| Research Abstract |
According to recent studies concerning the phenomena involved in transmitter release at nerve terminal, it has been shown that depolarization-dependent Ca^<2+> influx into the nerve terminal through Ca^<2+> channels triggers exocytosis of synaptic vesicles. Similar processes could occur on the occasion of exocytosis induced by Ca^<2+> influx into secretory cells such as adrenal chromaffin cells. However, the processes between Ca^<2+> influx and vesicle fusion to the plasma membranes are totally unknown. Of many cytoskeleton-and membrane-associated proteins, synapsis 1 was shown to be involved in the release of the transmitter at nerve terminals by a recent physiological study. Calpactin I, which exists in the adrenal medulla, is a Ca^<2+>-dependent phospholipid-binding protein, and aggregates phospholipid-containing liposomes in Ca^<2+>-dependent manner, suggesting that it may play a role in the process of exocytosis. Therefore, to elucidate the molecular mechanism of exocytosis, we ex
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amined the localization and function of synapsin 1 and calpactin I in presynaptic terminal and adrenal chromaffin cells, respectively.
We examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in presynaptic terminals using the low angle rotary shadowing technique, Quick-Freeze Deep Etch electron microscopy (QF-DE), immunoelectron microscopy. The synapsin 1, -47nm long, was composed of a head (-14nm diam) and a tail (-33nm long), having a tadpole-like appearance. The high resolution provided by QF-DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles forming -30nm short strands. Synapsis 1 also connected a microtubule to synaptic vesicles, forming -30nm strands. These data suggest that synapsis 1 could be a main element of short linkage between actin filaments and synaptic vesicles, and between microtubules and syanptic vesicles, and between synaptic vesicles. Because phosphoryaltion of synapsis 1 by Ca^<2+>/calmodulin-dependent kinase detaches synapsis 1 from vesicles, it could release synaptic vesicles, and thus increase mobility of synaptic vesicles to the presynaptic membrane upon depolarization-dependent Ca^<2+> flux into the presynaptic terminal.
Calpactin I from adrenal medulla was found to be a globular molecule with a diameter of -11nm on mica. When liposomes were aggregated by calpactin, QF-DE revealed a fine thin strand of 6.5 nm long cross-linking opposing membrane in addition to the globules on liposomes. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structure 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy showed that calpactin I was closely associated with the inner face of the plasma membrane and was especially conspicuous between plasma membrane and adjacent chromaffin vesicles. These data strongly suggest that clapactin I changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells and that it may play an important role in the binding of chromaffin vesicles to the plasma membrane during exocytosis. Less
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