Dynamics of Actin in Normal and Transformed Cells

描述(由申请人提供)：这个项目的长期目标是了解肌动蛋白细胞骨架执行的一系列功能的机制。这项拟议的研究将集中在控制细胞形状和迁移的基本原理上，这些原理在许多生理和病理过程中发挥着重要作用，包括胚胎发育、伤口愈合和转移侵袭。此外，细胞形状和迁移的控制是再生医学的一个瓶颈，几乎没有有效的策略。虽然在识别和表征分子组成方面取得了快速进展，但控制这些功能对物理环境的反应的基本规则仍然很大程度上是未知的。这项研究将结合强大的光学、机械、化学、微工程和数学方法来解决贴壁细胞对底物刚性和地形的响应，这些已知对细胞形状和迁移以及包括生长和分化在内的下游过程具有深远影响。第一个目标将通过确定传感器的大小来表征细胞对刚性的反应。这些实验还将测试一种假想的机械信号传感的通用机制，并研究刚性传感在癌症侵袭中的可能作用。第二个目标将确定决定细胞对粘合剂基材几何形状的反应的关键参数。此外，这些实验将测试潜在的几何传感机制，包括基于粘连位置的差异张力的机制，以及基于差异微管密度和/或动力学的机制。第三个目标将探索允许细胞区分3D环境和2D表面的机制。这些实验将解决地形要求以及可能涉及的收缩力、皮质刚性和微管定位。该项目将充分利用微细加工基板，以及从光调制、微操作到图像处理的其他创新方法。细胞将通过药理学和基因消融/沉默技术进行操作，并使用高分辨率共聚焦和全内反射荧光光学进行成像。这些实验揭示的基本原理将影响与细胞形状和迁移调节直接或间接相关的广泛的医学问题。 公共卫生评论：现在有广泛的证据表明，物理和地形信号，包括机械力、刚性和几何图案，对细胞的形状和迁移有深远的影响，进而影响从癌症侵袭到干细胞分化的广泛的正常和病理过程。然而，尽管细胞对化学相互作用的反应已经被广泛研究，但人们对它们对这些非化学信号的反应知之甚少，部分原因是传统实验方法的局限性。拟议的实验将应用高度创新的方法来克服障碍，并获得关键信息，以了解和治疗与细胞形状控制和细胞迁移有关的许多健康问题。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanisms of a wide range of functions performed by the actin cytoskeleton. The proposed research will focus on the basic principles that control cell shape and migration, which play an important role in numerous physiological and pathological processes including embryonic development, wound healing, and metastatic invasion. In addition, control of cell shape and migration represents a bottleneck in regenerative medicine with few effective strategies. While rapid advances have been made on identifying and characterizing the molecular components, the fundamental rules governing the responses of these functions to the physical environment remain largely unknown. The study will combine powerful optical, mechanical, chemical, microengineering, and mathematical approaches to address the responses of adherent cells to substrate rigidity and topography, which are known to have profound effects on cell shape and migration, as well as downstream processes including growth and differentiation. The first aim will characterize cellular responses to rigidity, by determining the size scale of the sensor. The experiments will also test a hypothetical universal mechanism for sensing mechanical signals, and investigate the possible role of rigidity sensing in cancer invasion. The second aim will identify key parameters that determine cellular responses to the geometry of adhesive substrates. In addition, the experiments will test potential mechanisms of geometry sensing, including one based on differential tension at adhesion sites and the other on differential microtubule density and/or dynamics. The third aim will probe the mechanism that allows cells to distinguish 3D environments from 2D surfaces. The experiments will address the topographic requirements and the possible involvement of contractile forces, cortical rigidity, and microtubule positioning. The project will take full advantage of microfabricated substrates, in addition to other innovative approaches from photo- modulation, micromanipulation, to image processing. Cells will be manipulated with pharmacological and gene ablation/silencing techniques, and imaged with high-resolution confocal and total internal reflection fluorescence optics. The fundamental principles revealed by these experiments will impact a broad range of medical issues related directly or indirectly to the regulation of cell shape and migration. PUBLIC HEALTH REVELANCE: There is now extensive evidence that physical and topographical signals, including mechanical forces, rigidity, and geometric patterns, have profound effects on cell shape and migration, which in turn affect a wide range of normal and pathological processes from cancer invasion to stem cell differentiation. However, while cellular responses to chemical interactions have been studied extensively, much less is known about their responses to these non-chemical signals, partially due to the limitations in conventional experimental methods. The proposed experiments will apply highly innovative approaches to overcome the barrier, and to obtain crucial information for understanding and treating many health issues related to cell shape control and cell migration.