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

Ena/VASP蛋白充当肌动蛋白聚合酶，其驱动膜突起（例如丝状伪足或板状伪足）中的丝状倒刺末端的进行性伸长。基于生物化学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使用其一个臂来procancellating跟踪生长的细丝倒刺末端，而其他臂上的三个G-肌动蛋白结合位点（GABs）可用于募集单体并将单体递送到细丝尖端，正式表明VASP在溶液中或牢固地聚集在珠表面上时作为单个四聚体起作用，尽管在两种条件下对加帽蛋白（CP）的持续合成能力和抗性显著不同。一致的是，通过寡聚化状态的变化和单个多肽链上GABA数量的增加，我们最近证实了VASP介导的肌动蛋白组装在本体溶液中的分子机制相比，在静态的表面拴系簇是显着不同的。值得注意的是，在生理背景下，Ena/VASP蛋白仅在质膜下的动态多蛋白簇中起作用。由于群集是中央了解VASP介导的肌动蛋白组装在细胞中，我们现在打算进一步推进一个重要的步骤，以模拟生理条件。因此，在这个后续的建议，我们的目标是重建和分析VASP介导的肌动蛋白组装和动力学的VASP集群SH 3结构域包含I-BAR蛋白的IRSp 53家族在支持脂质双层的ESTTIRF成像。I-BAR蛋白IRSp 53目前是驱动Cdc 42信号下游VASP聚集的主要候选者，但另外两个IRSp 53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-细胞骨架界面处的肌动蛋白组装复合物中聚集。因此，通过使用下拉与合适的Ena/VASP片段从细胞提取物，然后通过蛋白质组学，我们另外的目的是确定和表征新的因素，介导Ena/VASP聚集在膜-胞质溶胶界面。与Ena/VASP相比，IRSp 53也参与丝状伪足的形成，并被认为有助于簇集、膜变形和丝状伪足肌动蛋白丝组装，尽管对其在该过程中的特定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IRSp 53相关I-BAR蛋白的敲除和B16-F1衍生的小鼠黑色素瘤突变体细胞的综合分析，以评估这些I-BAR蛋白在VASP聚类，肌动蛋白组装，迁移细胞突起和运动性形成中的精确生理作用。

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.