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: 9058548
• 负责人: Daniel A Hammer
• 金额: $ 33.74万
• 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 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058548
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Adhesions; Adhesives; Affect; Architecture; Behavior; Biochemical; CCL19 gene; CCL21 gene; Caliber; Cell Adhesion; Cell Adhesion Molecules; Cells; Chemicals; Chemotaxis; Coupled; Cues; Cytoskeleton; Dendritic Cells; Device Designs; Devices; EEF1A2 gene; Elasticity; Ensure; F-Actin; Fibronectins; Filopodia; Generations; Goals; Homing; Image; Immune; Immune response; Immune system; Immunotherapy; In Vitro; Individual; Infiltration; Integrins; Intercellular adhesion molecule 1; Knockout Mice; Label; Length; Ligands; Malignant Neoplasms; Maps; Measures; Mechanics; Mediating; Methodology; Methods; Microfluidics; Modification; Molecular; Motion; Mus; Myosin Type II; Natural Immunity; Organ; Positioning Attribute; Publishing; Regulation; Role; Signal Transduction; Signaling Molecule; Small Interfering RNA; Stress; T-Lymphocyte; Technology; Time; Tissues; Traction; Work; adaptive immunity; cancer immunotherapy; cell motility; chemokine; chemokine receptor; detector; directional cell; elastomeric; genetic regulatory protein; in vivo; insight; knock-down; method development; migration; mutant; novel; response; spatiotemporal; tool; trafficking

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因子， 运动性对它们的正常功能至关重要。癌症免疫治疗等技术 主要依赖于DC迁移。DC具有多种趋化因子受体， 响应趋化因子梯度，其指导DC在整个免疫系统中的定位， 系统最终，DC必须集成多个信号，以便在单个 方向本计画的目标是使用一种新颖的生物介面工具-微柱阵列 检测器（mPAD），与微流体梯度室耦合，以施加时间- 恒定趋化因子梯度的细胞，并测量由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（0.5 nN/丝状足和20 nN/细胞）， 移民区。我们现在使用这些数组来了解细胞内的组件， 引起定向细胞运动，并了解DC如何整合趋化因子信号 并将其转化为牵引应力和定向运动。该委员会的具体目标 建议：1）使用新型微柱力检测器测量井中DC运动， 确定单一趋化因子在多个粘附配体上的梯度; 2）测量趋化因子的浓度， 调节蛋白HS 1和WASp对单个趋化因子中DC迁移的影响 梯度;以及3）当旋转时，测量旋转期间DC迁移的力。 梯度迅速改变方向。在所有目标中，将校准柱阵列，以确保 力地图是独立的岗位架构，我们将关联的方向 运动到DC施加的力的时空图。此外，通过改变 长度的职位，我们将确定之间的关系基板弹性和 定向运动这个项目得到了大量分子和细胞工具的帮助 包括敲除小鼠，其中趋化因子受体，肌动蛋白调节蛋白， 如WASp、HS 1和肌球蛋白II，以及各种分子敲除和药理学作用， 剂.我们在这里建立的方法将产生一个全面的图片的力量 在DC运动期间施加，并且这里建立的方法将具有显著的 对阐明其他快速移动变形虫运动机制的影响 免疫系统中产生低强度的细胞，包括T淋巴细胞。

项目成果

The Arp2/3 complex binding protein HS1 is required for efficient dendritic cell random migration and force generation.

• DOI: 10.1039/c7ib00070g
• 发表时间: 2017-08-14
• 期刊: Integrative biology : quantitative biosciences from nano to macro
• 作者: Bendell AC;Williamson EK;Chen CS;Burkhardt JK;Hammer DA
• 通讯作者: Hammer DA

Motile Dendritic Cells Sense and Respond to Substrate Geometry.

• DOI: 10.1007/s10439-018-2041-7
• 发表时间: 2018-09
• 期刊: Annals of biomedical engineering
• 影响因子: 3.8
• 作者: Bendell AC;Anderson N;Blumenthal D;Williamson EK;Chen CS;Burkhardt JK;Hammer DA
• 通讯作者: Hammer DA