Allosteric Regulation of Actin Capping Protein: Mechanism and Significance

项目总结/摘要 肌动蛋白组装是许多生物现象的基础和驱动力。倒刺端 的肌动蛋白丝，聚合的主要网站，是由异源二聚体控制， 肌动蛋白加帽蛋白（CP）。CP受CPI基序蛋白的直接结合调节 和蛋白质V-1这两类调节剂，CPI基序蛋白和V-1， CP的相对侧，它们诱导CP的构象变化， 变构地对抗另一类的结合。我们正在研究 这些变构调节剂的生物物理机制。我们也在研究 CP及其调节剂的生理功能，使用生化重建， 纯化的组分，沿着活细胞的分子遗传扰动。 我们的生化研究将测试一个新的假设， 在细胞中发挥作用。细胞中含有化学计量的V-1（微摩尔） 浓度，足以抑制几乎所有的细胞CP。V-1具有高度扩散性， V-1在空间上阻断CP为肌动蛋白丝加帽的能力。CPI基序蛋白是 靶向细胞膜，其CPI基序变构诱导V- 1，从而在膜上局部激活CP。 我们正在通过确定分子生物物理学来验证这一假设。 变构的机制，并通过测试CPI基序蛋白的功能， 关于细胞运动和迁移。我们已经发现了关键的差异， 不同家族的CPI基序蛋白的生化活性，我们现在正在使用 这些信息可以用来研究变构机制， FRET测量与分子动力学模拟。此外，我们正在使用 在诱导肌动蛋白的生化重建系统中纯化的蛋白质和脂质 组装在表面，从而模仿肌动蛋白聚合在细胞膜。 我们还利用我们对CPI基序的生物化学活性的发现来测试 CPI基序蛋白质的细胞功能，使用从不同CPI构建的嵌合体， 具有分子遗传扰动的基序蛋白和运动的实时电影 活细胞的现象。我们的细胞运动性测定采用内皮细胞系统， 具有迁移的免疫细胞和癌细胞的单层，模拟生理学上的 跨内皮迁移的过程。

Project Summary/Abstract Actin assembly underlies and drives many biological phenomena. Barbed ends of actin filaments, the major sites of polymerization, are controlled by the heterodimeric actin capping protein (CP). CP is regulated by the direct binding of CPI-motif proteins and the protein V-1. These two classes of regulators, CPI-motif proteins and V-1, bind to opposite sides of CP, and they induce conformational changes in CP that allosterically antagonize the binding of the other class. We are studying the molecular biophysical mechanism of these allosteric regulators. We are also studying the physiological function of CP and its regulators, using biochemical reconstitution with purified components, along with molecular genetic perturbations of living cells. Our biochemical studies will test a novel hypothesis for how CP regulators function in cells. Cells contain stoichiometric amounts of V-1 in micromolar concentrations, sufficient to inhibit nearly all of the cellular CP. V-1 is highly diffusible, and V-1 sterically blocks the ability of CP to cap actin filaments. CPI-motif proteins are targeted to membranes, and their CPI motifs allosterically induce the dissociation of V- 1, thus activating CP locally at the membrane. We are testing this hypothesis by determining the molecular biophysical mechanism of the allostery, and by testing the functions of the CPI-motif proteins with respect to cell motility and migration. We have discovered key differences in the biochemical activities of different families of CPI-motif proteins, and we are now using that information to investigate the allosteric mechanism, by combining single-molecule FRET measurements with molecular dynamics simulations. In addition, we are using purified proteins and lipids in a biochemical reconstitution system that induces actin assembly at a surface, thereby mimicking actin polymerization at cellular membranes. We are also using our discoveries about biochemical activities of CPI motifs to test cellular functions of CPI-motif proteins, using chimeras constructed from different CPI- motif proteins with molecular genetic perturbations and real-time movies of the motility phenomena of living cells. Our cell motility assays employ a system of endothelial cell monolayers with transmigrating immune and cancer cells, mimicking the physiological process of transendothelial migration.