Using the two-hybrid system for the identification of binding partners of SNAREs and secretory proteins

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CHAPTER 2 SIGNALING PATHWAYS REGULATING PROTEIN SECRETION FROM THE SALIVARY GLANDS OF UNFED FEMALE Ornithodoros savignyi

INTRODUCTION

 General anatomy of tick salivary glands

The salivary glands are the largest glands in the tick’s body. They are complex, heterogeneous organs. In both argasid and ixodid ticks, the salivary glands consist of a pair of grape-like clusters of acini (alveoli) comprising 2 major types, the agranular and granular acini (Sonenshine 1991). A myo-epithelial sheath surrounds the salivary glands of argasid ticks, while it is absent in ixodid ticks (Figure 2.1). The salivary glands surround the paired salivary ducts, which extend through the basis capituli and open into the salivarium (Sonenshine 1991). It is via these ducts that tick saliva gets secreted into the host’s bloodstream and allow for successful feeding.The organization of the salivary glands of ixodid ticks has been best described for the adult female Rhipicephalus appendiculatus. In these glands, there are about 1400 acini of three types (I, II, III) in contrast with the 1350 acini of four types (I, II, III, IV) found in males (Sauer and Hair 1986). In both the ixodid and argasid ticks, the agranular acini (type I) occur near the anterior end of the gland and are located adjacent to the main salivary duct. These acini open directly into the main duct. In contrast, the granular types II, III and IV acini occur more posteriorly and comprise grape-like clusters surrounding secondary ducts that ramify among lobes. Most of the granular acini open into secondary ducts (Sonenshine 1991). The most important features of these acini are summarized in Table 2.1.Type I acini consist of a single central cell and a number of peripheral cells, also called the pyramidal cells. Both of these cell types contain an abundance of mitochondria, an inconspicuous Golgi complex, and little or no endoplasmic reticulum. The cytoplasm is poor in ribosomes but contains widely dispersed α and β particles of glycogen. No granules are present but small dense lysosome-like bodies and lipid droplets of various sizes are common. This acinus has been reported to show little change in structure during tick feeding. Support for their function in osmoregulation came from studies by Needham and Coons who indicated significant differences in acini I between dehydrated and rehydrated ticks (Sauer and Hair 1986). Type II acini contain a bewildering diversity of glandular cell types. Binnington has designated these in 1978 as a, b, c1, c2, c3 and c4 in the tick Boophilus microplus (Sauer and Hair 1986). Their identification at light microscope level was largely based upon size, staining properties and histochemical reactions of their secretory granules. In 1983, Binnington reported that the gland of R. appendiculatus was similar to that of Boophilus with 6 granular cell types in acinus II (Sauer and Hair 1986). The most important features of these 6 cell types are summarized in Table 2.2. Type III acini are the best studied, since they are the site of sporogony of Theileria parva, the protozoan parasite that causes African East Coast fever. The acinus contains three glandular cell types (d, e and f), and their characteristics are summarized in Table 2.3.Classical studies performed in 1972 by Roshdy on the glands of Argas persicus indicated that at least three granular cell types (a, b and c) are present in the salivary glands of argasid ticks (Roshdy 1972). Roshdy and Coons confirmed this data in 1975 during ultra-structural studies of the salivary glands of Argas arboreus (Roshdy and Coons 1975). El Shoura described similar granular types in 1985 during ultra-structural studies of the salivary glands of Ornithodoros moubata (El Shoura 1985). During recent studies on the argasid tick Ornithodoros savignyi four cell types was described(Mans 2002a). Cell type ‘a’ contains dense core granules (diameter 3 – 5 µm), which stains positive for the proteins apyrase and savignygrin (Figure 2.2.i). The granular content of these cells are released during feeding. Type ‘b’ cells contain homogenous granules (diameter 4 – 10 µm), which could be immature precursors of the dense core granules.These granules also stained positive for apyrase and savignygrin (Figure 2.2.ii). Type ‘c’cells contain smaller granules (diameter 1 – 2 µm) with electron lucent cores. They stained positive for carbohydrates during the Thiery test. Finally, a fourth cell type (d) was described. These cells have a similar morphology and histochemical basis as type b-cells but stained negative for savignygrin. During feeding of O. savignyi, it could be shown that all cells that displayed granule release retained several granules. Also organelles such as mitochondria not previously observed in these cells became prominent. After feeding, the glands resumed the general morphology observed for unfed ticks within one day, except for a dilated lumen that was still visible (Mans 2002a).

 Extracellular stimuli

The group of Schmidt, Essenberg and Sauer described the first evidence for neuronal control of fluid secretion from tick salivary glands in 1981, when a D1-type dopamine receptor was identified in Amblyomma americanum (Schmidt et al. 1981). To date, 5 types of dopamine receptors are known (Table 2.4) (Watling 2001). The D1-dopamine receptor is known as a stimulating G-protein (Gs) that is associated with the activation of adenylyl cyclase (AC) and causes elevated levels of intracellular cAMP upon binding of its ligand, dopamine. Dopamine is a neurotransmitter synthesized from the amino acid tyrosine (Figure 2.3). It is also the substrate for dopamine β-hydroxylase for the synthesis of norepinephrine, which is converted to epinephrine by the enzyme phenylethanolamine N-methyltransferase. AC from the salivary glands of Amblyomma americanum is stimulated by several derivatives of phenylethylamine, dopamine, noradrenaline, adrenaline and isoproterenol (a β-adrenergic agonist). Octopamine and L-DOPA have no effect on basal adenylate cyclase activity.Dopamine has the highest potency and the lowest ka (0.4 µM), followed by adrenaline and noradrenaline (23 µM) and isoproterenol (0.15 mM). The most potent inhibitors of gland AC activity are the dopamine receptor antagonists. The phenothiazine drugs (thioridazine, chlorpromazine and fluphenazine) are more effective than the butyrophenone drug (haloperidol). The ki for the phenothiazine drugs are 60 nM for thioridazine, 1.9 µM for chlorpromazine and 2.3 µM for fluphenazine. The inhibition of AC activity is specific for the (+) enantiomer of butaclamol (a stereospecific dopamine receptor antagonist), suggesting that the Lone Star tick AC has a D1 type dopamine receptor (Schmidt et al. 1981).Apart from dopamine, other effectors of signal transduction pathways could also elicit oral secretion from the salivary glands of A. americanum. Overall, the volume of oral secretion produced by partially fed ticks in response to the effectors and pharmacological agents varied widely. The volume and rate of oral secretion stimulated by pharmacological agents were: pilocarpine > (dopamine and theophylline) = (dopamine, theophylline and GABA) > (dopamine, theophylline and phorbol 12 myristate 13-acetate; an activator of phospholipase C) (McSwain et al. 1992a).
Pilocarpine is often used to induce tick oral secretion, and it is believed to stimulate cholinergic receptors in the tick synganglion, which relay information to nerves innervating the salivary glands. Cholinergic agents fail to stimulate secretion in isolated salivary glands (McSwain et al. 1992a). Theophylline, GABA as well as the phorbol esters had no effect when injected individually. Co-injection of the phorbol ester PMA with dopamine and theophylline was inhibitory, indicating an essential role for PKC in eliciting oral secretion.SDS-PAGE analysis of the secreted proteins showed variable results in response to different agonists. The highest number of proteins was detected from ticks in the earliest stages of feeding. In some ticks, proteins were identified at one collection time but not at another time in secretions from the same tick stimulated by the same agent. One such an example is pilocarpine, which stimulated secretion of different proteins in the same tick after multiple injections. In the case of dopamine, theophylline and GABA, protein secretion was consistent (McSwain et al. 1992a). Other evidence indicates that Ca2+ is essential for secretion. Removal of Ca2+ from the support medium greatly inhibits dopamine and cAMP-stimulated secretion from isolated salivary glands (McSwain et al. 1992a). This is similar to results obtained in salivary duct cells of the cockroach, Periplaneta americana. In this study, dopamine evoked a slow and reversible dose-dependent elevation in [Ca2+]i in salivary duct cells. The dopamine-induced elevation in [Ca2+]i is absent in Ca2+-free saline and is blocked by La3+, indicating that dopamine induces an influx of Ca2+ across the basolateral membrane of the duct cells. Stimulation with dopamine depolarizes the basolateral membrane and is also blocked by 100
µM La3+ and abolished when the Na+ concentration in the solution is reduced from physiological concentrations to 10 mM (Lang and Walz 1999). GABA, as mentioned before, does not stimulate secretion by itself (McSwain et al. 1992a). The enhancing effect of GABA on dopamine-stimulated fluid secretion of isolated salivary glands occurred only at high concentrations (Lindsay and Kaufman 1986). Activation of another receptor by GABA may potentiate secretion by another, but poorly understood, mechanism. A model was proposed and experimentally investigated by Linday and Kaufman in 1996 (Figure 2.4). Their data indicated that GABA and spiperone binds to the same receptor, and that this reaction can be inhibited by sulpiride without diminishing the effect of dopamine. Another receptor, the so-called ergot alkaloid sensitive receptor, which can also be blocked by sulpiride without altering the response to dopamine, was also detected but its function remains unknown. In conclusion, the authors suggested that GABA might play an important neuromodulatory role in salivary fluid secretion (Lindsay and Kaufman 1986).Interestingly, spiperone is also a subtype selective antagonist for the 5-HT1A serotonin receptor, and it has been reported that serotonin, a known agonist of salivary secretion in the insect, Calliphora erythrocephala, inhibits basal cyclase activity (Schmidt et al. 1981;Watling 2001). Therefore, the possible inhibitory effect of serotonin on the GABA activated
signaling pathway should be investigated.

List of Abbreviations
List of Figures 
List of Tables 
Acknowledgements 
Chapter 1: Literature review
1.1. Ticks: An overview
1.2. Biogenesis of secretory granules 
1.3. The exocytotic pathways 
1.4. Protein-protein interactions: A target for therapy?
1.5. Aims of this thesis
1.6. References
Chapter 2: Signaling pathways regulating protein secretion from the salivary glands of unfed female Ornithodoros savignyi.
2.1. Introduction
2.1.1. General anatomy of tick salivary glands
2.1.2. Extracellular stimuli
2.1.3. Adenylyl cyclase and cAMP
2.1.4. Prostanoids
2.1.5. Phospholipase C and intracellular calcium
2.1.6. Current model for the control and mechanism of secretion in ixodid ticks
2.2. Hypothesis
2.3. Aims 
2.4. Materials 
2.5. Methods 
2.5.1. Tick salivary gland dissection
2.5.2. Apyrase activity assay
2.5.3. Agonist and antagonist treatment
2.5.4. Phosphorylation assay
2.6. Results and discussion
2.6.1. Dopamine / Isoproterenol / Carbachol
2.6.2. Intracellular calcium
2.6.3. Prostaglandins
2.6.4. cAMP
2.6.5. Verapamil
2.6.6. Ouabain
2.6.7. Extracellular and intracellular conditions (Membrane potential)
2.6.8. N-ethylmaleimide (NEM)
2.6.9. GTPγS
2.6.10. cAMP-Dependent phosphorylation
2.6.11. PI-3-Kinase inhibitor (Wortmannin)
2.6.12. Inositol (1, 4, 5) tri-phosphate (IP3)
2.6.13. PLC Inhibitor (U73,122)
2.6.14. Actin inhibitor (Cytochalasin D)
2.6.15. Tubulin Inhibitor (Colchicine)
2.7. Conclusion
2.8. References 
Chapter 3: Investigations into the conserved core machinery of regulated exocytosis in the salivary glands of O. savignyi
3.1. Introduction
3.1.1. Conserved core machinery for regulated exocytosis 
3.2. Hypothesis
3.3. Aims 
3.4. Materials 
3.5. Methods
3.5.1. Salivary gland fractionation
3.5.2. Protein gel electrophoresis
3.5.3. Western blotting
3.5.4. Immuno-fluorescent localization using confocal microscopy
3.5.5. Degenerative primer design
3.5.6. Total RNA isolation
3.5.7. Conventional cDNA synthesis
3.5.8. SUPER SMART™ cDNA synthesis
3.5.9. cDNA amplification by LD-PCR
3.5.10. Random amplification of 3’ cDNA ends (3’-RACE)
3.5.11. DIG- labelling of probes using PCR
3.5.12. DNA dot blotting
3.5.13. Agarose gel electrophoresis
3.5.14. PCR product purification
3.5.15. Quantification of nucleic acids
3.5.16. A/T cloning of PCR products into pGEM® T-Easy vector
3.5.17. Preparation of electrocompetent cells
3.5.18. Transformation by electroporation
3.5.19. Miniprep plasmid isolation
3.5.20. High pure plasmid isolation
3.5.21. Automated DNA sequencing and data analysis
3.6. Results and Discussion 
3.6.1. Western blotting of salivary glands with anti-SNARE and antiRab3a antibodies
3.6.2. Localization of SNAREs and cytoskeleton proteins using confocal microscopy
3.6.3. RNA isolation
3.6.4. 3’-RACE using ss cDNA
3.6.5. 3’-RACE using SUPER SMART™ ds cDNA
3.7. Conclusion
3.8. References
Chapter 4: Investigation into protein-protein interactions between rat brain secretory proteins and an O. savignyi cDNA library by means of the GAL4 two-hybrid system
4.1. Introduction
4.1.1. The yeast two hybrid system
4.1.2. Using the two-hybrid system for the identification of binding partners of SNAREs and secretory proteins
4.2. Hypothesis
4.3. Aims 
4.4. Materials 
4.5. Methods 
4.5.1. Full-length GAL4 AD/ library construction
4.5.2. Truncated GAL4 AD/ library construction
4.5.3. Verification of yeast host strains and control vectors
4.5.4. GAL4 DNA-BD/Bait construction
4.5.5. Small-scale yeast transformation
4.5.6. GAL4 DNA-BD/Bait test for autonomous reporter gene activation
4.5.7. Sequential library-scale transformation of AH109 yeast cells
4.5.8. Two-hybrid screening of reporter genes
4.5.9. Colony-lift β-galactosidase filter assay
4.5.10. Nested-PCR screening of positive clones
4.5.11. Plasmid isolation from yeast
4.5.12. AD/library plasmid rescue via transformation in KC8 E. coli
4.5.13. Sequencing of AD/library inserts
4.6. Results and Discussion
4.6.1. Full-length cDNA GAL4 AD / Plasmid library construction
4.6.2. Truncated GAL4 AD / Plasmid library construction
4.6.3. Bait construction
4.6.4. Transformation of bait/ GAL4 BD constructs into AH109
4.6.5. Library transformation and two-hybrid screening
4.6.6. Colony-lift β-galactosidase assay
4.6.7. Nested-PCR screening of β-galactosidase positive clones
4.6.8. Sequencing and analysis of positive AD/library inserts
4.7. Conclusion
4.8 References
Chapter 5: Investigating SNARE-interactions by functional complementation in Saccharomyces cerevisiae and pulldown assays with α-SNAP
5.1. Introduction
5.1.1. S. cerevisiae: A model organism for studying protein transport
5.1.2. Functional complementation.
5.1.3. Functional complementation of SNAREs and trafficking proteins in yeast
5.1.4. α-SNAP: Functional properties
5.2. Hypothesis
5.3. Aims 
5.4. Materials 
5.5. Methods 
5.5.1. O. savignyi salivary gland cDNA library construction
5.5.2. Growth and maintenance of SSO-mutated yeast cells
5.5.3. Transformation, selection and screening
5.5.4. Data analysis
5.5.5. Expression of rat brain α-SNAP
5.5.6. Salivary gland homogenate preparation
5.5.7. Affinity chromatography (Pull-down assays)
5.5.8. ELISA
5.5.9. SDS-PAGE
5.6. Results and Discussion 
5.6.1. cDNA library construction
5.6.2. Growth and maintenance of syntaxin knockout yeast
5.6.3. Transformation, selection and screening
5.6.4. Data analysis
5.6.5. Pull-down assays
5.7. Conclusion
5.8. References
Chapter 6: Concluding discussion
Summary
Appendix

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The mechanisms regulating exocytosis of the salivary glands of the soft tick, Ornithodoros savignyi