Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Chiba University|
OBINATA Takashi Chiba University, 理学部, 教授 (40012413)
BAMBURG James r. Colorado State University, 生化学教室, 教授
ABE Hiroshi Chiba University, 理学部, 助手 (00222662)
JAMES R Bamb Colorado State Univ, Dept of Biochem., 教授
|Project Period (FY)
1993 – 1994
Completed(Fiscal Year 1994)
|Budget Amount *help
¥6,000,000 (Direct Cost : ¥6,000,000)
Fiscal Year 1994 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1993 : ¥2,500,000 (Direct Cost : ¥2,500,000)
|Keywords||Cofilin / ADF / Actin-binding proteins / Actin cytoskeleton / Muscle cell / Nerve cell / Xenopus / Xenopus / Myofibrillogenesis|
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
4.Characterization of Xenopus ADF/cofilin
A 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