Role of Cytoskeletal System and its Regulation by Ca^<2+> in Exocytosis

最近关于神经末梢递质释放现象的研究表明，去极化依赖的Ca~(2+)通过Ca~(2+)通道进入神经末梢，触发突触小泡的胞吐。当Ca~(2+)流入肾上腺嗜铬细胞等分泌细胞时，也会发生类似的过程。然而，Ca~(2+)和gt~(2+)之间的内流和囊泡融合到质膜的过程是完全未知的。最近的一项生理学研究表明，在许多细胞骨架和膜相关蛋白中，突触1参与了神经末梢递质的释放。Calpactin I存在于肾上腺髓质中，是一种钙依赖的磷脂结合蛋白，以钙依赖的方式聚集含磷脂的脂质体，提示它可能在胞吐过程中发挥作用。因此，为了阐明胞吐作用的分子机制，我们进行了…进一步研究突触素1和钙化肌动蛋白I在突触前终末和肾上腺嗜铬细胞中的定位和功能。我们利用小角度旋转阴影技术、快速冷冻深蚀刻电子显微镜(QF-DE)、免疫电子显微镜观察突触前终末突触蛋白1的分子结构及其与肌动蛋白细丝、微管和突触小泡的关系。突触素1长-47 nm，由头(-14 nm直径)和尾(-33 nm长)组成，具有蝌蚪样外观。QF-DE提供的高分辨率显示，单个突触素1与肌动蛋白细丝和连接的肌动蛋白细丝相连，突触小泡形成-30 nm的短链。Synapsis 1还将微管连接到突触小泡，形成-30 nm的线条。这些数据表明，突触1可能是肌动蛋白细丝和突触小泡、微管和突触小泡之间以及突触小泡之间短连接的主要元件。由于突触1被钙调素依赖的激酶磷酸化使突触1从囊泡中分离出来，它可以释放突触小泡，从而增加突触小泡向突触前膜的迁移率，依赖去极化的钙离子流入突触前终末。肾上腺髓质中的Calpactin I被发现是一种直径为-11 nm的球形分子。当脂质体被Calpactin聚集时，QF-DE显示除了脂质体上的球状颗粒外，还有一条6.5 nm长的细小的交联对偶膜。在培养的嗜铬细胞中，经乙酰胆碱刺激后，嗜铬小泡与质膜之间有相似的交联短链(6~10 nm)。质膜表面也有许多直径为10 nm的球形结构。免疫电子显微镜显示，Calpactin I与质膜内面紧密相连，在质膜与相邻的嗜铬小泡之间尤为明显。这些数据有力地表明，clapactin I在刺激培养的嗜铬细胞后改变其构象以使小泡和质膜交联化，在胞吐过程中可能在嗜铬小泡与质膜的结合中发挥重要作用。较少

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 … More 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