Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces
Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces
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DOI：
10.1073/pnas.0405902101
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发表时间：
2004-10-12
影响因子：
11.1
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
Kovar, DR;Pollard, TD
形成蛋白是一种大的多结构域蛋白，用于组装肌动蛋白电缆，有助于动物和真菌细胞的极性和分裂。形成蛋白同源性-1（FH 1）结构域结合profilin，并且高度保守的FH 2结构域使肌动蛋白丝成核。我们的特点是两个formins，芽殖酵母Bni 1 p和裂殖酵母Cdc 12 p，肌动蛋白装配的影响。我们使用渐逝波荧光显微镜观察肌动蛋白丝的组装：（i）由可溶性α FH 1 FH 2结构域成核，（ii）与固定在显微镜载玻片上的α FH 1 FH 2结构域结合。Bni 1 p（FH 1FH 2）p和Cdc 12 p（FH 1FH 2）p使新的肌动蛋白丝成核，或捕获通过在固定的肌动蛋白和肌动蛋白丝的倒刺末端之间插入亚基而生长的预制肌动蛋白丝的倒刺末端。两种formin保持与生长的肌动蛋白丝倒刺末端结合> 1，000秒。在固定化肌动蛋白和第二锚点之间的细丝的伸长使细丝段短至0.7 μ m，表明单个肌动蛋白细丝的聚合产生> 1皮牛顿的力，接近理论最大值。屈曲后，进一步的增长产生了长的循环，没有超螺旋，这表明formins不阶梯沿着两个亚基暴露在不断增长的倒刺结束。在协议中，Arp 2/3复杂的分支丝不旋转，因为它们从附着在载玻片表面的formins生长。形成蛋白在机制上是不相同的，因为从Cdc 12（FH 1FH 2）p而不是Bni 1（FH 1FH 2）p延伸的倒刺末端需要profilin。然而，profilin增加了Bni 1（FH 1FH 2）p介导的倒刺末端伸长的速度从全速的75%到100%。
Formins are large multidomain proteins required for assembly of actin cables that contribute to the polarity and division of animal and fungal cells. Formin homology-1 (FH1) domains bind profilin, and highly conserved FH2 domains nucleate actin filaments. We characterized the effects of two formins, budding yeast Bni1p and fission yeast Cdc12p, on actin assembly. We used evanescent wave fluorescence microscopy to observe assembly of actin filaments (i) nucleated by soluble formin FH1FH2 domains and (it) associated with formin FH1FH2 domains immobilized on microscope slides. Bni1p(FH1FH2)p and Cdc12p(FH1FH2)p nucleated new actin filaments or captured the barbed ends of preformed actin filaments that grew by insertion of subunits between the immobilized formin and the barbed end of the filament. Both formins remained bound to growing actin filament barbed ends for >1,000 sec. Elongation of a filament between an immobilized formin and a second anchor point buckled filament segments as short as 0.7 mum, demonstrating that polymerization of single actin filaments produces forces of > 1 piconewton, close to the theoretical maximum. After buckling, further growth produced long loops that did not supercoil, suggesting that formins do not stair step along the two subunits exposed on the growing barbed end. In agreement, Arp2/3 complex branched filaments did not rotate as they grew from formins attached to the slide surface. Formins are not mechanistically identical because barbed end elongation from Cdc12(FH1FH2)p, but not Bni1(FH1FH2)p, requires profilin. However, profilin increased the rate of Bni1(FH1FH2)p-mediated barbed end elongation from 75% to 100% of full-speed.