CONTROL OF CYTOSKELETAL MECHANICS IN D. DISCOIDEUM

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需要的。尽管描述运动和f -肌动蛋白细胞骨架网络的作用的知识丰富，但产生细胞突起的力的来源却知之甚少。长期存在的模型尚未得到解决。我们已经开发了一种新的微观方法，激光偏转颗粒跟踪微流变学（LDPTM），可以通过完全表征活细胞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源于嵌入网络中的单个粒子的残余布朗运动。在重构网络中，LDPTM与传统流变学方法的一致性非常好。LDPTM快速提供了细胞骨架网络的全面物理表征，并提供了直接测试突出力模型的新参数。此外，LDPTM的速度足够快，可以解决吞噬过程中僵硬的15个“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运动变形虫，盘状齿状体。Dictyostelium具有发达的生物化学、遗传学和快速的行为反应，为了解细胞结构、吞噬作用和细胞运动提供了独特的优势。我们的具体目标是：1 .为了深入了解运动性，利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。使用零突变体和荧光定位的gfp标记的蛋白质。2。为了测试细胞突出模型，监测由伤口愈合或趋化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盘基骨柱。3。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。首先关注交联蛋白。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。

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.