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，我们建立了一种将生化技术与电镜超微结构分析相结合的分析方法。利用这种方法，我们研究了神经递质释放和激素分泌的分子机制。在神经递质释放过程中，我们利用低角度旋转阴影技术、速冻深蚀刻电镜（QF-DE）、免疫电镜研究突触素I的分子结构及其在突触前末端的分子组织。QFDE提供的高分辨率显示，单个突触蛋白1与肌动蛋白丝交联，并与突触囊泡形成30 nm的短链。突触蛋白1也将微管连接到突触囊泡，形成30纳米的链。这些数据表明突触蛋白1可能是肌动蛋白丝与突触囊泡之间、微管与突触囊泡之间以及突触囊泡之间的短桥的主要成分。由于Ca^<2+>/钙调素依赖性激酶磷酸化突触蛋白I使突触蛋白I脱离囊泡，从而释放突触囊泡，从而增加突触囊泡的移动性。去极化依赖的Ca^<2+>通量进入突触前末端。在肾上腺髓质中，发现calpactin I是一种球状分子，直径为1 μ m，位于云母上。当脂质体被calpacactin聚集时，QF-DE发现除了脂质体上的小球外，还有一条6.5 nm长的细链交联相反膜。在培养的染色质细胞中，乙酰胆碱刺激后，染色质囊泡与质膜之间出现了类似的交联短链（6-10 nm）。质膜细胞质表面显示大量直径为10nm的球状结构。免疫电镜显示calpactin I与质膜内表面密切相关，在质膜与相邻的染色质囊泡之间尤为明显。这些数据有力地表明，calpactin I在刺激培养的染色质细胞后改变其构象，使其与质膜交联，并可能在胞分泌过程中染色质囊泡与质膜的结合中发挥重要作用。因此，本文提出的方法对于阐明细胞骨架及其相关蛋白在各种细胞功能过程中的作用是非常有力的。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