Avery, Julia Catherine; (1996) The molecular mechanisms underlying exocytosis in sea urchin eggs. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Exocytosis is a ubiquitous cellular mechanism for the export of secretory products and the insertion of proteins and lipid into the plasma membrane. The sea urchin egg is an ideal system in which to study exocytosis. Fertilization in the sea urchin egg is characterized by a transient increase in intracellular calcium. This triggers the exocytosis of cortical secretory granules which lie immediately beneath the plasma membrane of unfertilized eggs, producing a structure called the fertilization envelope. Exocytosis can also be studied directly; the exocytotic apparatus, the egg cortex, can be isolated and responds to the physiological trigger - Ca2+ - in vitro. It has been suggested that the molecular mechanisms underlying exocytosis are evolutionarily conserved. I have used western blotting to identify whether homologues of proteins implicated in exocytosis in other secretory systems are present in sea urchin eggs. Using this approach, I have identified homologues of the synaptic proteins synaptobrevin and rab3A, three members of the annexin family of Ca2+/phospholipid-binding proteins and a calcineurin-like protein in sea urchin eggs. Using immunocytochemical confocal microscopy, I have localized the synaptobrevin homologue to the cortical granule membrane. I show that tetanus toxin light chain (TeTx LC), which inhibits neurotransmitter release by specifically cleaving synaptobrevin, cleaves the sea urchin synaptobrevin homologue in vitro. I demonstrate that preincubation of isolated cortices with TeTx LC causes a time-dependent inhibition of Ca2+-stimulated exocytosis which correlates with cleavage of the synaptobrevin homologue. These results suggest that a tetanus toxin-sensitive synaptobrevin homologue is required for exocytosis in sea urchin eggs. The Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin has been implicated in exocytosis in Paramecium. In sea urchin eggs, irreversible protein phosphorylation and antibodies to calmodulin block exocytosis in vitro. I have investigated the role played by calcineurin in exocytosis. Neither specific inhibitors nor functionally inhibitory antibodies to calcineurin affect calcium-stimulated exocytosis in vivo or in vitro. These results suggest that calcineurin is not involved in exocytosis in sea urchin eggs.

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