CONTROL OF CYTOSKELETAL MECHANICS IN D. DISCOIDEUM

我们的长期目标是了解细胞运动性，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必要的。 尽管有丰富的知识描述运动和F-肌动蛋白细胞骨架网络的作用，产生细胞突起的力的起源知之甚少。 长期存在的模式尚未得到解决。 我们已经开发了一种新的显微镜方法，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中的亚细胞力学特性来解决这个问题。 在这种非侵入性的方法中，细胞骨架的机械特性来自嵌入网络中的单个颗粒的残余布朗运动。 在重构网络中，LDPTM和传统的流变学方法之间的协议是优秀的。 LDPTM快速提供了细胞骨架网络的全面物理表征，并提供了直接测试膨胀力模型的新参数。 此外，LDPTM足够快，可以在吞噬过程中解决1s的刚度“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来研究哺乳动物细胞，但将把我们的努力集中在能动的变形虫，盘基网柄阿米巴。由于其发达的生物化学和遗传学及其快速的行为反应，Dictyosteoblasts为理解细胞结构，吞噬作用和细胞运动提供了独特的优势。 我们的具体目标是：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。 使用空突变体和GFP标记蛋白的荧光定位。 二.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。 检查活动的动物细胞、李斯特菌感染的细胞和活动的网骨细胞。 三.为了发展一个定量力学理论的F-肌动蛋白网络，使用Dictyosteoprotein比较在体外和体内的测量。 最初专注于交联蛋白质。这些研究应该提供一个范例，考虑在许多其他系统的细胞生物学的细胞骨架力学和重塑。

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.