Factor de Potencia

Using either HL60 or RBL cells, previous groups have found that both recombinant PI-TPa and PI-TPp enhanced secretion induced by GTPyS/calcium/ATP (G/C/A) and antigen (Thomas et al. 1993, Way. 1998). PI-TP is thought to promote secretion via enhancing PIP2 production (refer to section 7.1.1). In this study neither isoform of PI-TP (up to 100 ng/ml) modulated G/C/A induced

secretion. RPMC release 60% to 80% of their total hexosaminidase content in response to G/C/A. rPI-TP therefore has only a small window in which to enhance secretion.

These observations suggest that the signalling pathways of RPMC might be slightly different from those of RBL cells with regard to PI-TP. Significantly the secretory response of RPMC and RBL cells to G/C/A differs. RPMC secrete 60-80% of their total hexosaminidase content within ~1- 2min of exposure to G/C/A. This compares with RBL cells which secrete only 40% to 50% of their total hexosaminidase content within -10 to 15 min of exposure to this trigger. It was hypothesised in this study that the rapid and large response of RMPC to G/C/A maybe due to the cells being pre-primed, thereby avoiding the requirement for PI-TP: the role of PI-TP in priming was reported by Hay et al (1995). Depletion of ATP uncovered a requirement for PI-TPa in G/C/A induced secretion from RPMC (Fig 7.8): PI-TPa enhanced G/C/A triggered secretion by -20%. This suggests that RPMC are possibly already primed although as metabolic inhibition has substantial effects on cellular processes this is only a theory. The findings of this study show that PI-TPa present only prior to triggering induces a -10% increase in secretion. These observations suggest that in addition to the previously reported role of PI-TP in priming, it may also play a role in the final stages of exocytosis. This is in agreement with the observation that PI-TPa translocates to the plasma membrane during antigen induced exocytosis at a rate which parallels the time course of secretion (Way. 1998).

PI-TPa cannot by itself induce secretion, requiring GTPyS, calcium and ATP to be able to modulate this cellular function. This suggests that the signal transduction pathway by which PI- TPa enhances secretion involves a G-protein. The observations of both Fensome et al (1996) and Way (1998) suggest that this G-protein is probably not ARF, which has been shown to restore exocytosis by enhancing PIP2 production via PLD (refer to section 1.2.4.1). Possible candidates for this G-protein include Rac and Rho, which have been shown to enhance calcium- mediated secretion in RPMC (Price et al. 1995, refer to section 1.2.6). This is under investigation by other groups.

Conclusions

RPMC contain PI-TPa and almost certainly PI-TPp. This study has found that these proteins localise to the plasma membrane in RPMC in addition to their localisation at the previously reported sites. The localisation of PI-TPa is very similar to that of p-actin in RPMC. PI-TP did not enhance G/C/A induced secretion from permeabilised RPMC. However, when cells were metabolically inhibited before permeabilisation, the requirement for PI-TPa became apparent.

Acknowledgements

First and foremost I would like to thank my boss, Dr Anna Koffer, who has kept me going when times have been tough. As regards to people in the lab I would like to thank Dr Mark Holt who has taught me some great swear words to say when things are not going so well in the lab. To all the people that have made me smile - Gemma, Andy, Charlotte, Jef, Clive and a special thanks to Arnold Pizly, my soul mate in haematology.

This thesis is dedicated to my family, who have put up with me through it all. To my dad for the many packets of jelly tots (and for sitting with me casualty - two weeks prior to submission), to my mum for the magic wash basket and to my brother who designed a very simple reference manager for me.

A final thought I take from a article in Nature, which I spied while reading a report by Dr A Weeds - Bamboo hay, for example, has four times the protein content of hay from fodder grasses (the

Bibliography

Abe H, Nagaoka R, Obinata T {1993) Cytoplasmic localization and nuclear transport of cofilin in cultured myotubes. Exp Cell Res 206: pp1-10

Aktories K (1994) Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins. Mol Cell Biochem 138: ppl 67-76

Aktories K, Barmann M, Ohishi I, Tsuyama S, Jakobs KH, Habermann E (1986) Botulinum C2 toxin ADP-ribosylates actin. Nature 322: pp390-2

Aktories K, Wegner A (1992) Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins. Mol Microbiol 6: pp2905-8

Aib JG Jr, Gedvilaite A, Cartee RT, Skinner HB, Bankaitis VA (1995) Mutant rat phosphatidylinositol I phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity. Proc Natl Acad Sci U 8 A 92: pp8826-30

Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular biology of the cell. Garland Press Edition :pp563

Apgar JR (1995) Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization. Mol Biol Cell 6: pp97-108

A lte r 8, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard 0, Caroni P (1998)

Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase [see comments] Nature 393: pp805-9

Aridor M, Rajmilevich G, Beaven MA, Sagi-Eisenberg R (1993) Activation of exocytosis by the heterotrimeric G protein Gi3. Science 262: ppl 569-72

Atkinson TP, Lee CW, Rhee SG, Hohman RJ (1993) Orthovanadate induces translocation of phospholipase C-gamma 1 and -gamma 2 in permeabilized mast cells. J Immunol 151: ppl 448-55

Ayscough KR (1998) In vivo functions of actin-binding proteins. Curr Opin Cell Biol 10: ppl 02-11

Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG (1997) High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Biol 137: pp399-416

Ball, A (1997) Immunological investigations into the properties and function of the phosphaidylinositol transfer protein. Doctoral thesis, Senate House, London.

Bamburg JR, Bray D (1987) Distribution and cellular localization of actin depolymerizing factor. J Cell Biol 105: pp2817-25

Banerjee A, Barry VA, DasGupta BR, Martin TFJ (1996a) N-Ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2-i'activated exocytosis. J Biol Chem 271 : pp20223-6

Baneijee A, Kowalchyk JA, DasGupta BR, Martin TFJ (1996b) SNAP-25 is required for a late postdocking step in Ca2-Kdependent exocytosis. J Biol Chem 271 : pp20227-30

Bankaitis VA, Aitken JR, Cleves AE, Dowhan W (1990) An essential role for a phospholipid transfer protein in yeast Golgi function [see comments] Nature 347: pp561-2

Belmont LD, Patterson GM, Drubin DG (1999) New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites. J Cell Sci 112 ( Pt 9): ppl 325-36

Benhamou M, Gutkind JS, Robbins KC, Siraganian RP (1990) Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc Natl Acad Sci U S A 87:

PP5327-30

Benhamou M, Ryba NJ, Kihara H, Nishikata H, Siraganian RP (1993) Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation. J Biol Chem 268: pp23318-24

Bonder EM, Mooseker MS (1983) Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process. J Cell Biol 96: ppl 097-107

Borovikov YS, Norman JC, Price LS, Weeds A, Koffer A (1995) Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin. J Cell Sci 108 ( Pt2):pp657-66

Bôttinger H, Reuner KH, Aktories K (1987) Inhibition of histamine release from rat mast cells by botulinum C2 toxin. Int Arch Allergy AppI Immunol 84: pp380-4

Bovrman EP, Uhlinger DJ, Lambeth JD (1993) Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein. J Biol Chem

268:

PP21509-12

Brown HA, Gutowski S, Moomaw CR, Slaughter C, Sternweis PC (1993) ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity [see comments] Cell 75: ppl 137-44

Bubb MR, Senderowicz AM, Sausville EA, Duncan KL, Korn ED (1994) Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 269: ppl 4869-71

Bubb MR, Spector I, Bershadsky AD, Korn ED (1995) Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments. J Biol Chem 270: pp3463-6

Burgoyne RD, Cheek TR, Norman KM (1986) Identification of a secretory granule-binding protein as caldesmon. Nature 319: pp68-70

Buzzard KA, Giaccia AJ, Killender M, Anderson RL (1998) Heat shock protein 72 modulates pathways of stress-induced apoptosis. J Biol Chem 273: ppl 7147-53

earlier MF, Pantaloni D, Korn ED (1984) Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state. J Biol Chem 259: pp9983-6

earlier MF, Jean C, Rieger KJ, Lenfant M, Pantaloni D (1993) Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics. Proc Natl Acad Sci U S A 90: pp5034-8

earlier MF, Pantaloni D (1997) Control of actin dynamics in cell motility. J Mol Biol 269: pp459-67

Cassimeris L, Safer D, Nachmias VT, Zigmond SH (1992) Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J Cell Biol 119: ppl 261-70

Chamberlain LH, Roth D, Morgan A, Burgoyne RD (1995) Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis. J Cell Biol 130: ppl 063- 70

Chardin P, Paris S, Antonny B, Robineau S, Béraud-Dufour S, Jackson CL, Chabre M (1996) A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 384: pp481-4

Cheek TR, Burgoyne RD (1986) Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells. FEBS Lett 207: ppl 10-4

Cheek TR, Burgoyne RD (1987) Cyclic AMP inhibits both nicotine-induced actin disassembly and catecholamine secretion from bovine adrenal chromaffin cells. J Biol Chem 262: ppl 1663-6

Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA (1994) The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell 79: pp507-13

Church MK, Caulfield JP (1988) Mast cell and basophil functions in allergy. Editors: Holgate ST, Church MK. Gower Medical Publishing, London. pp5.1-5.12

CIssel DS, Fraundorfer PF, Beaven MA (1999) Phospholipase D and its role in mast cells. Editors Razin E, Rivera J, published by Springer, ppl 81-191

Cleves AE, McGee TP, Whitters EA, Champion KM, Aitken JR, Dowhan W, GoebI M, Bankaitis VA

(1991) Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64: pp789-800

Clubb BH, Locke M (1998) Peripheral nuclear matrix actin forms perinuclear shells. J Cell Biochem 70: pp240-51

Cockcroft S, Howell TW, Gomperts BD (1987) Two G-proteins act in series to control stimulus- secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis. J Cell Biol 105: pp2745-50

Cooper JA (1987) Effects of cytochalasin and phalloidin on actin. J Cell Biol 105: ppl 473-8

Costello PS, Turner M, Walters AE, Cunningham CN, Bayer PH, Downward J, Tybulewicz VL

(1996) Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells. Oncogene 13: pp2595-605

Coué M, Brenner SL, Spector I, Korn ED (1987) Inhibition of actin polymerization by latrunculin A. FEBS Lett 213: pp316-8

Cross MJ, Roberts S, Ridley AJ, Hodgkin MN, Stewart A, Claesson-Welsh L, Wakelam MJO (1996)

Stimulation of actin stress fibre formation mediated by activation of phospholipase D. Curr Biol 6: pp588-97

Cunningham E, Thomas GM, Ball A, Hiles I, Cockcroft S (1995) Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2. Curr Biol 5: pp775-83

De Career G, Lallena MJ, Correas I (1995) Protein 4.1 is a component of the nuclear matrix of mammalian cells. Biochem J 312 ( Pt 3): pp871-7

De La Cruz E, Pollard TD (1994) Transient kinetic analysis of rhodamine phalloidin binding to actin filaments. Biochemistry 33: ppl 4387-92

De La Cruz EM, Pollard TD (1996) Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species. Biochemistry 35: ppl 4054-61

de Vries KJ, Heinrichs AA, Cunningham E, Brunink F, Westerman J, Somerharju PJ, Cockcroft S, Wirtz KW, Snoek GT (1995) An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system. Biochem J 310 (R 2): pp643-9