Maurice E Müller Institute for Structural Biology, Biocenter, Basel, Switzerland.
J Mol Biol. 2010 Nov 5;403(4):529-45. doi: 10.1016/j.jmb.2010.09.017. Epub 2010 Oct 1.
Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.
小鼠 Dia 相关形态发生蛋白（mDia）是肌动蛋白聚合的形成蛋白家族的成员，通过单体添加到快速生长的帽状末端来促进丝状肌动蛋白（F-actin）的延伸。据推测，由于其富含脯氨酸的 FH1 结构域，mDia 优先募集与原肌球蛋白复合的肌动蛋白。在细丝延伸过程中，二聚体 mDia-FH2 结构域通过其 FH2 结构域附着在帽状末端，该结构域形成一个反平行的环状结构，包围着细丝帽状末端。mDia-FH2 结构域的二聚化依赖于其 N 端套索和连接子亚结构域（连接子）。在这里，我们使用 mDia1-FH2 结构域加连接子以及缺少连接子的核心 mDia1、mDia2 和 mDia3 研究了分离的 FH2 结构域对肌动蛋白聚合的影响，通过负染色后的共沉淀和电子显微镜进行分析。分析超速离心显示，mDia1 和 mDia2 的核心 FH2 结构域形成二聚体的程度较低，而 mDia3-FH2 减去连接子和 mDia1-FH2 加连接子则容易二聚化。只有核心 mDia3-FH2 能够成核肌动蛋白聚合。然而，所有测试的核心 FH2 结构域都能对 F-actin 进行装饰和束集，这可以通过负染色后的电子显微镜观察到。对于 mDia3-FH2，束集活性最高，mDia2-FH2 次之，mDia1-FH2 进一步降低。mDia1-FH2 结构域加连接子也能在没有原肌球蛋白的情况下诱导肌动蛋白聚合，但不能诱导 F-actin 变形和束集。我们还测试了 mDia1-FH2 是否能够在稳定球状肌动蛋白的不同蛋白质复合物中重新聚合肌动蛋白。所得数据表明，mDia1-FH2 仅从肌动蛋白/原肌球蛋白-1 复合物中诱导肌动蛋白重新聚合，但与肌动蛋白解聚因子、凝胶蛋白片段 1、维生素 D 结合蛋白或脱氧核糖核酸酶 I 复合时则不能。
