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，这对免疫防御和伤口愈合至关重要，但在转移性癌症中是不受欢迎的。尽管有丰富的知识描述了运动性和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来源却知之甚少。长期存在的模式尚未得到解决。我们开发了一种新的微观方法，激光偏转粒子跟踪微观流变学(LDPTM)，可以通过充分表征活细胞的亚细胞机械特性来解决这个问题。在这种非侵入性方法中，细胞骨架的力学性质是由嵌入网络中的单个粒子的残余布朗运动得出的。在重组网络中，LDPTM和传统流变学方法之间的一致性很好。LDPTM快速提供了细胞骨架网络的全面物理特征，并提供了直接测试模型的前进力的新参数。此外，LDPTM的速度足够快，可以在吞噬过程中分解1s的“尖峰”，吞噬过程中涉及许多与细胞运动相同的蛋白质。我们将继续使用LDPTM来研究哺乳动物细胞，但我们将把重点放在能动的阿米巴--盘基网柄菌上。由于其发达的生物化学和遗传学以及快速的行为反应，网柄菌在理解细胞结构、吞噬作用和细胞运动方面具有独特的优势。我们的具体目标是：1.对于与运动相关的见解，使用突然的机械变化来帮助解决吞噬过程中早期分子事件的顺序。使用零突变体和GFP标记蛋白的荧光定位。为了测试细胞突起的模型，监测由伤口愈合或趋化作用引起的爬行细胞内的机械变化。检查可移动的动物细胞、感染李斯特氏菌的细胞和可移动的网柄基菌。为了发展F-肌动蛋白网络的定量力学理论，使用Dictyostelium来比较体外和体内的测量结果。最初将重点放在交联蛋白质上。这些研究应该为考虑细胞骨架力学及其在许多其他细胞生物学系统中的重塑提供一个范例。