A New Method for the Analyses of Cytoskeletal and Their Related Proteins

为了阐明细胞骨架及其相关蛋白调控细胞功能的分子机制，我们发展了一种将生物化学技术与电镜超微结构分析相结合的新的分析方法。在神经递质释放的过程中，利用低角度旋转阴影技术、快速冷冻深蚀刻电镜（QF-DE）、免疫电镜等技术，观察突触前终末突触蛋白I（synapsin I）的分子结构及其分子组织结构。QFDE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30 nm短链。突触蛋白I还将微管连接到突触囊泡，形成30 nm的链。这些结果提示突触蛋白I可能是肌动蛋白丝之间短桥的主要成分 ...更多信息 的和突触小泡，和微管和突触小泡之间，和突触小泡之间。由于突触蛋白I被Ca^<2+>/钙调素依赖性激酶磷酸化后，突触蛋白I从囊泡中分离出来，从而释放突触囊泡，从而增加突触囊泡的运动性。在肾上腺髓质中，calpactin I是一个球状分子，在云母上的直径为1 Inm。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）。质膜也显示出许多球形结构的直径为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，calpactin I改变其构象，以交联囊泡和质膜刺激6 f培养的嗜铬细胞，它可能在嗜铬囊泡结合到质膜在胞吐过程中发挥了重要作用，因此，这里提出的方法是相当强大的阐明细胞骨架蛋白及其相关蛋白在各种细胞功能的过程中的作用。少

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 … More 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