Urbe, Sylvie; (1998) Molecular mechanisms of secretory granule biogenesis in neuroendocrine cells. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Secretory granules are storage organelles that form from the trans-Golgi network (TGN) as immature secretory granules (ISG) and subsequently undergo a maturation process characterised by a series of biochemical and morphological modifications. The first part of this thesis concerns the characterisation of the ISG with respect to the conversion of prohormones to active hormones and the progressive acidification of the granule lumen. Secretogranin II (Sgll) was used to study endoproteolytic processing by the endopeptidase PC2 in PC12/PC2 cells, a neuroendocrine cell-line. Processing could be observed in isolated ISGs but not in the TGN and was strongly dependent on the intragranular pH. Using processing of Sgll as a pH indicator for the granule interior, an intragranular pH of 6.3+/- 0.1 for ISGs in a physiological buffer in the presence of ATP was determined. The second part of this thesis describes the reconstitution of homotypic fusion of ISGs in a cell-free assay based on content mixing of two ISG populations isolated from PC12 or PC12/PC2 cells. Fusion was monitored by detecting the cleavage of 35S-sulphate labelled Sgll by PC2. Antibodies were generated and characterised for quantitation of the end product p18. Fusion of ISGs was shown to be temperature, time, ATP and cytosol dependent and was competed by the addition of unlabelled ISGs. Mature secretory granules and TGN derived from PC12/PC2 cells were unable to fuse with ISGs. The time window within which ISGs are fusion competent was characterised and fusogenicity was shown to decrease rapidly after budding from the TGN. Trypsin treatment of the ISGs abolished fusion whilst clostridial neurotoxins had no effect. N-ethylmaleimide treatment was inhibitory and fusion could be rescued with untreated cytosol and to some extent with recombinant NSF and alpha-SNAP.

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