Ena/VASP proteins in cell motility and adhesion

Ena/VASP 蛋白在细胞运动和粘附中的作用

• 批准号: 264240917
• 负责人: Professor Dr. Jan Faix
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/264240917?language=en
• 关键词: Ena; VASP; proteins; cell; motility

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.

细胞利用肌动蛋白聚合的力量形成突出的膜片，在前缘填充有致密的肌动蛋白丝网络，称为板状伪足，以驱动细胞迁移。根据我们目前的知识，肌动蛋白成核在lamellipodia和ruffles主要是完成的Arp 2/3复合物，而随后的延伸的肌动蛋白丝下质膜是驱动的肌动蛋白抑制性延长蛋白，如formins或启用/血管舒张刺激磷蛋白（Ena/VASP）。Ena/VASP蛋白是一个结构保守的蛋白家族，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性肌动蛋白组装的位点，如粘着斑、板状伪足或丝状伪足的尖端。它们涉及多种基本细胞过程，包括轴突导向、细胞迁移和病原体传播。用缺乏Mena和VASP（MVD 7）的小鼠胚胎成纤维细胞（MEF）进行的精液研究，另外假定缺乏可检测水平的EVL，发现细胞粘附没有显著变化，但令人惊讶地报告了更快的突起速率和增加的细胞运动性。然而，这一观点受到了挑战，我们最近的工作，这与预期相反，揭示了大量的EVL在该细胞系中的表达。鉴于这些发现，并基于所有三种Ena/VASP同种型在体外催化束缚和进行性肌动蛋白丝伸长的可比生物化学活性，我们假设在不存在Ena/VASP蛋白的情况下观察到细胞迁移和粘附位点形成的明显缺陷。因此，Ena/VASP的生理功能必须在缺乏所有三种亚型的细胞中重新审视和仔细解决。因此，我们打算通过使用锌指核酸酶的基因组编辑来破坏MVD 7细胞中的EVL基因，以获得真正的Ena/VASP无效细胞。该技术还将用于破坏高度运动和广泛使用的B16-F1小鼠黑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蛋白在板状伪足结构和突起以及细胞粘附中的确切生理作用，推测与其与粘着斑蛋白黏着斑蛋白的紧密相互作用有关，以更好地了解细胞迁移的分子机制。