Mechano-dynamics of the Transition to Firm Adhesion and MoIotility in Neutrophils

中性粒细胞是细胞炎症反应的第一道防线，研究它们的行为可以 导致治疗炎症性疾病的策略。使用定量工具和分析，我们建议 研究控制中性粒细胞粘附和运动的基本机械化学过程 从血液外渗到组织中。在aim 1中，我们将模拟整合素介导的 中性粒细胞通过将粘附动力学（一种用于建模粘附的机械精确方法）与 由内而外信号转导随机模拟。这些模型将预测， 嗜中性粒细胞的粘附由嗜中性粒细胞的内部分子机制控制。此外，我们将 使用随机信号方法来分析项目3中进行的实验，其中中性粒细胞保持 在移液管中通过与分子涂覆的珠碰撞来刺激。在目标2中，我们将测试预测 通过进行流动室粘附实验， 变量系统地变化，以确认我们对分子控制的定量理解， 粘合是正确的。与项目2和项目5密切合作，我们将使用药物干预， 抗体和sIRNA技术来调节中性粒细胞成分，并检查它们对转化的影响 以牢固粘附。在目标3中，我们将使用Core C并使用牵引力显微镜检查 中性粒细胞在化学引诱物的明确梯度中的运动。我们建造了一个结合了 微流体，以施加明确的化学引诱物梯度跨细胞，与牵引力显微镜。 目的是了解中性粒细胞运动的速度和方向如何与力的产生相关，以及 了解中性粒细胞中的分子成分如何控制收缩性。以前的工作表明， 嗜中性粒细胞的定向运动伴随着尾足动物中收缩牵引应力的强位点。使用 药理学抑制，抗体和sIRNA技术，我们将测量关键分子如何发挥作用， 影响中性粒细胞极性、牵引应力的产生和细胞运动。总之，我们的工作将 提供了关于分子成分如何控制炎症中中性粒细胞功能的基本见解。

Neutrophils are the first line of defense in the cellular inflammatory response, and studying their behavior can lead to strategies for treating inflammatory disorders. Using quantitative tools and assays, we propose to investigate the fundamental mechano-chemical processes of adhesion and motility that control neutrophil extravasation from blood into tissue. In aim 1, we will simulate integrin-mediated firm adhesion of a neutrophil by merging Adhesive Dynamics - a mechanically accurate method for modeling adhesion - with a stochastic simulation of inside-out signal transduction. These models will predict how the rate and extent of neutrophil firm adhesion are controlled by a neutrophil's internal molecular machinery. Furthermore, we will use stochastic signaling methods to analyze experiments performed in Project 3 in which a neutrophil held in a pipette is stimulated by impingement with a moleculariy-coated bead. In aim 2, we will test predictions from the modeling in aim 1 by performing flow chamber adhesion experiments in which external and internal variables are systematically varied, to confirm that our quantitative understanding of the molecular control of adhesion is correct. Working closely with Projects 2 and 5, we will use pharmacological intervention, antibodies, and sIRNA technology to adjust neutrophil components and examine their effect on the transition to firm adhesion. In aim 3, we will work with Core C and use traction force microscopy to examine the motility of neutrophils in well-defined gradients of chemoattractant. We have built a chamber that combines microfluidics, to impose well-defined chemoattractant gradients across cells, with traction force microscopy. The goal is to understand how speed and direction in neutrophil motility is related to force generation, and to understand how the molecular components in neutrophils control contractility. Previous work has shown that neutrophil directional motion is accompanied by strong loci of contractile traction stress in the uropod. Using pharmacological inhibition, antibodies and sIRNA technology, we will measure how key molecular players affect neutrophil polarity, the generation of traction stresses, and cell motion. In summary, our work will provide fundamental insights as to how molecular components control neutrophil function in inflammation.