Function and regulation of the Arp2/3 complex

描述（由申请人提供）：肌动蛋白细胞骨架对细胞形状和迁移至关重要，并与癌症和各种传染病等疾病有关。然而，在细胞皮层调节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耦合，以及肌动蛋白调节机制在细胞如成纤维细胞和神经元中如何不同，还没有很好地理解。肌动蛋白也存在于细胞核中，在那里它参与核形状、转录、染色质运动和染色质重塑。然而，人们对细胞骨架蛋白的核-胞质分布是如何调节的知之甚少，肌动蛋白是否起作用， 核中的单体或聚合物，以及它如何执行其核功能。我们将测试的整体假设，Arp 2/3复合物及其成核促进因子（NPFs），肌动蛋白成核和组织的关键监管机构，在调节细胞质和细胞核的肌动蛋白功能中发挥重要作用。我们将回答三个重要问题。Arp 2/3催化循环如何促进成纤维细胞的细胞迁移和神经元的神经突生长？NPFs如何影响细胞皮质的肌动蛋白聚合或细胞核的转录，从而控制神经突的形成和神经元的分化？Arp 2/3和肌动蛋白的核-胞质分布是如何调节的，它们在核中发挥什么样的功能？我们提出以下目标：（1）我们将通过结合生物化学和生物物理学方法来阐明Arp 2/3在体外肌动蛋白动力学和力产生中的作用，以及细胞生物学方法来阐明Arp 2/3在成纤维细胞和神经元中的功能，来确定Arp 2/3催化循环在细胞迁移和形状中的作用。这些研究将揭示Arp 2/3如何在不同细胞类型的形状和迁移中促进皮质肌动蛋白动力学。(2)我们将研究称为JMY的NPF的调节及其在神经元中的功能，测试它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调节神经突形成的假设。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中的新作用。(3)我们将使用杆状病毒AcMNPV作为研究核肌动蛋白调节和功能的工具。我们将研究AcMNPV如何通过操纵核转运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和驱动病毒运动和从细胞核出口的假设。这些研究将揭示核肌动蛋白的新功能。因为我们将揭示肌动蛋白在细胞形状，迁移和分化中的基本调节机制，以及核肌动蛋白在正常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.