Morphological era of discovery (1950s and 1960s) Visualization of the cell interior by electron microscopy catalysed both morphologists and biochemists to initiate experiments and make observations to identify, define and understand the complex compartmentalization of specialized cells, most notably including the morphologists G.E. Palade, C. DeDuve, D.W. Fawcett, K.R. Porter, J. Rhodin and F.S. Sjostrand. The exocrine pancreatic acinar was a specialized cell type favoured by GE Palade, and it was with this cell type that the first observations on cells with specialized granule compartments were made. 1956 1958 1962 1964 1967a b 1967a b 1966 1967a 1981 1 1978 1981 Fig. 1 ER CM SG SV VE LB  1978 + + 1977 1975 Many of the observations made were obtained using careful morphological approaches and expanded using biochemical techniques, and subcellular fractionation. However, these early biochemical approaches were limited by the inability to manipulate the cell systems in use, typically tissues from mice or rats. The arrival of molecular tools and genetic manipulation provided the next wave of advances bringing the field to our current level of understanding. Cell line model systems, the molecular age and the sorting problem 1985 1982 1985 1980 TGN and post-TGN sorting and processing in neuroendocrine cells 1986 1987 1985 1985 1984 1986 1977 1984 1985 2002 1983 1997 1997 1986 1989 1987 1998 1991 1998 1974 1993 1985b 1986 1987 1987 2006 2001 2006 2004 2003 1980 In vitro reconstitution and biochemical analysis of secretory granule biogenesis Cell-free reconstitution of ISG budding from TGN 1966 1990 1990 1992 1991 1996 2000 1997 1999 1966 2 Fig. 2 Step 1 Step 2 Step 3  Determination of the pH of ISGs using an in vitro approach 1995 35 1997 35 ISG-ISG homotypic fusion 1966 1991 35 1998 1998 2001 2006 ISG membrane remodelling In addition to ISG-ISG homotypic fusion, ISG content and membrane remodelling is another important step during granule maturation. This remodelling is performed via budding of clathrin-coated vesicle from the maturing granule membrane, and is the pathway for non-regulated constitutive-like secretion, or possibly sorting to endosomes. This clathrin-mediated remodelling step is a common feature in neuroendocrine and endocrine secretory granules, and is thought to provide a mechanism for proof-reading the content and membrane composition of the maturing ISG to ensure the production of MSGs which contain biologically active hormones and can undergo efficient exocytosis. 1985a 1986 1996 2000  1997 1999 2003 2001 2006 2001 2000 2007 2000 Recent developments in secretory granule formation and maturation 2006 2001 2006 2004 2003 Role of cholesterol in secretory granule biogenesis 2000 2006 Regulation of secretory granule maturation 2000 2005 1996 2004 2007 Role of Chromogranin A in secretory granule formation 2003 2004 2001 2004 2005 2005 2006 Role of prohormone convertases in secretory granule maturation Both in vitro and ex vivo the activity of the PC enzymes (notably PC1/3 and PC2 which will be the focus of the discussion here) have been extensively studied. PC null mice models have confirmed the in vivo function of the PCs and extended the characterization of their specific substrates and action in different tissues. 1997 1998 2002 1994 2001 1999 1997 1999 2002 Role of Rab3D in secretory granule maturation 1994 2000 1997 In contrast to Rab3A, Rab3B and Rab3C, Rab3D is predominantly expressed outside the nervous system, in peripheral tissues where the other isoforms either are expressed at low levels or are lacking. Originally identified in fat cells, Rab3D is present in several additional cell types including secretory cells such as pancreatic and parotid acinar cells, mast cells and peptide-secreting cell lines. In secretory cells Rab3D appears to be predominantly localized to secretory granules, thus mirroring the distribution of Rab3A in neurons and neuroendocrine cells. It was hypothesised that Rab3D could have the same function as Rab3A but in secretory cells. 2002 Conclusions and future perspectives The early morphological and biochemical characterization of the regulated secretory pathway provided key insights to the secretory process. More recent experiments have provided a more detailed understanding of the functional properties, and the regulation of the maturation of neuroendocrine secretory granules. Many important issues need to be resolved, for example how the cytoplasmic events, such as membrane remodelling are coupled with the intralumenal biochemical changes such as PC activation, prohormone cleavage, and acidification. In addition, the biological relevance of the maturation process, as it affects hormone processing and secretion, remains to be determined in light of the differences between endocrine and neuroendocrine granules.