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Title: A study of proteins involved in Ca²⁺-regulated exocytosis in AtT-20 cells
Author: Mackintosh, Jan Karen
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Regulated exocytosis has been studied mostly in neuronal or chromaffin cells, and several proteins that are thought to be involved have been identified. In this project proteins involved in vesicle docking and fusion in the ArT-20 (transformed mouse anterior pituitary) cell line were studied, as a comparison to these other cell types. Immunoblotting and immunofluorescence were used to detect and localize SNARE's (soluble NSF attachment protein receptors) and also cytosolic and cytoskeletal proteins. These results showed that the proteins present were similar to those in chromaffin and neuronal cells, with a similar intracellular distribution. Since AtT-20 cells secrete ACTH in a Ca2+-regulated manner, secretion could be measured using a radioimmunoassay for ACTH. This was used with a permeabilised cell system, allowing the introduction of recombinant proteins, drugs, antibodies and neurotoxins. Little effect was seen on introduction of some components, such as the synaptotagmin cytoplasmic domain and αSNAP, however a dramatic reduction in secretion was seen on introduction of the light chains of the neurotoxins Botulinum D and Tetanus toxin. These toxin subunits specifically cleave the v-SNARE synaptobrevin, demonstrating its importance in regulated secretion in AtT-20 cells as has been demonstrated in chromaffin and neuronal cells. The role of actin in exocytosis has also undergone much scrutiny, particularly in chromaffin cells. This was studied in AtT-20 cells using phalloidin, cytochalasin and a combination of the permeabilised cell assay and immunofluorescence. In contrast to chromaffin cells, the actin did not appear to form a distinct exocytotic barrier in AtT-20 cells. The introduction of actin-stabilizing and de-stabilizing factors also had no effect on the rate of exocytosis in AtT-20 cells. We conclude from this that actin may not have an essential role to play in Ca2+-regulated exocytosis from AtT-20 cells. The logical continuation of this project would be to use a molecular approach and transfect the AtT-20 cells with mutant forms of the proteins thought to be involved in exocytosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available