Abstract
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 AtT-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 (adrenocorticotrophic hormone) in a Ca -
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 aSNAP, 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.
Throughout this project ACTH secretion was measured using a rather laborious
radioimmunoassay. It was decided to look at an alternative method of measuring
regulated secretion, by stably transfecting the cells with a vector expressing a
secreted alkaline phosphatase (SEAP) reporter gene, fused to human growth
VIII
hormone (hGH). The logic behind this was that the hGH would direct the SEAP into
the regulated secretory pathway, allowing its release to be measured using
photometric or chemiluminescent methods of detection. The vector was found to be
very efficient in a traditional reporter gene role, but not sensitive enough to measure
the relatively small amounts of ACTH released on stimulation of secretion.
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. It would naturally focus on those producing dominant negative
phenotypes, leading to cells deficient in exocytosis.
