Quantitative Analysis of Chemotactic Motility Cycle of Ameboid Cells

阿米巴细胞趋化运动周期的定量分析

• 批准号: 7917518
• 负责人: RICHARD A FIRTEL
• 金额: $ 29.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917518
• 关键词: Actins; Address; Adhesions; Affect; Biochemical; Biochemical Process; Biomechanics; Biophysical Process; Cell Adhesion; Cell Shape; Cells; Chemotaxis; Cicatrix; Complex; Cytochalasin B; Cytometry; Cytoskeletal Modeling; Cytoskeletal Proteins; Defect; Dictyostelium discoideum; Dimensions; Disease; Embryonic Development; Eukaryotic Cell; Event; F-Actin; Frequencies; Future; Generations; Goals; Immune system; Individual; Inflammation; Knowledge; Lateral; Length; Leukocytes; Malignant Neoplasms; Maps; Measurement; Measures; Mechanics; Mediating; Mental Retardation; Methods; Modeling; Molecular; Molecular Motors; Motor; Motor Activity; Mutation; Myosin Type II; Osteoporosis; Phase; Physiological Processes; Play; Principal Component Analysis; Process; Production; Proteins; Regulation; Reporter; Research; Role; Shapes; Signal Pathway; Signal Transduction; Site; Staging; Stress; Testing; Therapeutic; Time; Tissues; Traction; Travel; base; cell growth regulation; cell motility; cell type; crosslink; genetic regulatory protein; migration; mutant; neglect; physical property; polymerization; public health relevance; research study; spatiotemporal; statistics

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.

描述（由申请人提供）：真核细胞运动对于许多生理过程，例如胚胎发育，组织更新和免疫系统的功能至关重要。事实证明，DICSYOSTELIUM DISCOIDEUM是变形虫细胞（如白细胞）趋化性迁移的绝佳模型。变形虫迁移是伪动物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指导的电机，F-肌动蛋白调节，肌动蛋白交联，运动蛋白蛋白介导的收缩率和细胞粘附的协调相互作用确定。但是，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受到限制。我们和其他人观察到，在变形虫迁移过程中发生的细胞形状变化的相当一部分是由于定期重复事件引起的，这使得使用条件统计方法可以分析涉及细胞运动涉及的生化过程的网络。 这项研究的主要目的是以统计稳定的定量方式来确定牵引力时空分布的生化基础以及运动循环的每个阶段的持续时间，通过研究候选细胞骨骼骨骼和调节分子的作用，并在不同阶段中涉及已知或过度的参与。我们假设肌球蛋白II不仅对运动循环的收缩阶段至关重要，而且对假脚产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作用。基于初步结果，我们进一步假设，牵引力的时空分布和每个周期的平均距离取决于肌动蛋白聚合。 为了检验上述假设，我们提出了三个具体目标。具体目的1是应用我们的新3D力细胞仪方法来测量细胞在底物上施加的力的三个成分。第二和第三目的旨在研究候选细胞骨架和调节分子的作用，通过对野生型细胞和肌动蛋白交联或运动蛋白蛋白收缩性缺损（特定AIM 2）以及F-肌动蛋白调节缺陷（特定的AIM AIM 3）进行系统比较（特定的AIM 2），并通过对野生型细胞和突变型菌株进行系统比较，从而与运动性有关。我们的方法包括同时测量荧光标记的信号传导（或细胞骨架）蛋白的分布的空间和时间变化，以及介导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。我们的研究将提供必要的构件，以开始构建控制细胞迁移的复杂生化过程网络。 公共卫生相关性：真核细胞的运动对于许多生理过程，例如胚胎发育，组织更新以及免疫系统的功能至关重要。不正确的运动性调节在许多疾病（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，未来的治疗方法将受益于对控制细胞运动的生物物理过程的精确定量理解。这项研究的目的是建立机制，即运动周期的每个单个阶段与特定的生化信号事件有关，并阐明这些信号传导途径对细胞运动的调节的影响，其最终目标是对足以预测和控制细胞运动的生物力学过程了解水平。