Dissecting the role of I-BAR proteins in VASP clustering and actin assembly

Ena/Vasp蛋白作为肌动蛋白聚合酶，驱动丝状足或片状纤毛等膜突起中细丝带刺末端的过程延长。基于生化数据和体外全内反射荧光显微镜(TIRFM)测量，我们先前已经表明，四聚体Vasp使用其一只手臂来跟踪生长的细丝带刺末端，而其他手臂上的三个G-肌动蛋白结合位点(GABs)可用于招募单体并将单体输送到丝尖，正式表明Vasp在溶液中或当牢固地聚集在珠子表面时作为单个四聚体运行，尽管在两种条件下对封顶蛋白(CP)的处理能力和抵抗力显著不同。最近，通过改变寡聚状态和增加单个多肽链上Gabs的数量，我们最近证实了Vasp介导的肌动蛋白在本体溶液中组装的分子机制与在静态表面拴系簇中的分子机制明显不同。值得注意的是，在生理环境中，Ena/Vasp蛋白仅在质膜下的动态多蛋白簇中起作用。由于群集是了解VASP介导的肌动蛋白在细胞中组装的核心，我们现在打算进一步深入研究，以模拟生理条件。因此，在这个后续的提案中，我们的目标是利用多色TIRF成像技术，通过含有IRSP53家族I-bar蛋白的SH3结构域在支持的脂质双层中重建和分析VASP介导的肌动蛋白组装和VASP聚集的动力学。I-bar蛋白IRSp53目前是推动CDC42信号下游Vasp聚集的主要候选蛋白，但另外两个与IRSp53相关的蛋白质IRTKS和Pinkbar或其他尚未鉴定的蛋白质也可能推动或至少帮助Vasp在膜-细胞骨架界面的肌动蛋白组装复合体中聚集。因此，通过使用从细胞提取液中提取合适的Ena/Vasp片段并随后进行蛋白质组学分析，我们另外的目标是识别和表征在膜-细胞质界面介导Ena/Vasp聚集的新因素。与EnA/Vasp类似，IRSP53也参与了丝足的形成，并被认为有助于聚集、膜变形和细丝组装，尽管对其在这一过程中的具体功能的明确分子理解仍然是未知的。因此，最终的主要目标是CRISPR/Cas9介导的IRSP53相关I-bar蛋白的敲除和对B16-F1小鼠黑色素瘤突变细胞的综合分析，以评估这些I-bar蛋白在VASP聚集、肌动蛋白组装、形成迁移细胞突起和运动中的确切生理作用。

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions such as filopodia or lamellipodia. Based on biochemical data and in vitro total internal reflection fluorescence microscopy (TIRFM) measurements, we have previously shown that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, formally suggesting that VASP operates as a single tetramer in solution or when firmly clustered on a bead surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Consistently, by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains, we recently confirmed that the molecular mechanisms of VASP-mediated actin assembly in bulk solution as compared in static surface-tethered clusters are markedly different. Of note, in the physiological context Ena/VASP proteins operate exclusively in dynamic multi-protein clusters beneath the plasma membrane. Since clustering is central to understand VASP-mediated actin assembly in cells we now intend to advance a significant step further in order to mimic physiological conditions. Thus, in this follow-up proposal we aim to reconstitute and analyze VASP-mediated actin assembly and dynamics of VASP clustering by SH3-domain containing I-BAR proteins of the IRSp53 family in supported lipid bilayers by multicolor TIRF imaging. The I-BAR protein IRSp53 is currently the major candidate to drive clustering of VASP downstream of Cdc42 signaling, but the two other IRSp53-related proteins IRTKS and Pinkbar or other and as yet uncharacterized proteins might also drive or at least assist VASP clustering in actin-assembly complexes at the membrane-cytoskeleton interface. Thus, by the use of pulldowns with suitable Ena/VASP fragments from cell extracts followed by proteomics we additionally aim to identify and characterize novel factors that mediate Ena/VASP-clustering at the membrane-cytosol interface. Comparable to Ena/VASP, IRSp53 has been also implicated in filopodium formation and is thought to contribute to clustering, membrane deformation and filopodial actin filament assembly although a clear molecular understanding of its specific functions in this process remains elusive. The final major objective is therefore the CRISPR/Cas9-mediated knockout of IRSp53-related I-BAR proteins and comprehensive analyses of B16-F1 derived mouse melanoma mutant cells to assess the precise physiological roles of these I-BAR proteins in VASP clustering, actin assembly, formation of migratory cell protrusion and motility.