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How differential distributions of actin-bindingproteins are formed in living cells.

肌动蛋白结合蛋白的差异分布如何在活细胞中形成。

基本信息

批准号：
15570153
负责人：
ISHIKAWA Ryoki
金额：
$ 2.18万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570153/
关键词：
actin tropomyosin actin-binding protein fascin drebrin

项目摘要

We examined the distribution and dynamics of actin-binding proteins in Ng108-15 cells. EGFP/fascin localized in filopodia. Pseudo-unphosphorylated mutant, in which 39-serine of fascin was replaced to asparatic acid, localized in filopodia while pseudo-phosphorylated mutant, in which 39-serine was replaced to alanine, showed dispersed distribution, suggesting that localization of fascin is regulated by phosphorylation. EGFP/smooth muscle type tropomyosin localized only in stress fibers, while EGFP/nonmusle type tropomyosin localized in stress fibers, filopodia, and lamellipodia. EGFP/caldesmon localized in all the actin structures.Reconstituted characterization of purified proteins revealed that fascin formed tight F-actin bundles with polarity. Drebrin bound to F-actin with a stoichiometry of 5 actin versus 1 drebrin molecule. Drebrin inhibited the actin-binding of caldesmon, actin-binding of tropomyosin, and actin-binding of fascin.These results suggest that competitive binding between drebrin and tropomyosin/caldesmon, between drebrin and fascin, and between fascin and tropomyosin/caldesmon may cause the differential localization fascin, drebrin, and tropomyosin/caldesmon, resulting in different organization of actin structure.

我们检测了Ng108-15细胞中肌动蛋白结合蛋白的分布和动态。EGFP/fascin定位于丝状足。伪未磷酸化突变体中，39-丝氨酸被替换为天冬氨酸，定位于丝足，而伪磷酸化突变体中，39-丝氨酸被替换为丙氨酸，分布分散，表明束蛋白的定位受磷酸化调节。EGFP/平滑肌型原肌球蛋白仅定位于应力纤维，而EGFP/非肌肉型原肌球蛋白定位于应力纤维、丝状足和板足。EGFP/caldesmon定位于所有肌动蛋白结构。纯化蛋白的重组表征显示，束状蛋白形成紧密的f -肌动蛋白束，具有极性。Drebrin与f -肌动蛋白结合，其化学计量为5个肌动蛋白对1个Drebrin分子。Drebrin抑制caldesmon的肌动蛋白结合、原肌球蛋白的肌动蛋白结合和筋膜蛋白的肌动蛋白结合。这些结果提示，由于drebrin与原肌凝蛋白/钙调蛋白、drebrin与fascin、fasin与原肌凝蛋白/钙调蛋白之间的竞争性结合，可能导致了fasin、drebrin和原肌凝蛋白/钙调蛋白定位的差异，从而导致了肌动蛋白结构的不同组织。