Using micropost arrays to measure traction forces during dendritic cell motility

使用微柱阵列测量树突状细胞运动过程中的牵引力

• 批准号: 8583289
• 负责人: Daniel A Hammer
• 金额: $ 35.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8583289
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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.

树突状细胞（DC）是哺乳动物免疫系统的重要调节剂， 运动对于其适当的功能至关重要。癌症免疫疗法等技术 严重取决于直流迁移。 DC具有多个趋化因子受体和爬网 响应趋化因子梯度，该趋化因子梯度将直流定位在整个免疫中 系统。最终，DC必须集成多个信号才能移动 方向。该项目的目的是使用一种新颖的生物界面工具Microstost Array 探测器（MPAD），再加上微流体梯度腔，以应用时间 不变的趋化因子梯度对细胞，并测量DC施加的牵引力 在迁移期间。我们最近发表的工作表明，MPAD阵列足够 敏感测量低牵引力的应力（每个纤维足动物0.5 nn和每个细胞20 nn） 迁移DC。现在，我们使用这些数组来了解单元格中的组件 引起定向细胞运动，并了解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。特定目标 提案是：1）使用新型的微型验证探测器来测量DC的良好性。 在多种粘合剂配体上定义的单个趋化因子的梯度； 2）测量 调节蛋白HS1和WASP对单个趋化因子中直流迁移的影响 梯度； 3）在转弯期间测量直流迁移的力 梯度迅速改变方向。总的来说，将校准后阵列以确保 力图独立于后建筑，我们将关联 向DCS发动的力的时空图映射运动。此外，通过改变 帖子长度，我们将确定底物弹性与 方向运动。该项目得到了大量分子和细胞工具的帮助 包括敲除趋化因子受体，肌动蛋白调节蛋白的小鼠 如WASP，HS1和肌球蛋白II，以及各种分子敲低和药理 代理商。我们在这里建立的方法将产生力量的全面图片 在直流运动期间施加的，此处建立的方法将具有重要的 对其他快速移动变形虫的运动机制阐明的影响 产生低力的免疫系统的细胞，包括T淋巴细胞。