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

根据最近关于神经末梢递质释放现象的研究，已经表明去极化依赖性Ca^<2+>通过Ca^<2 +>通道流入神经末梢触发突触囊泡的胞吐作用。类似的过程也发生在由Ca^2+内流进入肾上腺嗜铬细胞等分泌细胞引起的胞吐作用中。然而，Ca^2+内流和囊泡融合到质膜之间的过程是完全未知的。在许多与细胞膜和细胞外基质相关的蛋白质中，最近的生理学研究表明突触1参与神经末梢递质的释放。Calpactin I存在于肾上腺髓质，是一种Ca^<2+>依赖性的磷脂结合蛋白，并以Ca^<2+>依赖性方式聚集含磷脂的脂质体，表明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，我们从 ...更多信息 本研究采用低角度旋转阴影技术、快速冷冻深蚀刻电镜（QF-DE）、免疫电镜等方法，观察突触蛋白1（synapsin 1）的分子结构及其与突触前终末肌动蛋白丝、微管和突触囊泡的关系，并探讨突触蛋白1在突触前终末和肾上腺嗜铬细胞中的定位和功能。突触蛋白1长约47 nm，由头部（直径约14 nm）和尾部（长约33 nm）组成，具有蝌蚪状外观。QF-DE提供的高分辨率显示，单个突触蛋白1交联肌动蛋白丝，并将肌动蛋白丝与突触囊泡连接形成约30 nm的短链。突触1还将微管连接到突触囊泡，形成约30 nm的链。这些数据表明，突触1可能是肌动蛋白丝和突触小泡之间，微管和突触小泡之间，以及突触小泡之间的短连接的主要元素。由于突触1被Ca^<2+>/钙调素依赖性激酶磷酸化，使突触1从囊泡中分离出来，它可以释放突触囊泡，从而增加突触囊泡向突触前膜的迁移率，当去极化依赖性Ca^<2+>流入突触前末梢时，肾上腺髓质的Calpactin I在云母上被发现是一个直径为-11 nm的球状分子。当脂质体被calpactin聚集时，QF-DE显示除了脂质体上的小球之外，还有6.5nm长的交联相对膜的细链。在培养的嗜铬细胞中，经乙酰胆碱刺激后，在嗜铬小泡和质膜之间发现类似的交联短链（6-10 nm）。质膜也揭示了许多球状结构，直径10 nm的细胞质表面。免疫电镜显示，calpactin I与质膜的内面密切相关，尤其是质膜和相邻的嗜铬小泡之间的显着。这些数据有力地表明，acetactin I改变其构象，以交联囊泡和质膜刺激培养的嗜铬细胞后，它可能发挥了重要作用，在结合的嗜铬囊泡胞吐过程中的质膜。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