Actin dynamics and the roles of ADF and cofilin : studies with muscle and nerve as model systems

以肌肉和神经细胞为模型系统，研究了ADF/cofilin对肌动蛋白细胞骨架的调节作用，主要内容如下：1.小鼠肌肉型cofilin的鉴定克隆了编码肌肉型cofilin的cDNA，并测定了其全序列。北方杂交和原位杂交结果表明，肌丝切蛋白主要在肌细胞中表达，而另一种非肌丝切蛋白，cofilin/ADF的功能结构域分析，在其N端发现了一个新的肌动蛋白结合位点。3. ADF/cofilin在细胞中的功能活性分析，已知ADF/cofilin的活性与细胞的肌动蛋白结合位点有关，而ADF/cofilin的活性与细胞的肌动蛋白结合位点有关。cofilin受磷酸化和PIP 2结合调节。我们发现1）去磷酸化以细胞周期依赖的方式发生，2）N端附近的Ser 2被磷酸化，3）导入细胞的外源性cofilin是有活性的，但其活性被PIP 2-bindin抑制。 ...更多信息 g.4.非洲爪蟾ADF/cofilin的鉴定用编码鸡cofilin和ADF的cDNA筛选非洲爪蟾胚胎cDNA文库，获得两个cDNA，并测定其全序列，它们编码168个氨基酸的蛋白质，在整个序列中有14个残基不同。这两种蛋白质与鸡丝切蛋白约有77%的同源性，与鸡ADF约有66%的同源性。每种蛋白质都在细菌中表达为GST融合蛋白，经过纯化，并显示与肌动蛋白的相互作用与鸡丝切蛋白相同。制备了对非洲爪蟾ADF/cofilin具有特异性的单克隆和多克隆抗体。组织提取物的2D凝胶免疫印迹显示蛋白质的磷酸化和非磷酸化形式。磷酸化的种类在早期胚胎发育期间以最高量表达。原位杂交与地高辛的反义核糖核酸探针显示，ADF/cofilin的消息是本地化的整体胚胎组织，但在早期发展的神经和肌组织中相当丰富。少

Regulation of actin cytoskeleton by ADF/cofilin was studied with muscle and nerve cells as model systems as follows.1.Characterization of a novel muscle-type cofilin of mouse.The cDNA encoding the muscle-type cofilin was cloned and the entire sequence was determined. Northern blot and in situ hybridization showed that expression of muscle cofilin predominates in muscle cells, while the other type, non-muscle cofilin, is expressed in variety of tissues except muscle cells.2.Analysis of functional domains of cofilin/ADF.A new actin-binding site was detected near the N-terminus of the molecules.3.Analysis of functional activity of ADF/cofilin in the cells.It is known that the activity of ADF/cofilin is regulated by phosphorylation and PIP2-binding. Here, we found that 1) de-phosphorylation occurs in a cell cycle-depending manner, 2) Ser 2 near N-terminus is phosphorylated, and 3) the exogenous cofilin which is introduced in the cells is active but the activity is suppressed by PIP2-bindin … More g.4.Characterization of Xenopus ADF/cofilinA Xenopus embryo cDNA library was screened with cDNAs encoding chick cofilin and ADF.Two cDNAs were obtained and the entire sequences were determined, which encode proteins of 168 amino acids and differ in 14 residues spread throughout the sequence. Both proteins are about 77 % homologous to chick cofilin and 66 % homologous to chick ADF.Each protein was bacterially expressed as a GST-fusion protein, purified, and shown to interact with actin identically to chick cofilin. Monoclonal and polyclonal antibodies specific for the Xenopus ADF/cofilin was prepared. 2D-gel immunoblots of tissue extracts showed phosphorylated and non-phosphorylated forms of the proteins. The phsphorylated species is expressed in highest amount during early embryonic development. In situ hybridization with digoxigenin-containing antisense riboprobes showed that the message for ADF/cofilin was localized in overall embryonic tissues but rather abundantly in neural and myogenic tissues during early development. Less