Mechanical Regulation of Actin Networks

描述(申请人提供)：体内的爬行细胞必须穿过被细胞-细胞连接、细胞外基质和其他细胞阻挡的复杂景观。中性粒细胞优雅而看似毫不费力地在组织中穿行，一会儿处于张力状态，一会儿处于压缩状态，这引发了人们对运动细胞如何应对外部和自我施加的负荷的疑问。更具体地说，在运动过程中产生细胞突起的肌动蛋白细丝网络是如何受到作用力的影响的？通常认为动态肌动蛋白网络的性质只受肌动蛋白和结合蛋白浓度的控制，这不足以解释细胞如何适应其物理环境；必须考虑力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，如细丝密度和交联度，来机械地调节肌动蛋白网络的性质，以响应负载条件的变化。这种对肌动蛋白网络机械特性和生长速度的“调整”或适应可能是引导爬行细胞的重要机制，并在其他基于肌动蛋白的过程中发挥关键作用，包括内吞、吞噬和机械转导。然而，关于动态肌动蛋白网络的物理行为的基本问题仍然没有答案。这一建议侧重于Arp2/3分支肌动蛋白网络对力的反应。使用肌动蛋白网络生长的体外模型，我们提出了几个关于机械特性、生长速度和肌动蛋白结合蛋白在组织网络结构中的作用的基本问题。我们开发了一种带有时移荧光显微镜的双悬臂原子力显微镜(AFM)，它具有在受控负载条件下测量静态和动态肌动蛋白网络属性的独特能力。在使用这项技术的初步实验中，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力学行为，这些行为取决于加载历史而不是瞬时负载。这项建议将使用一个最小的纯化蛋白质系统，这些蛋白质形成不断增长的肌动蛋白网络，以(I)确定载荷对机械性能的影响，(Ii)跟踪生长速度对力的响应的变化，以及(Iii)检查肌动蛋白细丝切断蛋白对网络性能的瞬时影响。这项拟议的工作将通过揭示肌动蛋白网络机制来影响健康，肌动蛋白网络机制在细胞突起中发挥基础性作用，对细胞发育和转移过程中的细胞运动至关重要。与公共健康相关的动态肌动蛋白网络不仅对细胞运动至关重要，而且对内吞作用、胞吞作用、病原体入侵、T细胞信号转导、内生生物形成和细胞分裂都是必不可少的。更好地了解机械微环境是如何改变肌动蛋白网络行为的，包括它的弹性性质以及它产生和引导力量改变形状的能力，有可能揭示新的疾病机制，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.