Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin
Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin
复制标题
DOI：
10.1091/mbc.e18-08-0500
复制
发表时间：
2019-03-21
影响因子：
3.3
通讯作者：
Kovar, David R.
中科院分区：
文献类型：
--
作者：
Harker, Alyssa J.;Katkar, Harshwardhan H.;Kovar, David R.
Ena/Vasp四聚体是一种进行性肌动蛋白延长因子，定位于不同的F-肌动蛋白网络中，这些F-肌动蛋白网络由不同的交联蛋白组成，如丝状伪足(Fasin)、片状脂膜(Fimbrin)和应激纤维(α-Actinin)。以前，我们发现EnA在束状F-肌动蛋白的拖尾带刺末端上进行的过程运行时间大约是原来的三倍。在这里，我们使用单分子全内反射荧光显微镜(TIRFM)，并建立了动力学模型，进一步剖析了Ena/Vasp对束状纤维的推进机制。我们发现，Ena在拖尾带刺末端的处理能力增强是针对束素束的，而对Fimbrin或α-Actinin束没有增强作用。值得注意的是，Ena/Vasp的进程长度随着束素束细丝和Ena“臂”的数量增加而增加，表明亲和力有助于增强进程。在果蝇培养细胞中，Ena四聚体始终比突变的二聚体和三聚体形成更多的丝状足。此外，束素束状细丝拖尾带刺末端的加工能力增强是Ena/Vasp同源物的进化保守特性，包括人Vasp和秀丽线虫UNC-34。这些结果表明，ENA四聚体是为增强束素束的加工能力而量身定做的，多臂与多丝结合的亲和力对这一过程至关重要。此外，我们发现了一种新的调控过程，通过这种过程，束大小和捆绑蛋白特异性控制着一个过程组装因子的活动。
Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (alpha-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. We discovered that Ena's enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or alpha-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena "arms," revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.