Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.
Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.
复制标题
DOI：
10.1128/mbio.02259-17
复制
发表时间：
2018-04-10
期刊：
影响因子：
6.4
通讯作者：
Guttman JA
中科院分区：
文献类型：
--
作者：
Dhanda AS;Vogl AW;Albraiki SE;Otey CA;Beck MR;Guttman JA
Palladin是肌动蛋白丰富的运动结构的重要组成部分，并在体外使分支肌动蛋白丝阵列成核。在这里，我们研究了palladin在单核细胞增生李斯特菌感染过程中的作用，以梳理出palladin的新功能。我们表明，palladin是增选的单核细胞增生李斯特菌在其细胞进入和细胞内运动。帕拉丁的耗尽导致更短和畸形的彗星尾巴，当肌动蛋白或VASP结合的帕拉丁突变体在细胞中过表达时，彗星尾巴解体或变得更薄。彗星尾部变薄导致结构内的平行肌动蛋白束。为了确定palladin是否可以补偿Arp 2/3复合物，我们在用Arp 2/3抑制剂CK-666处理的细胞中过表达palladin。在处理过的细胞中，当palladin水平增加时，细菌运动性可以启动和维持。为了证实这些发现，我们使用了一个细胞系耗尽的多个Arp 2/3复合物亚基。在这些细胞中，单核细胞增生李斯特菌未能产生彗星尾。当palladin在该Arp 2/3功能无效细胞系中过表达时，单核细胞增生李斯特菌产生彗星尾的能力恢复。使用纯化的蛋白质组分，我们证明，单核细胞增生李斯特菌肌动蛋白云和彗星尾可以产生（在无细胞系统）的Arp 2/3复合物的情况下，由palladin。总的来说，我们的研究结果表明，palladin可以在功能上取代Arp 2/3复合物在细菌肌动蛋白为基础的运动。含有分支肌动蛋白丝的结构需要Arp 2/3复合物。研究由基于Arp 2/3的肌动蛋白运动产生的细胞内运动的最常用的系统之一利用由李斯特菌产生的富含肌动蛋白的彗星尾。使用这些感染与活成像和无细胞蛋白质重建实验，我们表明，另一种蛋白质，palladin，可以用来代替Arp 2/3，形成肌动蛋白丰富的结构。此外，我们表明，帕拉丁是必要的彗星尾巴的结构完整性，因为其耗尽或突变的关键区域造成巨大的变化，彗星尾巴的组织。这些发现首次确定了一种可以在功能上取代Arp 2/3复合物的蛋白质，并对所有被认为专门使用该复合物的肌动蛋白结构产生了影响。
Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.