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The control of exocytosis in sea urchin eggs

Whalley, Timothy David; (1990) The control of exocytosis in sea urchin eggs. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Fertilisation in the sea urchin egg is characterised by a transient increase in intracellular free calcium. This triggers the exocytosis of the cortical secretory granules which lie immediately beneath the plasma membrane, producing a structure called the fertilisation envelope. The exocytotic apparatus or cortex can be isolated and responds to the physiological trigger — Ca2+ — in vitro, it is an ideal system in which to study exocytosis directly. I have developed a method of reconstituting the exocytotic apparatus. This is a very useful technique because it allows manipulation of individual components of the system. I have used this technique to study the effects of calcium ion treatment of each component of the system. I show that this does not affect the responsiveness of subsequent reconstitution. I have investigated the mechanisms by which various compounds activate eggs and cause the increase in Ca2+, which triggers exocytosis. Using a permeabilised egg preparation I show that neither guanosine 3',5'-cyclic monophosphate (cGMP) nor guanosine 5'-O-(3-thio-triphosphate) (GTPyS—a compound which causes irreversible activation of GTP-binding proteins), directly stimulate the release of calcium from intracellular stores. Using an indirect single cell assay for DAG by measuring pH changes due to activation of a Na+/H+ antiport, I show that GTIyS but not cGMP can stimulate the enzyme phosphoinositidase C in the absence of an increased intracellular free calcium. I also show that, under the conditions of this assay, insemination fails to activate the Na+/H+ antiport. This suggests that sperm do not trigger the array of events which occur at fertilisation by interaction with GTP-binding proteins. It has been suggested that a change in the phosphorylation state is important in secretory control in many cells. I have investigated whether this also the case in the sea urchin egg by microinjecting into eggs the compound adenosine-5'-O-(3-thiotriphosphate) (ATPyS) which can be used to irreversibly phosphorylate proteins. ATPyS prevents fertilisation envelope elevation after insemination but does not prevent the sperm induced rise in intracellular free calcium. I present evidence to show that ATPyS requires the presence of cytoplasm in order to elicit its inhibitory effects. I also show that the effect is irreversible and that ATPyS is not itself the inhibitor. The results with this compound strongly suggest that a protein, when phosphorylated inhibits exocytosis in the sea urchin egg.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The control of exocytosis in sea urchin eggs
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Fertilization
URI: https://discovery.ucl.ac.uk/id/eprint/10124734
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