CONTROL OF CYTOSKELETAL MECHANICS IN D. DISCOIDEUM

盘状 D. 细胞骨架力学的控制

• 批准号: 6045573
• 负责人: SCOT CHARLES KUO
• 金额: $ 28.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6045573
• 关键词: Dictyostelium; actin binding protein; actins; biomechanics; cell motility; chemotaxis; confocal scanning microscopy; crosslink; cytoskeleton; electron microscopy; green fluorescent proteins; nonblood rheology; phagocytosis; technology /technique development; tissue /cell culture; viscosity

Our long-term goals are to understand cell motility, which is critical for immunological defense and wound healing, but unwanted during metastatic cancers. Despite the wealth of knowledge describing motility and the role of F-actin cytoskeletal networks, the origin of forces for generating cell protrusions is poorly understood. Long-standing models have yet to be resolved. We have developed a new microscopic approach, laser-deflection particle-tracking microrheology (LDPTM), that can address this problem by fully characterizing subcellular mechanical properties in living cells. In this noninvasive approach, mechanical properties of the cytoskeleton are derived from the residual Brownian motion of individual particles embedded in the network. In reconstituted networks, agreement between LDPTM and traditional rheological approaches is excellent. LDPTM quickly provides a thorough physical characterization of cytoskeletal networks and provides new parameters that directly test models for protrusive forces. In addition, LDPTM is fast enough to resolve 1s "spikes" in stiffness during phagocytosis, which involves many of the same proteins as cell motility. We will continue to use LDPTM to study mammalian cells, but will focus our efforts on the motile amoeba, Dictyostelium discoideum. Because of its well-developed biochemistry and genetics and its fast behavioral response, Dictyostelium offers unique advantages for understanding both cell structure, phagocytosis, and cell motility. Our specific aims are: I. For insights related to motility, use abrupt mechanical changes to help resolve the order of early molecular events during phagocytosis. Use null-mutants and fluorescent localization of GFP-tagged proteins. II. To test models of cell protrusion, monitor mechanical changes within crawling cells caused by wound-healing or chemotaxis. Examine motile animal cells, Listeria-infected cells and motile Dictyostelium. III. To develop a quantitative mechanical theory of F-actin networks, use Dictyostelium to compare in vitro and in vivo measurements. Initially focus on crosslinking proteins. These studies should provide a paradigm for considering cytoskeletal mechanics and its remodeling in many other systems of cell biology.

我们的长期目标是了解细胞运动，这对于免疫防御和伤口愈合至关重要，但在转移性癌症中不需要。 尽管有大量的知识描述运动性和F-肌动蛋白细胞骨架网络的作用，但对产生细胞突起的力的起源却很少了解。 长期模型尚未解决。 我们已经开发了一种新的显微镜方法，即激光反射颗粒跟踪微流变学（LDPTM），可以通过充分表征活细胞中的亚细胞机械性能来解决此问题。 在这种非侵入性方法中，细胞骨架的机械性能源自网络中嵌入的单个颗粒的残留布朗运动。 在重构的网络中，LDPTM与传统流变学方法之间的一致性非常好。 LDPTM快速提供了细胞骨架网络的彻底物理表征，并提供了直接测试突出力模型的新参数。 此外，LDPTM足够快，可以在吞噬作用期间刚度1S“尖峰”，这涉及许多与细胞运动相同的蛋白质。我们将继续使用LDPTM研究哺乳动物细胞，但将我们的精力集中在Motile Amoeba Distyostelium Discoideum上。由于其发达的生物化学和遗传学及其快速行为反应，Dictyostelium为了解细胞结构，吞噬作用和细胞运动提供了独特的优势。 我们的具体目的是：I。对于与运动有关的见解，请使用突然的机械变化来帮助解决吞噬作用期间早期分子事件的顺序。 使用无效的GFP标记蛋白的荧光定位。 ii。为了测试细胞突起的模型，监测由伤口愈合或趋化性引起的爬行细胞内的机械变化。 检查运动动物细胞，李斯特菌感染细胞和动杆菌。 iii。为了开发F-肌动蛋白网络的定量机械理论，请使用dictyostelium比较体外测量。 最初专注于交联蛋白。这些研究应提供一个用于考虑细胞骨架力学及其在许多其他细胞生物学系统中进行重塑的范式。