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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，如下：1.新型小鼠肌肉型cofilin的表征。克隆了编码肌肉型cofilin的cDNA并确定了其全序列。 Northern blot和原位杂交结果表明，肌肉cofilin主要在肌肉细胞中表达，而另一种类型非肌肉cofilin在除肌肉细胞外的多种组织中表达。2.cofilin/ADF功能域分析。在分子N端附近检测到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众所周知，ADF/cofilin 的活性受磷酸化和 PIP2 结合的调节。在这里，我们发现 1) 去磷酸化以细胞周期依赖性方式发生，2) N 末端附近的 Ser 2 被磷酸化，3) 引入细胞中的外源 cofilin 具有活性，但其活性被 PIP2-bindin 抑制… 更多 g.4. 非洲爪蟾 ADF/cofilin 的表征用编码小鸡的 cDNA 筛选非洲爪蟾胚胎 cDNA 文库获得了两个cDNA并确定了整个序列，其编码168个氨基酸的蛋白质并且在整个序列中分布有14个残基不同。这两种蛋白与鸡丝切蛋白约 77% 同源，与鸡 ADF 同源性约 66%。每种蛋白均通过细菌表达为 GST 融合蛋白，进行纯化，并显示与肌动蛋白的相互作用与鸡丝切蛋白相同。制备了爪蟾 ADF/cofilin 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。磷酸化物种在早期胚胎发育期间表达量最高。与含有地高辛配基的反义核糖核酸探针的原位杂交表明，ADF/cofilin 的信息位于整个胚胎组织中，但在早期发育过程中大量存在于神经和肌源性组织中。较少的