Distinct actin oligomers modulate differently the activity of actin nucleators
Distinct actin oligomers modulate differently the activity of actin nucleators
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DOI：
10.1111/febs.13381
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发表时间：
2015-10-01
期刊：
影响因子：
5.4
通讯作者：
Mannherz, Hans Georg
中科院分区：
文献类型：
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作者：
Qu, Zheng;Silvan, Unai;Mannherz, Hans Georg
肌动蛋白单体聚合成丝需要首先形成由几个肌动蛋白亚基组成的核；然而，它们的不稳定性阻碍了对它们的详细研究。因此，我们使用化学交联的肌动蛋白寡聚物来分析它们对肌动蛋白聚合的影响。通过凝胶过滤分离出沿遗传螺旋（Lys199 和 Cys374 之间）通过亚苯基双马来酰亚胺进行分子间交联的肌动蛋白二聚体（上二聚体，UD）、三聚体和四聚体，并发现如芘测定和全内反射荧光显微镜所示，它们越来越刺激肌动蛋白聚合。相比之下，以 Cys374-Cys374 交联为特征的所谓低肌动蛋白二聚体 (LD) 仅在低浓度下刺激肌动蛋白聚合，但在高浓度下抑制肌动蛋白聚合。 UD和三聚体刺激肌动蛋白与胸腺肽β4（Tβ4）或肌动蛋白复合物的再聚合，而LD仅刺激肌动蛋白：肌动蛋白的再聚合，而不是肌动蛋白：Tβ4复合物的再聚合。在体内，肌动蛋白聚合受到成核因子的刺激。因此，分析了纯化的 LD、UD 和三聚体对凝溶胶蛋白、小鼠透明相关 (mDia) 福明和肌动蛋白相关蛋白 2/3 (Arp2/3) 复合物的肌动蛋白成核活性的相互作用和影响。天然凝胶电泳证明 LD、UD 和三聚体与凝溶胶蛋白及其片段 G1-3、formins mDia1 和 mDia3 的 FH2 结构域以及 Arp2/3 复合物结合。 UD 和三聚体增加了凝溶胶蛋白和 G1-3 的成核活性，但不增加 mDia-FH2 结构域和 Arp2/3 复合物的成核活性。相反，与 Arp2/3 复合物等摩尔浓度的 LD 刺激其成核活性，但抑制 mDia-FH2 结构域、凝溶胶蛋白和 G1-3 的成核活性，表明含有不同方向肌动蛋白亚基的二聚体对其成核活性进行差异调节。
Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin beta 4 (T beta 4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : T beta 4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits.