Late events in regulated exocytosis

To understand the intracellular mechanisms that control exocytosis it is necessary to have access to the cell interior. This is achieved by plasma membrane permeabilisation or by application of patch-pipettes. These conditions permit control over the cytosol composition and also allow leakage of soluble factors that may have roles in the exocytotic mechanism. Different permeabilisation methods allow different extents of leakage and therefore provide complementary data. The exocytotic machinery itself remains intact and can be activated by providing Ca2+ and/or a guanine nucleotide. In some cells there is evidence for the participation of two guanine nucleotide-binding proteins (GP and GE), as well as a Ca(2+)-binding protein. In others Ca2+ is the only requirement. In a number of cell types, ATP is not required for the late steps in the secretory pathway.     

Freeze-fracture studies of chemotactic peptide-induced exocytosis in neutrophils: evidence for two patterns of secretory granule fusion

Rabbit neutrophils were fixed in 2% glutaraldehyde and then either quick-frozen and freeze-fractured or embedded and thin-sectioned. Cells exposed to 10(8) M N-formylmethionyl-leucylphenylalanine (FMLP) and 5 micrograms/ml cytochalasin B at 22 degrees C underwent a rapid, compound exocytosis. Within 10 sec after stimulation, narrow pores were seen joining individual peripheral granules with the plasma membrane. Sequential fusion of interior granules occurred between 20 and 60 sec and took on two different patterns. The first consisted of a linearly directed series of fusion events resulting in a radial array of tapering invaginations directed toward the cell center. The second pattern consisted of an undirected fusion of larger granules to form highly branched structures. These granules were often connected by narrow tubules and in some cases a series of fused granules would end in a small, vesiclelike tip. This suggested that sequential fusion may involve a set of smaller vesicles as well as the granule membranes themselves.     

The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca²⁺ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca²⁺ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca²⁺ and GTP) are substantially enhanced.