MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.

中文翻译：

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的聚合枯草杆菌上的脂质膜MREB揭示MREB旁系同源物的侧向共聚合和阳离子对细丝形成强烈影响

MreB是肌动蛋白的细菌直系同源物，在细胞膜下方形成垂直于细胞长轴的活动细丝，对维持细胞形状起关键作用。我们希望在体外可视化枯草芽孢杆菌MreB，因此建立了获得单体蛋白的方案，该蛋白可以在平面膜系统上聚合或与大膜囊泡结合。使用平面膜系统和电子显微镜，我们显示枯草芽孢杆菌MreB形成细丝束，其可以分支和融合，平均宽度为70 nm。未聚合的YFP-MreB，CFP-Mbl和mCherry-MreBH蛋白的荧光显微镜显示出与膜的均匀结合，表明沿膜的2D扩散可促进细丝形成。加入二价镁和钙离子后，这三种蛋白质均形成了高度无序的细丝片，这些细丝片可能分裂或融合，因此在高蛋白质浓度下，MreB及其旁系同源物产生了一个细丝网络，该细丝网络远离膜。长丝的形成受到二价离子的正影响，而受到一价离子的负影响。YFP-MreB或CFP-Mbl还在两个相邻膜之间形成细丝，这些细丝通常具有弯曲的外观。新的MreB，Mbl或MreBH单体可能会添加到预先存在的长丝的侧面，并且MreB旁系同源物共聚，表明MreB旁系同源物之间存在直接的侧向相互作用。我们的数据表明，枯草芽孢杆菌MreB旁系同源物在体外可能不易形成有序细丝，这可能是由于广泛的侧向接触所致，但可以共聚。单体MreB，Mbl和MreBH均匀结合到膜上，