Using micropost arrays to measure traction forces during dendritic cell motility

树突状细胞(DC)是哺乳动物免疫系统的重要调节者 运动性对它们的正常功能至关重要。癌症免疫治疗等技术 严重依赖数据中心迁移。树突状细胞具有多种趋化因子受体并爬行 作为对趋化因子梯度的反应，趋化因子梯度引导DC在整个免疫系统中定位 系统。最终，DC必须集成多个信号才能在单个 方向。这个项目的目标是使用一种新的生物界面工具，微柱阵列 探测器(MPAD)，与微流控梯度室相结合，以施加时间- 不变趋化因子向细胞的梯度，并测量DC施加的牵引力 在迁移过程中。我们最近发表的工作表明，MPAD阵列足以 敏感地测量低牵引应力(每丝脚0.5nN，每单元20nN) 迁移中的DC。现在，我们使用这些数组来了解单元内的组件 引起定向的细胞运动并理解DC如何整合趋化因子信号 并将其转换为牵引力和方向运动。《公约》的具体目标 建议：1)利用新型微柱测力仪测量井下直流动量； 单个趋化因子在多个黏附配体上的定义梯度；2)测量 调节蛋白HS1和WASP对单个趋化因子中DC迁移的影响 梯度；以及3)在旋转过程中测量DC迁移力 渐变会迅速改变方向。在所有AIMS中，将校准柱子阵列以确保 力图独立于帖子架构，我们将关联 运动到DC所施加的力的时空图。此外，通过改变 桩的长度，我们将确定底物弹性和 定向运动。这个项目得到了丰富的分子和细胞工具的帮助。 包括剔除小鼠体内的趋化因子受体、肌动蛋白调节蛋白等 如黄蜂、HS1和肌球蛋白II，以及各种分子敲除和药理作用 探员们。我们在这里建立的方法将产生对部队的全面了解 在DC运动过程中施加的，并且这里建立的方法将具有重要的意义 对阐明其他快速运动阿米巴运动机制的影响 免疫系统中产生低力的细胞，包括T淋巴细胞。

Dendritic cells (DCs) are important regulators of the mammalian immune system and motility is critical to their proper function. Technologies such as cancer immunotherapy critically depend on DC migration. DCs possess multiple chemokine receptors and crawl in response to chemokine gradients, which direct DC positioning throughout the immune system. Ultimately, DCs must integrate multiple signals in order to move in a single direction. The goal of this project is to use a novel biointerfacial tool, micropost array detectors (mPADs), coupled with microfluidic gradient chambers, to apply a time- invariant chemokine gradient to cells, and measure the traction forces exerted by DCs during migration. Our recently published work shows that mPAD arrays are sufficiently sensitive to measure the low traction stresses (0.5 nN per filopod and 20 nN per cell) of migrating DCs. We now use these arrays to understand the components within cells that give rise to directed cell motion and to understand how DCs integrate chemokine signals and convert them to traction stresses and directional motion. The specific aims of the proposal are: 1) to use novel micropost force detector to measure DC motility in well- defined gradients of single chemokines on multiple adhesive ligands; 2) to measure the effects of regulatory proteins HS1 and WASp on DC migration in single chemokine gradients; and 3) to measure the forces of DC migration during turning when the gradient rapidly changes direction. In all aims, post arrays will be calibrated to ensure force maps are independent of post architecture, and we will correlate the direction of motion to the spatio-temporal map of forces that DCs exert. Furthermore, by varying the length of posts, we will determine the relationship between substrate elasticity and directional motion. This project is aided by a wealth of molecular and cellular tools including knock out mice in which chemokine receptors, actin regulatory proteins such as WASp, HS1 and myosin II, and various molecular knockdowns and pharmacological agents. The methods we establish here will yield a comprehensive picture of the forces exerted during DC motility, and the methods established here will have a significant impact on the elucidation of the mechanisms of motility of other fast moving amoeboid cells of the immune system that generate low forces, including T-lymphocytes.