Faculty of Medicine, Department of Biophysics, University of Pécs, Szigeti Str. 12, Pécs H-7624, Hungary.
J Biol Chem. 2010 Apr 23;285(17):13154-69. doi: 10.1074/jbc.M109.093914. Epub 2010 Feb 21.
We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.
我们通过使用各种生物物理方法和体内实验，研究了果蝇 DAAM-FH2 和 DAAM-FH1-FH2 片段的特性及其与肌动蛋白和 Profilin 的相互作用。结果表明，尽管 DAAM-FH2 片段在体内对肌动蛋白组装没有任何明显的影响，但在表达 DAAM-FH1-FH2 片段的细胞中，观察到 Profilin 依赖性肌动蛋白结构形成增加。DAAM-FH1-FH2 在气管中的特异性表达也会引起表型效应，导致气管管腔塌陷和幼虫期的致死性。在体外，这两个 DAAM 片段都能催化肌动蛋白成核，但严重降低肌动蛋白丝的延伸和解聚速率。Profilin 是 DAAM 功能的分子开关。DAAM-FH1-FH2 与带刺末端结合，驱动 Profilin-肌动蛋白的连续组装，而 DAAM-FH2 与带刺末端形成无支持 Profilin-肌动蛋白组装的无活性复合物。这两个 DAAM 片段还结合到肌动蛋白丝的侧面并诱导肌动蛋白束形成。这些观察结果表明，果蝇 DAAM 形成因子代表了由 Profilin 门控的极端带刺末端调节因子。
