REGULATION OF ACTIN CAPPING PROTEIN

我们的目标是阐明控制组装和 细胞中肌动蛋白细丝的分解和了解肌动蛋白组装动力学如何有助于 细胞运动。肌动蛋白细丝的游离带刺末端的产生是肌动蛋白的关键决定因素 为了在膜上产生力和运动，需要对这些末端进行封口。这里, 我们研究了异二聚体带刺末端封端蛋白(CP)的功能和一组 含有保守的CP结合基序的膜相关蛋白，称为CPI，但 否则就没有关系了。 CARMIL包含CPI基序和第二个CP结合基序，称为CSI(CARMIL- 特定的相互作用)，是一种有效的CP抑制物，能够通过去封顶来产生自由的带刺末端 有帽的长丝。去封顶很重要，因为细胞中CP和肌动蛋白细丝的周转率 比在体外观察到的纯化蛋白快几个数量级。其他CPI-主题 CD2AP、Cin85、CKIP-1、WASHCAP(FAM21)和CapZIP等蛋白与CP结合，但抑制作用较小 这表明它们可能将活性CP定位于细胞膜隔室。 目标1：封顶蛋白的调节因子是如何发挥作用的？为了了解CP监管是如何运作的，我们 定义了肌动蛋白在CP上的结合部位，并生成了CP与CP-的络合物的晶体结构。 结合蛋白。CPI基序与CP上的一个公共位点结合，距离肌动蛋白结合位点有一段距离。 CARMIL结合引起肌动蛋白结合位点构象的变构变化。现在，我们 问各种CPI基序蛋白的细胞功能是抑制CP还是招募活性CP 到细胞中的某个位置。在体外，我们将比较这些蛋白质结合和抑制CP的能力。 用提纯的成分进行生化分析。在细胞中，我们将确定靶向和 CP在肌动蛋白细胞骨架中的掺入依赖于CP相互作用的蛋白质及其如何 与CP的相互作用影响局部肌动蛋白的组装和运动。 目标2：CARMIL1在细胞中是如何发挥作用的？CARMIL对细胞迁移和其他肌动蛋白- 后生动物细胞中的相关功能。脊椎动物表达三个保守的CARMIL基因。我们 发现CARMIL1和CARMIL2对细胞迁移都很重要，而且它们 在单个迁移细胞中具有不同的非重叠功能。CARMIL1与双重全球环境基金三重奏互动， 激活rac1，并刺激片状脂血症的形成。我们计划研究它的分子基础。 这些表型和相互作用通过本地化、生化、基因敲除和 表达方式接近。 作者：John A. 1R01 GM095509-01A1

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. COOPER, John A. 1R01 GM095509-01A1