Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.
Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.
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DOI：
10.1371/journal.pone.0026200
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yumura S
中科院分区：
文献类型：
--
作者：
Uyeda TQ;Iwadate Y;Umeki N;Nagasaki A;Yumura S
为了验证肌球蛋白II运动结构域（S1）在体内优先结合肌动蛋白细丝的特定亚群的假设，我们表达了gfp融合的S1，突变增强了其对Dictyostelium细胞中肌动蛋白的亲和力。与假设一致，GFP-S1突变体定位在细胞皮层的特定部分。与固定细胞的罗丹明-phalloidin染色比较表明，GFP-S1探针优先结合后皮层和卵裂沟中的肌动蛋白丝，其中肌动蛋白丝通过与内源性肌球蛋白II丝的相互作用而拉伸。GFP-S1探针在没有肌球蛋白II的情况下被动拉伸的皮质中也同样富集。GFP-S1突变体与拉伸的肌动蛋白丝的优先结合并不依赖于皮质素I或PTEN，这两种蛋白先前与肌球蛋白II丝向拉伸的皮质募集有关。这些结果表明，是肌动蛋白丝本身的拉伸增加了它们对肌凝蛋白II运动结构域的亲和力。相比之下，gfp融合的肌球蛋白I运动结构域没有定位到拉伸的肌动蛋白丝，这表明运动结构域对不同结构的肌动蛋白丝的不同偏好在肌球蛋白I和肌动蛋白II的不同细胞内定位中起作用。我们提出了一种方案，其中肌动蛋白丝的拉伸，肌凝蛋白II丝与拉伸的肌动蛋白丝的优先结合，以及肌凝蛋白II依赖的收缩形成一个正反馈回路，有助于细胞极性的稳定和细胞对外部机械刺激的反应性。
To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.
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