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

肌动蛋白动力学以及 ADF 和丝切蛋白的作用：以肌肉和神经为模型系统的研究

基本信息

批准号：
05044122
负责人：
OBINATA Takashi
金额：
$ 3.84万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05044122/
关键词：
Cofilin ADF Actin-binding proteins Actin cytoskeleton Muscle cell Nerve cell Xenopus Xenopus Myofibrillogenesis

项目摘要

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

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。1.一种新的小鼠肌型cofilin的特征，克隆了编码肌型cofilin的cDNAs并测定了其全序列。Northern印迹和原位杂交表明，肌胶原蛋白主要在肌肉细胞中表达，而非肌胶原蛋白在除肌肉细胞外的多种组织中都有表达。2.分析肌胶蛋白/ADF的功能结构域，在分子的N端附近发现了一个新的肌动蛋白结合部位。3.细胞内ADF/Cofilin的功能活性分析。在这里，我们发现1)去磷酸化以一种依赖于细胞周期的方式发生，2)N端附近的Ser2被磷酸化，3)引入细胞内的外源cofilin是活性的，但活性被pip2-bindin…抑制利用编码鸡Cofilin和ADF的cDNAs对非洲爪哇胚胎cdna文库进行筛选，得到两个cDNA，并测定了它们的全序列，这两个cDNA编码168个氨基酸的蛋白质，在整个序列中有14个残基的差异。这两种蛋白与鸡Cofilin的同源性约为77%，与鸡ADF的同源性约为66%。每种蛋白都以GST融合蛋白的形式在细菌中表达，纯化后与肌动蛋白的相互作用与鸡Cofilin相同。制备了针对非洲爪哇ADF/cofilin的单抗和多克隆抗体。组织提取液的2D-Gel免疫印迹显示蛋白质的磷酸化和非磷酸化形式。磷酸化物种在早期胚胎发育中的表达最高。与含有地高辛的反义核糖核酸探针的原位杂交表明，ADF/cofilin的信息定位于整个胚胎组织中，但在发育早期的神经和肌源性组织中含量较高。较少