Mechanical Regulation of Actin Networks

描述（由申请人提供）：体内爬行的细胞必须穿过由细胞-细胞连接、细胞外基质和其他细胞阻碍的复杂景观。中性粒细胞在某一时刻处于拉伸状态、下一时刻处于压缩状态的组织中优雅且明显毫不费力地导航，引发了关于运动细胞如何响应外部以及自身施加的负荷的问题。更具体地说，在运动过程中产生细胞突起的肌动蛋白丝网络如何受到作用在其上的力的影响？人们普遍认为动态肌动蛋白网络具有仅受肌动蛋白和结合蛋白浓度控制的特性，这不足以解释细胞如何适应其物理环境。必须考虑力量在重组或重塑肌动蛋白网络中的作用。我们假设外部负载通过改变网络结构（例如丝密度和交联）来机械调节肌动蛋白网络特性，以响应负载条件的变化。这种肌动蛋白网络机械特性和生长速率响应负载的“调整”或适应可能是引导细胞爬行的重要机制，并在其他基于肌动蛋白的过程（包括内吞作用、吞噬作用和力转导）中发挥关键作用。然而，有关动态肌动蛋白网络物理行为的基本问题仍未得到解答。该提案重点关注 Arp2/3 分支肌动蛋白网络对力的响应。使用肌动蛋白网络生长的体外模型，我们提出了关于机械特性、生长速率以及肌动蛋白结合蛋白在组织网络结构中的作用的几个基本问​​题。我们开发了一种带有延时荧光显微镜的双悬臂原子力显微镜 (AFM)，它具有在受控加载条件下测量静态和动态肌动蛋白网络特性的独特能力。在使用该技术的初步实验中，我们已经确定了肌动蛋白网络在高负载和增长率下的复杂机械行为，这些行为取决于负载历史而不是瞬时负载。该提案将使用形成生长肌动蛋白网络的最小纯化蛋白系统来（i）识别负载对机械性能的影响，（ii）跟踪响应力的生长速率的变化，以及（iii）检查肌动蛋白丝切断蛋白对网络性能的瞬时影响。拟议的工作将通过揭示肌动蛋白网络机制来影响健康，该机制在发育和转移过程中细胞运动所必需的细胞突出中发挥着基础作用。公共健康相关性 动态肌动蛋白网络已被确定不仅对细胞运动至关重要，而且对胞吞作用、胞吐作用、病原体入侵、T 细胞信号传导、侵袭伪足形成和细胞分裂也至关重要。更好地了解机械微环境如何改变肌动蛋白网络行为，包括其弹性特性及其产生和引导形状变化力的能力，有可能揭示疾病的新机制并确定药物发现的新靶点。

DESCRIPTION (provided by applicant): Crawling cells in the body must traverse a complex landscape obstructed by cell-cell junctions, extracellular matrix, and other cells. The elegant and apparently effortless navigation of neutrophils through tissue in tension at one moment and in compression the next raises questions about how motile cells respond to external as well as self-imposed loads. More specifically, how are the actin filament networks that generate cellular protrusions during motility influenced by the forces on them? The common view of dynamic actin networks as having properties governed only by concentrations of actin and binding proteins is not sufficient to explain how cells adapt to their physical environment; the role of forces in restructuring or remodeling actin networks must be considered. We hypothesize that external loads mechanically regulate actin network properties by altering network architecture, such as filament density and crosslinking, in response to changes in loading conditions. This `tuning' or adaptation of actin network mechanical properties and growth rates in response to load may be an important mechanism for guiding crawling cells and play critical roles in other actin-based processes including endocytosis, phagocytosis, and mechanotransduction. However, basic questions about the physical behavior of dynamic actin networks remain unanswered. This proposal focuses on the response of Arp2/3-branched actin networks to forces. Using an in vitro model of actin network growth, we ask several basic questions about mechanical properties, growth rates, and the role of actin binding proteins in organizing network architecture. We have developed a dual-cantilever atomic force microscope (AFM) with time-lapse fluorescence microscopy that has the unique ability to measure both static and dynamic actin network properties under controlled loading conditions. In preliminary experiments using this technique, we have identified complex mechanical behavior of actin networks under high loads and growth rates that depend on loading history rather than instantaneous load. This proposal will use a minimal system of purified proteins that form growing actin networks to (i) identify the effect of loading on mechanical properties, (ii) track changes in growth rates in response to force, and (iii) examine the transient effect of an actin filament severing protein on network properties. The proposed work will impact health by revealing actin network mechanics that play a fundamental role in cellular protrusion essential for cell movements during development as well as metastasis. PUBLIC HEALTH RELEVANCE Dynamic actin networks have been identified as essential for not only cell motility, but also endocytosis, exocytosis, pathogen invasion, T-cell signaling, invadopodia formation, and cell division. Better understanding of how the mechanical microenvironment alters actin network behavior, including its elastic properties and its ability to generate and direct forces for shape change, has the potential to reveal new mechanisms of disease and identify new targets for drug discovery.