Actin filament attachments for sustained motility in vitro are maintained by filament bundling.
Actin filament attachments for sustained motility in vitro are maintained by filament bundling.
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DOI：
10.1371/journal.pone.0031385
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kuhn JR
中科院分区：
文献类型：
--
作者：
Hu X;Kuhn JR
我们从单个蛋白质在体外重建了细胞的运动性，以研究肌动蛋白细丝是如何组织在前沿的。利用肌动蛋白细丝的全内反射荧光显微镜，我们测试了Profilin、Arp2/3和Capping Protein(CP)如何共同作用于包裹有N-WASP WCA结构域的薄玻璃纳米纤维或微珠。细纳米纤维产生了宽大的彗星尾巴，比珠子基质在肌动蛋白细丝组织中显示出更多的结构变化。在持续的运动中，生理浓度的镁离子产生肌动蛋白细丝束，这些细丝束连续地附着在纳米纤维上。总镁离子的减少消除了颗粒的运动性和肌动蛋白附着在颗粒表面，而不影响肌动蛋白聚合、Arp2/3成核或细丝封顶。对微球类似运动性的分析表明，失去丝束不会影响肌动蛋白外壳的形成或对称性破坏，但会消除彗星尾巴和粒子表面之间的持续附着。在低镁缓冲液中，添加镁离子、赖氨酸-赖氨酸或发酵素可以恢复彗星的尾部附着和持续的粒子运动。TIRF显微分析表明，在没有Arp2/3、Profilin和CP的情况下，WCA包被微球捕获的微丝通过聚阳离子或法菌素添加的微丝捆绑增加了WCA结构域对带刺末端的捕获。我们提出了一个模型，在该模型中，CP将带刺末端引导到前沿，而聚阳离子诱导的丝束维持顺序性的带刺末端附着到前沿。
We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg2+, Lys-Lys2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg2+ buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.
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影响因子：
3.4
作者：
Dickinson, RB;Purich, DL
通讯作者：
Purich, DL
影响因子：
9.2
作者：
Cameron, LA;Svitkina, TM;Borisy, GG
通讯作者：
Borisy, GG
影响因子：
64.5
作者：
Co, Carl;Wong, Derek T.;Taunton, Jack
通讯作者：
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DOI：
10.1083/jcb.146.6.1319
发表时间：
1999-09-20
期刊：
The Journal of cell biology
影响因子：
--
作者：
Egile C;Loisel TP;Laurent V;Li R;Pantaloni D;Sansonetti PJ;Carlier MF
通讯作者：
Carlier MF
影响因子：
9.8
作者：
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通讯作者：
Mullins, R. Dyche