Ena/VASP proteins in cell motility and adhesion

细胞利用肌动蛋白聚合的力量形成突出的膜片，在边缘填充密集的肌动蛋白丝网络，称为板足，以驱动细胞迁移。根据我们目前的知识，肌动蛋白在板足和褶边的成核主要是由arp2 /3复合物完成的，而肌动蛋白丝在质膜下的延伸则是由肌动蛋白丝延长蛋白如成形蛋白或激活/血管扩张刺激磷酸化蛋白（Ena/VASP）驱动的。Ena/VASP蛋白是一个结构保守的家族，在运动细胞中高度表达。脊椎动物含有三种VASP相关蛋白Mena（小鼠Ena）， VASP和EVL （Ena-VASP样），它们都被证明定位于活跃的肌动蛋白组装部位，如局灶粘连，板足或丝状足的尖端。它们涉及多种基本的细胞过程，包括轴突引导、细胞迁移和病原体的传播。对缺乏Mena和VASP （MVD7）的小鼠胚胎成纤维细胞（MEF）进行的开创性研究，也被认为缺乏可检测到的EVL水平，发现细胞粘附没有显著变化，但令人惊讶的是报告了更快的突出率和细胞运动性增加。然而，这一观点受到了我们最近工作的挑战，与预期相反，我们发现EVL在该细胞系中大量表达。根据这些发现，并基于所有三种Ena/VASP异构体在体外催化系系和进展性肌动蛋白丝伸长的相似生化活性，我们假设在缺乏Ena/VASP蛋白时，在细胞迁移和粘附位点的形成方面观察到明显的缺陷。因此，在缺乏所有三种同种异构体的细胞中，必须重新审视和仔细定位Ena/VASP的生理功能。因此，我们打算通过使用锌指核酸酶进行基因组编辑来破坏MVD7细胞中的EVL基因，以获得真正的Ena/VASP-null细胞。该技术还将用于破坏高运动和广泛使用的B16-F1小鼠黑色素瘤细胞中的所有三个Ena/VASP成员。这项工作的目的是评估Ena/VASP蛋白在板状基结构和突出以及细胞粘附中的精确生理作用，可能与它们与局灶粘附蛋白vinculin的紧密相互作用有关，以更好地了解细胞迁移的分子机制。

Cells exploit the power of actin polymerization for the formation of protruding membrane sheets filled with a dense actin filament network at the leading edge referred to as lamellipodia to drive cell migration. According to our current knowledge, actin nucleation in lamellipodia and ruffles is mainly accomplished by the Arp2/3-complex, while subsequent elongation of actin filaments beneath the plasma membrane is driven by actin filament-elongating proteins such as formins or Enabled/vasodilator-stimulated phosphoproteins (Ena/VASP). Ena/VASP proteins are a structurally conserved family, which are highly expressed in motile cells. Vertebrates contain three VASP-related proteins Mena (mouse Ena), VASP and EVL (Ena-VASP-like), which all were shown to localize in sites of active actin assembly such as focal adhesions, the tips of lamellipodia or filopodia. They are implicated in a variety of fundamental cellular processes including axon guidance, cell migration and dissemination of pathogens. Seminal studies carried out with mouse embryonic fibroblasts (MEF) devoid of Mena and VASP (MVD7), additionally assumed to lack detectable levels of EVL, found no significant changes in cell adhesion, but surprisingly reported faster protrusion rates and increased cell motility. However, this view has been challenged by our recent work, which contrary to expectations, revealed substantial expression of EVL in this cell line. In light of these finding and based on the comparable biochemical activities of all three Ena/VASP isoforms catalyzing tethered and processive actin filament elongation in vitro, we assume to observe marked defects in the absence of Ena/VASP proteins on cell migration and the formation of adhesion sites. For that reason, the physiological functions of Ena/VASP must be revisited and carefully addressed in cells lacking all three isoforms. We therefore intend to disrupt the EVL gene in MVD7 cells through genome editing employing zinc finger nucleases to obtain bona fide Ena/VASP-null cells. This technology will also be employed to disrupt all three Ena/VASP members in the highly motile and widely used B16-F1 mouse melanoma cells. The objective of this work is to assess the precise physiological roles of Ena/VASP proteins in lamellipodium architecture and protrusion as well as in cell adhesion, presumably linked to their tight interaction with the focal adhesion protein vinculin, to better understand the molecular mechanisms underlying cell migration.