Systematic Analysis of the Actin Cytoskeleton and Directed Cell Migration

项目摘要 细胞运动可以说是细胞生物学中最古老的问题，因为细胞的运动是最早的事情之一 当显微镜在17世纪被开发时就被注意到了。过去60年的工作表明， 运动性的分子基础涉及对细胞骨架的主动控制。然而，许多问题 仍未得到答复。虽然我们已经确定了细胞骨架的许多成分，并知道 关于它们的生化和结构特征，我们缺乏对它们是如何 各部分相互作用，以产生协调的细胞骨架功能，例如在细胞迁移期间。也许是最多的 细胞运动学中的重要问题是了解细胞如何感知环境中的各种提示和 将此信息转换为定向迁移响应。加深对这两个相互关联的认识 问题将告知更高级的生物过程，如胚胎发生、免疫反应和创伤 治愈，以及癌症转移等疾病。2012年，我们发表了一篇分水岭论文 从那以后塑造了我们工作进程的研究计划(Wu等人，细胞)。在这篇文章中，我们 发现哺乳动物成纤维细胞可以在没有RNAi的情况下几乎完全耗尽Arp2/3 严重损害了生存能力。这使我们能够深入问题的核心并研究 Arp2/3复合体直接表达。使用这些细胞和微流控设备来产生持久的梯度，我们制造了 令人惊讶的是，细胞不需要Arp2/3复合体来进行趋化，但绝对需要它 用于触觉趋向性。受到RNAi成功的启发，我们转向了有条件的基因敲除小鼠模型，其中 编码Arp2/3关键的Arpc2(P34)亚单位的基因可以在命令下被删除。使用这些工具，我们 我追求两个目标：1)研究细胞如何在没有Arp2/3复合体的情况下构建肌动蛋白细胞骨架 (由RO1 GM111557资助)和2)询问细胞定向迁移的分子机制 间充质细胞和其他细胞类型(由RO1 GM110155资助)。在过去五年取得进展的基础上再接再厉 几年来，我们建议通过定义Arp2/3和非Arp2/3肌动蛋白的角色来扩展我们在这两个领域的工作 有助于细胞行为的网络，如定向迁移到化学物质(趋化性)，底物- 绑定(触觉趋性)和机械(趋性)线索。

PROJECT ABSTRACT Cell motility is arguably the oldest problem in cell biology as the movement of cells was one of the first things noted when the microscope was developed in the 17th century. The last sixty years of work revealed that the molecular underpinnings of motility involve the active control of the cytoskeleton. However, many questions remain unanswered. While we have identified many of the components of the cytoskeleton and know a great deal about their biochemical and structural characteristics, we lack a systematic understanding of how the parts interact to produce coordinated cytoskeletal function such as during cell migration. Perhaps the most important problem in cell motility is understanding how cells perceive various cues in their environment and convert this information into a directed migration response. A deeper understanding of these two inter-related problems will inform higher order biological processes such as embryogenesis, immune response and wound healing, as well as diseases such as cancer metastasis. In 2012, our published a watershed paper for our research program that has shaped the course of our work ever since (Wu et al, Cell). In this paper, we discovered that mammalian fibroblasts could be nearly completely depleted of Arp2/3 by RNAi without significantly compromising viability. This allowed us to go the heart of the problem and study the function of the Arp2/3 complex directly. Using these cells and microfluidic devices to produce long-lasting gradients, we made the surprising observation that cells did not need the Arp2/3 complex for chemotaxis, but absolutely required it for haptotaxis. Inspired by our success with RNAi, we shifted to a conditional knockout mouse model where the gene encoding the critical Arpc2 (p34) subunit of Arp2/3 could be deleted on command. Using these tools, we have pursued two goals: 1) Investigate how cells can build an actin cytoskeleton without the Arp2/3 complex (funded by RO1 GM111557) and 2) Interrogate the molecular mechanisms of directed cell migration of mesenchymal cell and other cell types (funded by RO1 GM110155). Building on our progress in the last five years, we propose to extend our work in these two areas by defining the roles of Arp2/3 and non-Arp2/3 actin networks in contributing to cell behaviors such as directed migration towards chemical (chemotaxis), substrate- bound (haptotaxis) and mechanical (durotaxis) cues.