ERATO Actin Filament Dynamics Project, Japan Science and Technology Corporation, RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Biochem Biophys Res Commun. 2010 Jan 22;391(4):1598-603. doi: 10.1016/j.bbrc.2009.12.078. Epub 2009 Dec 22.
The actin homolog ParM plays a microtubule-like role in segregating DNA prior to bacterial cell division. Fluorescence and cryo-electron microscopy have shown that ParM forms filament bundles between separating DNA plasmids in vivo. Given the lack of ParM bundling proteins it remains unknown how ParM bundles form at the molecular level. Here we show using time-lapse TIRF microscopy, under in vitro molecular crowding conditions, that ParM-bundle formation consists of two distinct phases. At the onset of polymerization bundle thickness and shape are determined in the form of nuclei of short helically disordered filaments arranged in a liquid-like lattice. These nuclei then undergo an elongation phase whereby they rapidly increase in length. At steady state, ParM bundles fuse into one single large aggregate. This behavior had been predicted by theory but has not been observed for any other cytomotive biopolymer, including F-actin. We employed electron micrographs of ParM rafts, which are 2-D analogs of 3-D bundles, to identify the main molecular interfilament contacts within these suprastructures. The interface between filaments is similar for both parallel and anti-parallel orientations and the distribution of filament polarity is random within a bundle. We suggest that the interfilament interactions are not due to the interactions of specific residues but rather to long-range, counter ion mediated, electrostatic attractive forces. A randomly oriented bundle ensures that the assembly is rigid and that DNA may be captured with equal efficiency at both ends of the bundle via the ParR binding protein.
肌动蛋白同源物 ParM 在细菌细胞分裂前对 DNA 进行分离时发挥类似于微管的作用。荧光和冷冻电子显微镜已经表明 ParM 在体内将分离的 DNA 质粒之间形成丝状束。鉴于缺乏 ParM 束形成蛋白，因此仍然不知道 ParM 束如何在分子水平上形成。在这里，我们使用延时 TIRF 显微镜，在体外分子拥挤条件下，显示 ParM 束形成由两个不同的阶段组成。在聚合的起始阶段，束的厚度和形状以短螺旋无序纤维核的形式确定，这些纤维核排列在液态晶格中。然后，这些核经历伸长阶段，从而迅速增加长度。在稳定状态下，ParM 束融合成一个大的聚集体。这种行为已经被理论预测，但尚未观察到任何其他细胞运动生物聚合物，包括 F-肌动蛋白。我们利用 ParM 筏子的电子显微镜图像，这是 3D 束的 2D 类似物，来识别这些超结构内主要的分子间纤维接触。平行和反平行取向的纤维之间的界面相似，并且在束内纤维极性的分布是随机的。我们认为，纤维间的相互作用不是由于特定残基的相互作用，而是由于长程、抗衡离子介导的静电吸引力。随机取向的束确保了组装是刚性的，并且通过 ParR 结合蛋白，DNA 可以以相同的效率在束的两端被捕获。
