Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.
Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.
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DOI：
10.7554/elife.06585
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发表时间：
2015-08-21
期刊：
影响因子：
7.7
通讯作者：
Mullins RD
中科院分区：
文献类型：
--
作者：
Hansen SD;Mullins RD
使能/血管扩张（Ena/VASP）蛋白在多个位置促进肌动蛋白丝组装，包括：前缘膜，局灶性粘连和细胞内病原体的表面。一种重要的Ena/VASP调节剂是mig-10/Lamellipodin/RIAM家族的衔接子，其促进成纤维细胞中的片足形成并驱动神经元中的神经突生长和轴突导向。为了更好地了解MRL蛋白如何促进肌动蛋白网络的形成，我们研究了Lamellipodin（LPD），肌动蛋白和VASP之间的相互作用，在体内和体外。我们发现，LPD直接结合到肌动蛋白丝，这种相互作用调节其亚细胞定位和增强其对VASP聚合酶活性的影响。我们建议，LPD提供Ena/VASP蛋白的不断增长的倒钩结束，并增加其聚合酶活性的束缚他们的细丝。这种相互作用代表了另一种途径，通过这种途径，生长的肌动蛋白丝产生正反馈，以控制蛋白质的定位和活性，从而调节它们的组装。http://dx.doi.org/10.7554/eLife.06585.001肌动蛋白是大多数真核细胞中最丰富的蛋白质，它组装成一个横跨细胞长度和宽度的细丝网络。就像动物的骨架一样，这种“肌动蛋白细胞骨架”赋予细胞形状和强度，并使细胞能够积极地在环境中移动。为了开始运动，许多细胞开始在细胞膜旁组装肌动蛋白丝。这些细丝的生长推动细胞膜向前，并形成一个称为“片状体”的二维结构，它探索细胞周围的空间并引导其运动。片足动物的肌动蛋白丝是动态的，并经历重复的组装和拆卸循环。这些过程受到各种其他蛋白质的严格调控。例如，Ena/VASP蛋白家族的成员收集肌动蛋白丝的构建模块，并将它们快速堆叠在细丝快速生长的末端。Ena/VASP蛋白的活性在形成片状伪足肌动蛋白网络和驱动细胞运动中发挥特别重要的作用。之前的工作表明，一种名为Lamellipodin的蛋白质与Ena/VASP蛋白结合，并帮助将它们招募到细胞膜上。然而，目前还不清楚Lamellipodin是否会影响Ena/VASP蛋白的活性或它们与肌动蛋白丝的相互作用。汉森和马林斯现在已经分析了Ena/VASP，Lamelliodin和肌动蛋白之间的相互作用。实验表明，Lamelliopodin不只是拴Ena/VASP蛋白的膜，但也直接结合到肌动蛋白丝，通过结合位点，这是从接触Ena/VASP的网站不同。进一步的实验表明，Lamellipodin可以同时与肌动蛋白丝和Ena/VASP蛋白相互作用。汉森和马林斯还发现，纯化的Lamellipodin与VASP蛋白相互作用，形成成簇的蛋白质复合物，加上肌动蛋白丝与膜的束缚，这种成簇大大增加了VASP延长肌动蛋白丝的能力。通过在活细胞中观察标记有绿色荧光蛋白的片状脂质蛋白，汉森和马林斯证明它与肌动蛋白丝的相互作用足以将片状脂质蛋白定位到细胞膜上。最后，由于除了Ena/VASP蛋白外，Lamellipodin还与多种信号分子相互作用，因此下一个重大挑战是了解Lamellipodin本身是如何调节的。未来的研究还可以探索细胞如何利用肌动蛋白细胞骨架的力量来进行这些基本活动。DOI：http://dx.doi.org/10.7554/eLife.06585.002网站
Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 Actin—the most abundant protein in most eukaryotic cells—assembles into a network of filaments that spans the length and breadth of the cell. Like the skeleton of an animal, this ‘actin cytoskeleton’ gives the cell its shape and strength, and enables the cell to actively move through its environment. To start moving, many cells begin assembling actin filaments next to the cell membrane. The growth of these filaments pushes the membrane forward and creates a two-dimensional structure called a ‘lamellipod’, which explores the space around the cell and steers its movement. The actin filaments in a lamellipod are dynamic and undergo repeated cycles of assembly and disassembly. These processes are tightly regulated by a variety of other proteins. Members of the Ena/VASP protein family, for example, collect the building blocks of an actin filament and rapidly stack them in place on the fast-growing end of a filament. The activities of Ena/VASP proteins play an especially important role in creating lamellipodial actin networks and in driving cell movement. Previous work showed that a protein called Lamellipodin binds to Ena/VASP proteins and helps recruit them to the cell membrane. However, it was unclear whether Lamellipodin could affect the activity of Ena/VASP proteins or their interaction with the actin filaments. Hansen and Mullins have now analyzed the interactions between Ena/VASP, Lamellipodin and actin. The experiments demonstrate that Lamellipodin does not simply tether Ena/VASP proteins to the membrane but also binds directly to actin filaments, via a binding site that is distinct from the site that contacts Ena/VASP. Further experiments with purified proteins revealed that Lamellipodin could interact with both actin filaments and Ena/VASP proteins at the same time. Hansen and Mullins also found that purified Lamellipodin interacted with VASP proteins to form clustered protein complexes, and that together with the tethering of actin filaments to the membrane, this clustering greatly increased VASP's ability to lengthen actin filaments. By visualizing Lamellipodin tagged with a green fluorescent protein in living cells, Hansen and Mullins then showed that its interaction with actin filaments was sufficient to localize Lamellipodin to the cell membrane. Finally, since Lamellipodin interacts with a multitude of signaling molecules in addition to Ena/VASP proteins, the next big challenge is to understand how Lamellipodin itself is regulated. Future studies could also explore how cells harness the power of the actin cytoskeleton to carry out these essential activities. DOI: http://dx.doi.org/10.7554/eLife.06585.002