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，驱动丝状足或板足等膜突起的丝倒钩末端的持续伸长。基于生化数据和体外全内反射荧光显微镜（TIRFM）测量，我们之前已经表明，四聚体VASP使用它的一个臂来渐进地跟踪生长的丝倒钩末端，而其他臂上的三个g -肌动蛋白结合位点（GABs）可用于招募和运送单体到丝尖端，正式表明VASP在溶液中作为单个四聚体或牢牢聚集在珠表面时起作用。尽管在这两种条件下，对capping蛋白（CP）的加工性和抗性存在显著差异。一致地，通过寡聚化状态的变化和单个多肽链上GABs数量的增加，我们最近证实了vasp介导的肌动蛋白在散装溶液中组装的分子机制与在静态表面拴系簇中组装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。由于聚类是理解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推进重要的一步，以模拟生理条件。因此，在这一后续提案中，我们的目标是通过多色TIRF成像重建和分析VASP介导的肌动蛋白组装和支持脂质双分子层中含有IRSp53家族的I-BAR蛋白的sh3结构域VASP聚集的动力学。I-BAR蛋白IRSp53目前是驱动Cdc42信号下游VASP聚集的主要候选蛋白，但另外两种IRSp53相关蛋白IRTKS和Pinkbar或其他尚未表征的蛋白也可能驱动或至少协助VASP在膜-细胞骨架界面的肌动蛋白组装复合物中聚集。因此，通过使用从细胞提取物中提取合适的Ena/VASP片段的下拉菜单，然后使用蛋白质组学，我们还旨在鉴定和表征在膜-细胞质界面介导Ena/VASP聚集的新因子。与Ena/VASP相比，IRSp53也与丝足形成有关，并被认为有助于聚集，膜变形和丝足肌动蛋白丝组装，尽管对其在这一过程中的具体功能的明确分子理解仍然难以捉摸。因此，最终的主要目标是CRISPR/ cas9介导的irsp53相关I-BAR蛋白敲除，并对B16-F1衍生的小鼠黑色素瘤突变细胞进行全面分析，以评估这些I-BAR蛋白在VASP聚集、肌动蛋白组装、迁移细胞突起形成和运动中的精确生理作用。

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.