also found that the formation of these microdomains is necessary for calcium concentrations to exceed the binding threshold of downstream sensory protein CCaMK. This provides new insight into the possible mechanism of generation of the nuclear calcium signature, suggesting a potential multi-functional role for cluster formation. The mathematical method through which this was achieved involved solving the heat equation semi-analytically in order to obtain a series of Green's functions. This provides us with a useful 3-dimensional framework which allows us to perform simulations significantly faster than could be achieved through traditional finite-element methods. An experimental methodology, using plant lines expressing GCAMP suspended in a FlowCell device, was developed in order to successfully image calcium signalling over longer times, with easier stimulus application. This was used to compare the calcium response in \textit{Arabidopsis thaliana}, hereby Arabidopsis, with \textit{Nicotiana benthamiana} highlighting the difference in calcium signalling between species with and without nodulation abilities. Original data on the geometry of the nucleus was collected for parameterisation of our model. Analysis of this data revealed statistically significant differences in nuclear morphology between cortical and root hair cells and also revealed a correlation between the nuclear morphology of cells in the elongation zone and their distance from the root apex

Modulation of calcium signalling in human sperm by nitric oxide

Nitric oxide (NO) generation by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilization. In vitro studies were undertaken to assess the ability of human sperm and cumulus cells (surrounding the oocyte) to generate NO, investigate the mechanism of action of NO, the NO-mediated [Ca$^{2+}$]$_i$ signalling pathways, the possible interaction of NO with progesterone (a product of cumulus) and its impact in the regulation of human sperm functions. Immunofluorescent staining revealed constitutive NOS in human cumulus. DAF-FM diacetate staining demonstrated NO production by cumulus cells. Human sperm exposure to NO donors caused mobilization of stored Ca$^{2+}$ by a mechanism not requiring guanylate cyclase activation but mimicked by S-nitrosoglutathione (GSNO; an S-nitrosylating agent). Dithiothreitol application, to reduce protein –SNO groups, rapidly reversed the actions of NO and GSNO on [Ca$^{2+}$]$_i$. The effects of NO, GSNO and dithiothreitol on protein S-nitrosylation, assessed using the biotin-switch assay, closely paralleled their actions on [Ca$^{2+}$]$_i$. Progesterone mobilizes stored Ca$^{2+}$ in human sperm, by a mechanism involving ryanodine receptor (RyR) activation. Pre-treatment with NO reduced the amplitude of the Ca$^{2+}$ response to ryanodine (a RyR agonist), suggesting convergence of the actions of NO and ryanodine. Sperm pre-treatment with NO greatly enhanced the progesterone effect on [Ca$^{2+}$]$_i$, causing a prolonged increase in flagellar excursion. We conclude that NO regulates mobilization of stored Ca$^{2+}$ in human sperm by protein (possibly RyRs) S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilization

Mode of Action of Farnesol, the “Noble Unknown” in Particular in Ca2+ Homeostasis, and Its Juvenile Hormone-Esters in Evolutionary Retrospect

Farnesol, the sesquiterpenoid precursor of insect juvenile hormones (JH) that itself has JH activity, existed already long before animals and their hormones came into being. Although it is omnipresent in all eukaryotes, this molecule remains a “noble unknown” in cell physiology. It is neither documented as a hormone nor as another type of signaling molecule. To date, its function as an intermediate in the synthesis of squalene-cholesterol-steroids in chordates/vertebrates, and of the insect/arthropod JHs, esters of farnesol, in the mevalonate biosynthetic pathway is assumed to be the only one. This assumption neglects that already two decades ago, farnesol has been shown to be a potent endogenous inhibitor of N-type voltage-gated Ca2+ channels in some mammalian cell types. The tandem mevalonate pathway and Ca2+ channels originated early in eukaryotic evolution, and has since been well conserved, “promoting” it as a ubiquitous player in Ca2+ homeostasis in all eukaryotes. This paper accentuates how this drastic change in thinking gained momentum after the discovery by Paroulek and Sláma that the huge amounts of JH I in male accessory glands of the Cecropia moth, are actually synthesized in these glands themselves and not in the corpora allata, the hitherto assumed unique synthesis site of such compounds. In addition, MAG-JHs have no hormonal- but an exocrine function. Here we hypothesize that MAG-JHs may function in protecting the spermatozoa against toxic Ca2+ concentrations, and in enabling their flagellum to undulate. They may do so by acting through membrane receptors. Our novel paradigm assigns to farnesol/JHs a function of flexible hydrophobic molecular valves for restricting untimely Ca2+-passage through some types of canonical Ca2+channels, using covalently bound farnesyl- or geranyl-geranyl group attachment as well as GPCRs-G proteins all containing a prenyl group. The high rotatable bond count, and their horseshoe-shape are instrumental to their valve function. In our paradigm, Met/Tai and Gce, to date generally thought to be the (only) functional (nuclear) receptors for JHs, are classified as probable Ca2+-sensitive transcription factors. Some theoretical and practical considerations for possible applications in a medical context will be discussed

Single-Channel Properties in Endoplasmic Reticulum Membrane of Recombinant Type 3 Inositol Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca2+ signaling in most cell types. A family of InsP3Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP3Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP3R-3), a widely expressed isoform also implicated in plasma membrane Ca2+ influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP3R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP3R-3 are strikingly similar to those of Xenopus type 1 InsP3R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP3R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP3R isoforms is a mechanism to modify the temporal and spatial features of [Ca2+]i signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP3Rs that have relatively similar Ca2+ permeation properties

REGULATION OF CALCIUM INFLUX AND REACTIVE OXYGEN SPECIES PRODUCTION DURING INFECTION OF LEGUMES BY RHIZOBIA

Nod factor (NF) can induce two separate calcium responses in legume root hairs. Nuclear-associated calcium spiking is central to the symbiosis signalling (Sym) pathway, which is necessary for the activation of genes required for nodule formation and bacterial infection. In addition NF activates a tip-focused calcium influx, which is less-well studied but is thought to be involved in bacterial infection. NF also activates ROS transient production at the tip of root hair cells. In this thesis I used fluorescent probes (Ca2+-sensitive Cameleons YC2.1 and YC3.6 and the ROS-sensitive CM-H2DCFDA dye) to characterise the NF-induced calcium influx and ROS transient responses in Medicago truncatula. Along with being spatially and temporally co-incident, the responses require similar concentrations of NF to be activated, are inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and are dependent on the NF receptor NFP but independent of the Sym pathway components DMI1 and DMI2. These results suggest the NF-induced calcium influx and ROS transient are part of a common signalling pathway during bacterial infection. ROP signalling is associated with ROS production and calcium influx during developmental root hair elongation. I assessed the role of ROPs during rhizobial infection in M. truncatula and found a ROP-activating protein, MtGAP1, was upregulated in root hairs during bacterial infection and is involved in normal root hair curling and infection thread development. Two pieces of evidence directly link ROP signalling with the NF-induced calcium influx: gap1 mutants were hypersensitive for induction of the calcium influx, and there was a reduction in the number of calcium influx responses in ROP9 RNAi knockdown lines. Drawing parallels between developmental root hair elongation and bacterial infection I propose a model for the regulation of ROP signalling by NF leading to root hair curling, the activation of the calcium influx and ROS transient, and infection thread formation

Two types of calcium signalling in legume-rhizobia symbiosis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo