REGULATION OF ACTIN CAPPING PROTEIN

我们的目标是阐明控制组装的分子机制 以及细胞中肌动蛋白丝的分解， 动力学有助于细胞运动。肌动蛋白末端自由倒钩的形成 细丝是肌动蛋白组装的关键决定因素，而封盖这些末端是 在膜上产生力和运动所必需的。在这里，我们调查了 异二聚体倒钩末端加帽蛋白（CP）和一组膜- 相关蛋白含有保守的CP结合基序，称为CPI，但 其他方面无关。 CARMIL含有CPI基序和第二个CP结合基序，称为 CSI（CARMIL特异性相互作用）是一种有效的CP抑制剂，能够产生 通过不加帽的加帽的细丝的自由的有倒钩的末端。取消封顶很重要，因为 细胞中CP和肌动蛋白丝的周转率比细胞中的细胞快几个数量级。 在体外用纯化的蛋白质观察到的那些。其他CPI基序蛋白，包括 CD 2AP、Cin 85、CKIP-1、WASHCAP（FAM 21）和CapZIP结合CP，但抑制效果较差， 这表明它们可以将活性CP靶向膜区室。 目标1：加帽蛋白的调节剂如何工作？了解CP监管如何 工作，我们确定了CP上的肌动蛋白结合位点，我们产生了晶体结构， CP与CP结合蛋白复合。CPI基序结合到CP上的共同位点， 与肌动蛋白结合位点的距离CARMIL结合引起细胞内的变构变化。 肌动蛋白结合位点的构象。现在，我们要问的是， 各种CPI基序蛋白的作用是抑制CP或将活性CP募集到细胞中的位置。 在体外，我们将比较蛋白质结合和抑制CP的能力， 用纯化的组分进行测定。在细胞中，我们将确定如何靶向和 CP掺入肌动蛋白细胞骨架取决于CP相互作用蛋白 以及它们与CP的相互作用如何影响局部肌动蛋白的组装和运动。 目标2：CARMIL 1在细胞中如何发挥作用？CARMIL对细胞迁移很重要 和其他与肌动蛋白相关的功能。脊椎动物表达三种保守的 CARMIL基因。我们发现，CARMIL 1和CARMIL 2对细胞生长都很重要， 它们在单个迁移细胞中具有不同的非重叠功能。 CARMIL 1与双GEF Trio相互作用，激活Rac 1并刺激板状伪足 阵我们计划研究这些表型的分子基础， 通过定位、生物化学、敲除和表达的组合进行相互作用 在人类培养细胞和斑马鱼中的方法。

Our goals are to elucidate the molecular mechanisms that control the assembly and disassembly of actin filaments in cells and to understand how actin assembly dynamics contribute to cell movement. The creation of free barbed ends of actin filaments is a critical determinant of actin assembly, and capping those ends is necessary to produce force and movement at membranes. Here, we investigate the function of the heterodimeric barbed-end capping protein (CP) and a set of membrane- associated proteins that contain a conserved CP-binding motif, called CPI, but are otherwise unrelated. CARMIL, which contains the CPI motif and a second CP-binding motif, called CSI (CARMIL-specific interacting), is a potent inhibitor of CP with the ability to create free barbed ends by uncapping capped filaments. Uncapping is important because the turnover rates of CP and actin filaments in cells are faster by orders of magnitude than those observed with purified proteins in vitro. Other CPI-motif proteins, including CD2AP, Cin85, CKIP-1, WASHCAP(FAM21) and CapZIP bind CP but inhibit less well, suggesting that they may target active CP to membrane compartments. Aim 1: How Do Regulators of Capping Protein Work? To understand how CP regulation works, we defined the actin-binding sites on CP, and we produced crystal structures for CP in complex with CP-binding proteins. CPI motifs bind to a common site on CP at a distance from the actin-binding sites. CARMIL binding causes an allosteric change in the conformation of the actin-binding sites. Now, we ask whether the cellular function of the various CPI-motif proteins is to inhibit CP or to recruit active CP to a location in the cell. In vitro, we will compare the abilities of the proteins to bind and inhibit CP in biochemical assays with purified components. In cells, we will determine how targeting and incorporation of CP into the actin cytoskeleton depends on the CP-interacting proteins and how their interaction with CP affects local actin assembly and movement. Aim 2: How Does CARMIL1 Function in Cells? CARMIL is important for cell migration and other actin-related functions in metazoan cells. Vertebrates express three conserved CARMIL genes. We discovered that CARMIL1 and CARMIL2 are both important for cell migration and that they have distinct non-overlapping functions in a single migrating cell. CARMIL1 interacts with the dual-GEF Trio, activates Rac1, and stimulates lamellipodia formation. We plan to investigate the molecular basis of these phenotypes and interactions by a combination of localization, biochemical, knockdown, and expression approaches in human cultured cells and zebrafish.