Integrative Experimental and Theoretical Studies of the Mechanics of Phagocytosis

描述(申请人提供)：病原体的吞噬中和在宿主抵抗感染和从感染中恢复方面起着核心作用。该计划旨在通过将单细胞实验与先进的“虚拟免疫细胞”理论模型紧密结合起来，揭示吞噬作用的主要机械决定因素。这一跨学科的努力与建立对自主细胞运动性及其在先天性免疫反应中的作用的坚实的量化理解的长期目标密切相关。首先将使用综合实验/理论方法来确定吞噬作用关键模型(目标1)和不同吞噬途径(目标2)的基线。然后将其应用于基因改变的模型系统，以阐明正常吞噬功能的主要分子成分的机械作用(目标3)。该方法作为研究先天免疫功能的有价值的单细胞分析的相关性将通过检测感染性病原体的吞噬作用来验证(目标4)。最适合与理论模型紧密结合的实验构型包括一个最初的球形细胞，它吞没了一个球形珠子。将使用定制的视频显微镜/微吸管操作装置来监测球形靶的单细胞吞噬作用。数据分析将提供每个实验的细胞形态、皮质张力和珠子运动的时间进程。这些定量测量将作为验证有限元计算机模型的标准，这些模型基于物理上现实的和生物学上看似合理的规则。这些模型成功地再现了观察结果，这将对吞噬作用中起作用的力量施加强有力的限制。适应基因改变或疾病状态所需参数值的变化将非侵入性地暴露受影响的纳米结构对分子马达活性和细胞骨架特性的影响。对标准化的单细胞/单靶构型的关注提出了对吞噬细胞表型的独特观点，该构型具有高度的重复性和足够简单的定量分析。实验和理论的结合详细揭示了免疫吞噬反应是如何失败或成功的，这取决于吞噬细胞和病原体的性质。这是从人类健康的传染病挑战中恢复过来的决定性因素。

DESCRIPTION (provided by applicant): The phagocytic neutralization of pathogens plays a central role in host protection against, and recovery from infection. The proposed project aims to expose the major mechanical determinants of phagocytosis by firmly integrating single-cell experiments with advanced theoretical models of "virtual immune cells". This interdisciplinary effort ties closely into the long-term objective of establishing a solid quantitative understanding of autonomous cell motility and its role in the innate immune response. An integrative experimental/theoretical approach will first be used to establish a baseline of key models of phagocytosis (Aim 1) and of different phagocytic pathways (Aim 2). It will then be applied to genetically altered model systems in order to elucidate the mechanical roles of major molecular components for normal phagocytic function (Aim 3). The method's relevance as a valuable single-cell assay in studies of the innate immune function will be validated by examining the phagocytosis of infectious pathogens (Aim 4). The experimental configuration most suitable for close integration with theoretical models consists of an initially spherical cell that engulfs a spherical bead. Single-cell phagocytosis of spherical targets will be monitored using a custom-built videomicroscopy/micropipette-manipulation setup. Data analysis will provide the time courses of cellular morphology, cortical tension, and bead movement for each experiment. These quantitative measurements will serve as standards for the validation of finite-element computer models that are based on physically realistic and biologically plausible rules. The models' success in reproducing the observations will place strong constraints on the forces at play in phagocytosis. The changes in parameter values that are required to accommodate genetically altered or diseased states will noninvasively expose the impact of the affected nanoscale structures on molecular motor activity and cytoskeletal properties. The focus on a standardized single cell/single target configuration that is highly reproducible and simple enough to be quantitatively analyzed presents a unique viewpoint on the phagocytic phenotype. The integration of experiment and theory reveals in detail how the immune phagocytic response fails or succeeds depending on the nature of both phagocyte and pathogen. This is a determining element for the recovery from infectious challenges in human health.