Quantitative Analysis of Chemotactic Motility Cycle of Ameboid Cells

描述(由申请人提供)：真核细胞的运动对许多生理过程是必不可少的，如胚胎发育、组织更新和免疫系统的功能。盘基网柄菌已被证明是白细胞等阿米巴细胞趋化迁移的极佳模型。变形虫的迁移是伪足类突起和缩回的顺序重复的结果，并由牵引力的产生驱动。牵引力的强度和时空组织由肌动蛋白导向的运动、F-肌动蛋白调节、肌动蛋白交联、运动蛋白介导的收缩和细胞黏附的协调相互作用决定。然而，由于缺乏定量信息和分析，对这些过程的生物物理协调的精确知识一直受到限制。我们和其他人观察到，在阿米巴迁移过程中，相当一部分细胞形状的变化是由于周期性的重复事件，这使得使用条件统计方法来分析涉及细胞运动的生化过程网络。这项研究的主要目的是通过研究已知或怀疑参与运动周期不同阶段的候选细胞骨架和调节分子的作用，以统计、稳健、定量的方式确定牵引力时空分布的生化基础和运动周期每个阶段的持续时间。我们假设，肌球蛋白II不仅对运动周期的收缩阶段是必不可少的，而且对假足突起阶段也是必不可少的。牵引力的产生不仅取决于肌球蛋白II的收缩作用，还取决于其肌动蛋白的交联性。根据初步结果，我们进一步假设牵引力的时空分布和细胞每周期的平均行程取决于肌动蛋白聚合。为了验证上述假设，我们提出了三个具体目标。具体目标1是应用我们的新的三维力细胞术方法来测量细胞对底物施加的力的三个分量。第二和第三个目的是通过对野生型细胞和具有肌动蛋白交联性或运动蛋白收缩缺陷的突变株(特异性目标2)和F-肌动蛋白调控缺陷(特异性目标3)进行系统比较，研究已知或怀疑参与运动性的候选细胞骨架和调控分子的作用。我们的方法包括同时测量荧光标记的信号(或细胞骨架)蛋白分布的空间和时间变化，以及调节细胞运动周期每个阶段的3D牵引力，同时还记录细胞形状的变化。我们将应用条件统计和主成分分析(PCA)将特定的生化过程与运动周期中的每个物理事件联系起来。我们的研究将为开始构建控制细胞迁移的复杂生化过程网络提供必要的基础。 公共卫生相关性：真核细胞的运动性对于胚胎发育和组织更新等许多生理过程以及免疫系统的功能都是必不可少的。不正确的运动调节在许多疾病(癌症、破坏性炎症、骨质疏松症、智力低下等)中起着重要作用，因此，对控制细胞运动的生物物理过程的准确定量了解将有助于未来的治疗方法。本研究的目的是建立运动周期的各个阶段与特定的生化信号事件相关的机制，并阐明这些信号通路的调节对细胞运动的影响，最终目的是发展对足以预测和控制细胞运动的生物力学过程的理解水平。

DESCRIPTION (provided by applicant): Eukaryotic cell motility is essential for many physiological processes such as embryonic development, tissue renewal, and the function of the immune system. Dictyostelium discoideum has proven to be an excellent model for the chemotactic migration of amoeboid cells such as leukocytes. Amoeboid migration is the result of the sequential repetition of pseudopod protrusions and retractions and is driven by the generation of traction forces. The strength and spatiotemporal organization of traction forces is determined by the coordinated interactions of actin-directed motors, F-actin regulation, actin crosslinking, motor protein-mediated contractility, and cell adhesions. However, precise knowledge of the biophysical coordination of these processes has been limited by the lack of quantitative information and analysis. We and others have observed that a considerable portion of the changes in cell shape occurring during amoeboid migration are due to periodic repetitive events, which enables the use of conditional statistics methods to analyze the network of biochemical processes involved in cell motility. The primary goal of this research is to determine in a statistically-robust, quantitative manner the biochemical basis for the spatiotemporal distribution of traction forces and the duration of each phase of the motility cycle by studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the different stages of the motility cycle. We hypothesize that Myosin II is essential not only to the contractility phase of the motility cycle but also to the pseudopod protrusion phase. The generation of the traction forces depends not only on the contractile action of Myosin II, but also in its actin crosslinking effect. Based on preliminary results, we further hypothesize that the spatiotemporal distribution of traction forces and the average distance a cell travels per cycle depend on actin polymerization. To test the above hypotheses, we propose three Specific Aims. Specific Aim 1 is to apply our new 3D force cytometry method to measure the three components of the forces exerted by the cells on the substrate. The second and third aims are aimed at studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the motility by undertaking systematic comparison of wild type cells and mutant strains with actin crosslinking or motor protein contractility defects (Specific Aim 2), and F-actin regulation defects (Specific Aim 3). Our method consists of simultaneously measuring the spatial and temporal changes in the distribution of fluorescently tagged signaling (or cytoskeletal) proteins and the 3D traction forces that mediate each stage of the cell motility cycle, while also recording the changes in cell shape. We will apply conditional statistics and Principal Component Analysis (PCA) to connect specific biochemical processes to each of the physical events in the motility cycle. Our studies will provide the necessary building blocks to begin constructing the complex network of biochemical processes controlling cell migration. PUBLIC HEALTH RELEVANCE: Motility of eukaryotic cells is essential for many physiological processes such as embryonic development, and tissue renewal, as well as for the function of the immune system. Incorrect regulation of motility plays an important part in many diseases (cancer, destructive inflammation, osteoporosis, mental retardation, etc.), and therefore, future therapeutic approaches will benefit from a precise quantitative understanding of the biophysical processes controlling cell motility. The aim of this study is to establish the mechanisms whereby each individual stage of the motility cycle is related to specific biochemical signaling events, and to elucidate the effects that the regulation of these signaling pathways has on cell motility, with the ultimate goal of developing a level of understanding of the biomechanical processes sufficient to predict and control cell motility.