Systematic analysis of the actin cytoskeleton and directed cell migration

摘要： 细胞运动可以说是细胞生物学中最古老的问题，因为细胞的运动是细胞生物学中最重要的问题之一。 这是在世纪显微镜发展起来的时候注意到的。过去60年的工作表明， 运动性的分子基础包括细胞骨架的主动控制。然而，许多问题 仍然没有答案。虽然我们已经确定了许多细胞骨架的组成部分，并知道一个伟大的 关于它们的生物化学和结构特征，我们缺乏系统的了解， 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。也许最 细胞运动中重要问题是理解细胞如何感知其环境中的各种线索， 将此信息转换为定向迁移响应。深入了解这两个相互关联的 问题将告知更高级的生物过程，例如胚胎发生、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， 从那时起，我们的研究计划就形成了我们的工作过程（Wu et al，Cell）。本文主要 发现哺乳动物成纤维细胞可以通过RNAi几乎完全耗尽Arp 2/3， 严重损害生存能力。这使我们能够深入问题的核心，研究 Arp 2/3复合物。使用这些细胞和微流体装置来产生持久的梯度，我们 令人惊讶的发现，细胞不需要Arp 2/3复合物进行趋化，但绝对需要它 对于触觉。受到RNAi成功的启发，我们转向了条件性基因敲除小鼠模型， 编码Arp 2/3关键亚基的基因Arpc 2（p34）可根据指令被删除。利用这些工具，我们 我追求两个目标：1）研究细胞如何在没有Arp 2/3复合物的情况下构建肌动蛋白细胞骨架 （由RO 1 GM 111557资助）和2）探究定向细胞迁移的分子机制， 间充质细胞和其他细胞类型（由RO 1 GM 110155资助）。在过去五年取得的进展的基础上， 在接下来的几年里，我们建议通过定义Arp 2/3和非Arp 2/3肌动蛋白的作用来扩展我们在这两个领域的工作 网络有助于细胞行为，如定向迁移到化学（趋化性），底物， 束缚（触变性）和机械（硬脊膜扩张）线索。

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.