A novel mode of capping protein-regulation by twinfilin.
A novel mode of capping protein-regulation by twinfilin.
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DOI：
10.7554/elife.41313
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发表时间：
2018-10-23
期刊：
影响因子：
7.7
通讯作者：
Goode BL
中科院分区：
文献类型：
--
作者：
Johnston AB;Hilton DM;McConnell P;Johnson B;Harris MT;Simone A;Amarasinghe GK;Cooper JA;Goode BL
肌动蛋白丝的成核组装受肌动蛋白丝的倒刺端控制，封端蛋白（CP）在那里限制聚合作用。双联丝蛋白是CP在体内的一种保守结合伴侣，但这种相互作用的意义一直是个谜。在此，我们发现双联丝蛋白的C末端尾部含有一个CP相互作用（CPI）基序，从而确定它是一种新型的CPI基序蛋白。双联丝蛋白和CPI基序蛋白CARMIL在CP上有重叠的结合位点。此外，双联丝蛋白与CARMIL竞争性结合CP，保护CP不被CARMIL从倒刺端置换下来。双联丝蛋白还加速CP抑制剂V - 1的解离，使CP恢复到有活性的封端状态。双联丝蛋白和CP的敲低在细胞形态上都会导致类似的缺陷，并且双联丝蛋白表达升高可挽救由CARMIL活性过高导致的缺陷。总之，这些观察结果将双联丝蛋白定义为CP的第一种“促封端”配体，并促使我们对CP调节周期的理解提出重要修正。
植物和动物细胞由类似骨架的结构支撑，这些结构可以在细胞膜下生长和收缩，对细胞边缘产生推拉作用。这种支架网络——被称为细胞骨架——包含由一种叫做肌动蛋白的蛋白质的许多相同拷贝组成的长链或细丝。肌动蛋白的形状使它们能够首尾相连，并且使这些链能够按需生长和收缩。当链达到合适长度时，细胞用一种叫做封端蛋白的蛋白质封住生长端。这有助于稳定细胞骨架，防止链变得更长或更短。
干扰封端蛋白活性的蛋白质会使肌动蛋白链生长或收缩。一些蛋白质，比如一种叫做V - 1的蛋白质，附着在封端蛋白上并产生阻碍，使其无法位于肌动蛋白链的末端。其他的，比如CARMIL，与封端蛋白结合并改变其形状，使其更有可能从链上脱落。到目前为止，还没有人发现一种能帮助封端蛋白限制肌动蛋白细胞骨架生长的伴侣。
一种叫做双联丝蛋白的蛋白质经常与封端蛋白一起出现，但这两种蛋白质似乎对彼此没有影响，并且似乎有不同的作用。封端蛋白阻止生长并稳定肌动蛋白链，而双联丝蛋白加速其末端的解聚。但是约翰斯顿、希尔顿等人现在揭示这两种蛋白质实际上是协同工作的。双联丝蛋白帮助封端蛋白抵抗CARMIL和V - 1的影响，并且封端蛋白将双联丝蛋白置于链的末端。因此，当封端蛋白最终被CARMIL移除时，双联丝蛋白继续解聚肌动蛋白链。
双联丝蛋白的尾部看起来像CARMIL蛋白质的一部分，这表明它们可能以相同的方式与封端蛋白相互作用。在双联丝蛋白尾部连接一个荧光标签显示，这两种蛋白质竞争附着在封端蛋白的同一部位。当小鼠细胞产生额外的双联丝蛋白时，它阻断了CARMIL的作用，有助于肌动蛋白链的生长。V - 1附着在封端蛋白的不同位置，但双联丝蛋白也能够干扰其活性。当双联丝蛋白附着在CARMIL结合位点时，它没有直接阻断V - 1的结合，但使该蛋白质更有可能脱落。
理解肌动蛋白细胞骨架如何移动是细胞生物学中的一个关键问题，但它在医学上也有应用。双联丝蛋白在某些血癌细胞的扩散以及内耳中帮助我们听力的精细结构的形成中起作用。理解双联丝蛋白和封端蛋白如何相互作用可能为一系列医学病症开辟新的治疗途径。
Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first ‘pro-capping’ ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle. Plant and animal cells are supported by skeleton-like structures that can grow and shrink beneath the cell membrane, pushing and pulling on the edges of the cell. This scaffolding network – known as the cytoskeleton – contains long strands, or filaments, made from many identical copies of a protein called actin. The shape of the actin proteins allows them to slot together, end-to-end, and allows the strands to grow and shrink on-demand. When the strands are the correct length, the cell caps the growing ends with a protein known as Capping Protein. This helps to stabilize the cell’s skeleton, preventing the strands from getting any longer, or any shorter. Proteins that interfere with the activity of Capping Protein allow the actin strands to grow or shrink. Some, like a protein called V-1, attach to Capping Protein and get in the way so that it cannot sit on the ends of the actin strands. Others, like CARMIL, bind to Capping Protein and change its shape, making it more likely to fall off the strands. So far, no one had found a partner that helps Capping Protein limit the growth of the actin cytoskeleton. A protein called Twinfilin often appears alongside Capping Protein, but the two proteins seemed to have no influence on each other, and had what appeared to be different roles. Whilst Capping Protein blocks growth and stabilizes actin strands, Twinfilin speeds up their disassembly at their ends. But Johnston, Hilton et al. now reveal that the two proteins actually work together. Twinfilin helps Capping Protein resist the effects of CARMIL and V-1, and Capping Protein puts Twinfilin at the end of the strand. Thus, when Capping Protein is finally removed by CARMIL, Twinfilin carries on with disassembling the actin strands. The tail of the Twinfilin protein looks like part of the CARMIL protein, suggesting that they might interact with Capping Protein in the same way. Attaching a fluorescent tag to the Twinfilin tail revealed that the two proteins compete to attach to the same part of the Capping Protein. When mouse cells produced extra Twinfilin, it blocked the effects of CARMIL, helping to grow the actin strands. V-1 attaches to Capping Protein in a different place, but Twinfilin was also able to interfere with its activity. When Twinfilin attached to the CARMIL binding site, it did not directly block V-1 binding, but it made the protein more likely to fall off. Understanding how the actin cytoskeleton moves is a key question in cell biology, but it also has applications in medicine. Twinfilin plays a role in the spread of certain blood cancer cells, and in the formation of elaborate structures in the inner ear that help us hear. Understanding how Twinfilin and Capping Protein interact could open paths to new therapies for a range of medical conditions.