Function and regulation of the Arp2/3 complex

描述(申请人提供)：肌动蛋白细胞骨架对细胞形态和迁移是必不可少的，与癌症和各种传染病等疾病有关。然而，调节细胞皮质中肌动蛋白动力学以控制形状和迁移的分子机制，肌动蛋白动力学如何与力的产生相结合，以及肌动蛋白调控机制在成纤维细胞和神经元中的不同，还不是很清楚。肌动蛋白也存在于细胞核中，参与核的形状、转录、染色质运动和染色质重塑。然而，目前尚不清楚细胞骨架蛋白的核-胞质分布是如何被调节的，肌动蛋白是否起着 原子核中的单体或聚合物，以及它如何发挥其核功能。我们将检验Arp2/3复合体及其成核促进因子(NPF)这一总体假设，它们是肌动蛋白成核和组织的关键调节因子，在调节肌动蛋白的细胞质和核功能方面发挥重要作用。我们将回答三个重要问题。Arp2/3催化循环如何促进成纤维细胞中的细胞迁移和神经元中的突起生长？NPF如何影响细胞皮质的肌动蛋白聚合，或细胞核内的转录，以控制神经元的轴突形成和分化？Arp2/3和肌动蛋白的核浆分布是如何调节的，它们在核内有哪些功能？我们提出了以下目标：(1)我们将结合生化和生物物理方法来确定Arp2/3催化周期在细胞迁移和形态中的作用，以阐明Arp2/3在体外肌动蛋白动力学和力产生中的作用，并通过细胞生物学方法来阐明Arp2/3在成纤维细胞和神经元中的功能。这些研究将揭示Arp2/3如何在不同类型细胞的形状和迁移中对皮质肌动蛋白动力学做出贡献。(2)我们将研究一种名为JMY的NPF的调节及其在神经元中的功能，测试其通过直接影响皮质肌动蛋白和/或通过调节神经元分化过程中的转录来负向调节轴突形成的假设。这些研究将揭示NPF调节的新特征，以及NPF在调节皮质和核肌动蛋白以控制细胞形状和分化方面的新作用。(3)我们将利用杆状病毒AcMNPV作为研究核肌动蛋白调控和功能的工具。我们将研究AcMNPV如何通过操纵核运输途径诱导核肌动蛋白积累，以及核肌动蛋白是否调节病毒和宿主的转录。我们还将测试核肌动蛋白组织病毒复制结构并驱动病毒运动和核外排出的假设。这些研究将揭示核肌动蛋白的新功能。由于我们将揭示肌动蛋白调节细胞形态、迁移和分化的基本机制，以及核肌动蛋白在正常细胞和感染细胞中的作用，我们的研究可能会导致诊断和治疗疾病的新方法。

DESCRIPTION (provided by applicant): The actin cytoskeleton is essential for cell shape and migration, and is implicated in illnesses including cancer and various infectious diseases. Nevertheless, the molecular mechanisms that regulate actin dynamics at the cell cortex to control shape and migration, how actin dynamics are coupled with force generation, and how the mechanisms of actin regulation differ in cells such as fibroblasts and neurons, are not well understood. Actin is also present in the nucleus, where it participates in nuclear shape, transcription, chromatin movement and chromatin remodeling. However, it is poorly understood how the nuclear-cytosolic distribution of cytoskeletal proteins is regulated, whether actin acts as a monomer or polymer in the nucleus, and how it performs its nuclear functions. We will test the overall hypothesis that the Arp2/3 complex and its nucleation promoting factors (NPFs), key regulators of actin nucleation and organization, play important roles in regulating both cytoplasmic and nuclear functions of actin. We will answer three important questions. How does the Arp2/3 catalytic cycle contribute to cell migration in fibroblasts and neurite outgrowth in neurons? How do NPFs influence actin polymerization at the cell cortex, or transcription in the nucleus, to control neurite formation and differentiation of neurons? How are the nuclear-cytosolic distributions of Arp2/3 and actin regulated, and what nuclear functions do they perform? We propose the following aims: (1) We will determine the role of the Arp2/3 catalytic cycle in cell migration and shape by combining biochemical and biophysical approaches to elucidate how Arp2/3 acts in actin dynamics and force generation in vitro, with cell biological approaches to elucidate Arp2/3 functions in fibroblasts and neurons. These studies will reveal how Arp2/3 contributes to cortical actin dynamics in shape and migration of different cell types. (2) We will examine the regulation of an NPF called JMY and its function in neurons, testing the hypotheses that it negatively regulates neurite formation by directly impacting cortical actin and/or by modulating transcription during neuronal differentiation. These studies will reveal new features of NPF regulation, and new roles for NPFs in regulating cortical and nuclear actin to control cell shape and differentiation. (3) We will use the baculovirus AcMNPV as a tool to study nuclear actin regulation and function. We will examine how AcMNPV induces nuclear actin accumulation by manipulating nuclear transport pathways, and whether nuclear actin regulates viral and host transcription. We will also test the hypotheses that nuclear actin organizes viral replication structures and drives viral motility and egress from the nucleus. These studies will reveal new functions for nuclear actin. Because we will uncover basic mechanisms of actin regulation in cell shape, migration and differentiation, and roles for nuclear actin in normal and infected cells, our studies may result in new approaches for diagnosing and treating disease.