We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca2+ at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca2+-dependent manner dur- ing 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimula- tion. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.

Regulated exocytosis involves the tightly controlled fusion of a transport vesicle with the plasma membrane. It includes processes as diverse as the release of neurotransmitters from presynaptic nerve endings and the sperm-triggered deposition of a barrier preventing polyspermy in oocytes. Cell-free model systems have been developed for studying the biochemical events underlying exocytosis. They range from semi-intact permeabilized cells to the reconstitution of membrane fusion from isolated secretory vesicles and their target plasma membranes. Interest in such cell-free systems has recently been reinvigorated by new evidence suggesting that membrane fusion is mediated by a basic mechanism common to all intracellular fusion events. In this chapter, we review some of the literature in the light of these new developments and attempt to provide a critical discussion of the strengths and limitations of the various cell-free systems.

The annexins are a major class of calcium-binding proteins with unknown functions. In an attempt to define novel model systems in which to study members of the annexin family, we have investigated the expression of annexins in eggs from the sea urchin Lytechinus pictus. Western blot analysis of L. pictus eggs using antisera raised against human annexins I, V and VI revealed the presence of immunoreactive proteins of approximately 34 kDa, 35 kDa and 68 kDa respectively. The sea urchin annexins behaved similarly to their mammalian counterparts, both during purification and in their ability to bind calcium-dependently to anionic phospholipids. Of the three sea urchin annexins, the 34 kDa form was most abundant, yielding sufficient quantities for peptide microsequencing. The amino acid sequences derived in this way showed the L. pictus annexin to be closely related both to mammalian annexin I and to annexins IX, X and XII from Drosophila and Hydra. However, N-terminal sequence from the L. pictus annexin showed it to be a novel member of the annexin super-gene family. The results are interesting in view of the complex evolution of the annexin gene family, and also point to the potential usefulness of echinoderm eggs as a model system in which to study annexin function.

Sea urchin eggs provide an efficient in vitro model of exocytosis. We have identified proteins in sea urchin eggs that cross-react with antibodies to mammalian synaptobrevin, synaptotagmin, SNAP-25, syntaxin and rab3a. We show that these proteins are localized to the sea urchin egg cortex, using western blotting and immunocytochemistry. Tetanus toxin light chain cleaves the synaptobrevin-related protein in vitro and inhibits calcium-induced exocytosis. These data demonstrate a conservation between phyla of protein sequence and molecular mechanisms thought to facilitate exocytosis and show that the sea urchin egg provides a unique in vitro exocytotic model with which to study the conserved protein machinery of membrane fusion during secretion.

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