Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy.
Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy.
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DOI：
10.7554/elife.04806
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发表时间：
2015-02-02
期刊：
影响因子：
7.7
通讯作者：
Uyeda TQ
中科院分区：
文献类型：
--
作者：
Ngo KX;Kodera N;Katayama E;Ando T;Uyeda TQ
采用高速原子力显微镜观察由丝切蛋白结合诱导的肌动蛋白丝的结构变化。与先前的电子显微镜和荧光显微镜研究一致，丝切蛋白沿肌动蛋白丝形成簇，与对照丝相比，此处的丝粗2纳米，螺旋螺距短约25%。有趣的是，缩短的螺旋螺距传播到簇的尖端侧相邻的无蛋白区，而有刺端侧的螺距与对照相似。因此，丝切蛋白簇在丝中诱导出明显不对称的构象变化。与丝切蛋白有利于具有较短螺旋螺距的肌动蛋白结构这一观点一致，丝切蛋白簇向丝的尖端单向生长。经常在无蛋白区和簇之间的边界附近观察到切割，但不一定就在边界处。
DOI: http://dx.doi.org/10.7554/eLife.04806.001
肌动蛋白是在包括植物、动物和真菌细胞在内的所有真核细胞中发现的最丰富的蛋白质之一。这种蛋白质参与对生物体生存至关重要的广泛的生物学过程。肌动蛋白形成长丝，帮助细胞维持其形状，并为细胞分裂和/或移动提供所需的力。
肌动蛋白丝呈螺旋状，由许多连接在一起的肌动蛋白亚基组成。肌动蛋白丝是可变结构，随着新的肌动蛋白亚基在丝的末端添加或移除，其不断生长和收缩。肌动蛋白丝的一端比另一端生长得更快；生长快的一端称为有刺端，而生长慢的一端称为尖端。
已知有100多种其他蛋白质与肌动蛋白结合并协同作用，以调节其在细胞中的作用以及它如何形成丝。丝切蛋白就是这样一种蛋白质，它与肌动蛋白丝结合并在其上形成簇，它还能切割肌动蛋白丝。切割肌动蛋白丝可促使丝解聚，或者它可帮助产生新的有刺端，然后这些有刺端可生长成新的丝。先前的研究表明，丝切蛋白在肌动蛋白丝上有丝切蛋白覆盖的区域和没有覆盖的区域之间的连接处切割肌动蛋白丝。也已知丝切蛋白与丝的结合会导致丝改变形状，并且形状变化会传播到丝上没有丝切蛋白覆盖的相邻部分。然而，丝切蛋白在哪里结合以及形状变化如何沿肌动蛋白丝传播的细节尚不清楚。此外，这些先前研究的结果主要基于对肌动蛋白丝的静态图像的检查，这与活细胞中不断变化的丝不同。
Ngo、Kodera等人现在利用一种最近开发的称为高速原子力显微镜的成像技术，分析了丝切蛋白与肌动蛋白丝结合并沿其形成簇时所发生的情况。这种技术可用于直接观察单个蛋白质的活动。与先前的发现一致，Ngo、Kodera等人观察到有丝切蛋白覆盖的丝比没有丝切蛋白的丝粗；并且丝切蛋白的结合也大大降低了丝的螺旋扭曲。Ngo、Kodera等人还发现这些形状变化沿丝仅在一个方向——向尖端传播。此外，丝切蛋白簇也仅向肌动蛋白丝的尖端生长——并且丝经常在有丝切蛋白覆盖和无覆盖区域之间的连接处附近被切割，但不完全在连接处。肌动蛋白丝这种单向的形状变化可能有助于调节其他肌动蛋白结合蛋白如何与丝相互作用，从而调节丝本身的作用。
DOI: http://dx.doi.org/10.7554/eLife.04806.002
High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001 Actin is one of the most abundant proteins found inside all eukaryotic cells including plant, animal, and fungal cells. This protein is involved in a wide range of biological processes that are essential for an organism's survival. Actin proteins form long filaments that help cells to maintain their shape and also provide the force required for cells to divide and/or move. Actin filaments are helical in shape and are made up of many actin subunits joined together. Actin filaments are changeable structures that continuously grow and shrink as new actin subunits are added to or removed from the ends of the filaments. One end of an actin filament grows faster than the other; the fast-growing end is known as the barbed-end, while the slow-growing end is referred to as the pointed-end. Over 100 other proteins are known to bind to and work with actin to regulate its roles in cells and how it forms into filaments. Cofilin is one such protein that binds to and forms clusters on actin filaments and it can also sever actin filaments. Severing an actin filament can encourage the filament to disassemble, or it can help produce new barbed ends that can then grow into new filaments. Previous work had suggested that cofilin severs actin filaments at the junction between regions on the filament that are coated with cofilin and those that are not. It was also known that cofilin binding to a filament causes the filament to change shape, and that the shape change is propagated to neighboring sections of the filaments not coated with cofilin. However, the details of where cofilin binds and how changes in shape are propagated along an actin filament were not known. Furthermore, the findings of these previous studies were largely based on examining still images of actin filaments, which are unlike the constantly changing filaments of living cells. Ngo, Kodera et al. have now analyzed what happens when cofilin binds to and forms clusters along actin filaments using a recently developed imaging technique called high-speed atomic force microscopy. This technique can be used to directly visualize individual proteins in action. Consistent with previous findings, Ngo, Kodera et al. observed that filaments coated with cofilin are thicker than those filaments without cofilin; and that cofilin binding also substantially reduces the helical twist of the filament. Ngo, Kodera et al. also found that these changes in shape are propagated along the filament but in only one direction—towards the pointed-end. Moreover, cofilin clusters also only grew towards the pointed-end of the actin filament—and the filaments were often severed near, but not exactly at, the junctions between cofilin-coated and uncoated regions. Such one-directional changes in shape of the actin filaments presumably help to regulate how other actin binding proteins can interact with the filament and consequently regulate the roles of the filaments themselves. DOI: http://dx.doi.org/10.7554/eLife.04806.002