| Research Abstract |
To elucidate the molecular mechanisms by which the cell functions are regulated by the cytoskeletal and their related proteins, we developed a new analyzing method combining the biochemical techniques with ultrastructural analyses by electron microscopy. Using this method, we examined the molecular mechanisms of neurotransmitter release and hormone secretion.
In the process of neurotransmitter release, we exarffined the molecular structure of synapsin I and its molecular organization in presynaptic terminals using the low angle rotary shadowing technique, quick-freeze deep etch electron microscopy(QF-DE), immunoelectron microscopy. The high resolution provided by QFDE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles forming 30 nm short strands. Synapsin I also connected a microtubule to synaptic vesicles, forming 30 nm strands. These data suggest that synapsin I could be a main element of short bridge between actin filament
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s and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles. Because phosphorylation of synapsin I by Ca^<2+>/calmodulin-dependentkinase detaches synapsin I 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.
In adrenal medulla, calpactin I was found to be a globular molecule with a diameter of I Inm 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 crosslinking 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 of 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 calpactin I changes its conformation to cross-link vesicles and the plasma membrane after stimulation 6f cultured chromaffin cells and that it may play an important role in the binding of chromaffin vesicles to the plasma membrane during exocytosis.
Thus, the developed method presented here is quite powerful to elucidate a role of cytoskeletal and their related proteins in the processes of various cell functions. Less
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