CONTROL OF CELL ELASTICITY BY THE ACTIN CORTEX

肌动蛋白皮层对细胞弹性的控制

• 批准号: 6362474
• 负责人: JOSEF A KAS
• 金额: $ 10.04万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-18 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6362474
• 关键词: PC12 cells; actins; cell component structure /function; cell motility; cell transformation; crosslink; cytoskeleton; elasticity; fluorescence microscopy; lasers; measurement; microfilaments; myosins; nonblood rheology; viscosity

Eukaryotic cells reversibly assemble the actin protein into filaments which are then arrayed by accessory proteins into the actin cytoskeleton (AC). Disruption of the AC is associated with significant cell softening and in the case of migrating cells it also results in loss of motility. The exact role of the AC in the mechanical strength of cells is not fully understood. The AC can be roughly divided into two superimposing elastic elements: a homogeneous network of protein filaments predominantly underlying the plasma membrane and stress fibers which span the cell interior. This proposal will focus on the homogeneous cortical part of the AC. The proposed research takes two novel approaches: (1) in vitro measurements will investigate how the elastic response of actin networks is generated on the microscopic level of a single actin filament in a network and (2) in vivo measurements of the specific elasticity of the cortical AC will determine the role of actin networks for whole cell elasticity. Simultaneous measurements of the elastic properties of F-actin solutions/gels and fluorescence microscopy of individual constituent actin filaments under shear will provide detailed characterization of the interactions between actin filaments which cause the elastic behavior. These data will distinguish between recently suggested models and lead to quantitative predictions of the strength of the AC as a function of its composition. This knowledge will improve the understanding of cell motility. A particular focus will be on how transient crosslinkers and molecular motors modulate the strength of these networks. Preliminary results indicate that inactive myosin II acts as a crosslinker, whereas active myosin liquefies F-actin networks in the absence of other crosslinking proteins. The dependence of the mechanical strength on filament stiffness will be investigated as an additional possibility in modulating the strength of actin networks. To accurately measure cell elasticity of large, statistically significant numbers of cells, an optical tool has been developed to non- destructively stretch single cells between two beams of a laser. Measurements of cells in suspension, which show a peripheral AC but no stress fibers, will allow the PI to quantify the contribution of actin networks to cell elasticity. The contribution of these networks to the overall mechanical strength of cells will be determined by measuring the elasticity of cells with varying ACs. This also allows determination of the relevance of in vitro research on homogeneous actin networks for the cell cytoskeleton. A primary focus will be the extent to which malignant transformation of cells by oncogenic vectors -which is correlated with changes of the AC - can be monitored by cell elasticity measurements.

真核细胞可逆地将肌动蛋白组装成细丝 然后由辅助蛋白排列进入肌动蛋白细胞骨架 (Ac)。AC的中断与显著的细胞相关 软化，在迁移细胞的情况下，它还会导致 能动性。AC在细胞机械强度中的确切作用 还没有完全被理解。空调大致可以分为两个部分 叠加弹性元件：蛋白质的均质网络 主要位于质膜和应力纤维下面的细丝 横跨整个细胞内部。这项提案将重点放在 AC的同质皮质部分。拟议中的研究需要两项 新方法：(1)体外测量将调查 肌动蛋白网络的弹性响应是在微观水平上产生的 和(2)在体内测量 大脑皮层AC的特定弹性将决定 肌动蛋白网络的整个细胞弹性。 F-肌动蛋白弹性性质的同步测量 溶液/凝胶和单个组分的荧光显微镜 剪切下的肌动蛋白细丝将提供详细的特征 肌动蛋白细丝之间的相互作用导致弹性 行为。这些数据将区分最近建议的模型 并导致了对AC作为一种 其构成的功能。这一知识将提高 对细胞运动的理解。特别关注的将是如何 瞬时交联剂和分子马达调节细胞的强度 这些网络。初步结果表明，失活的肌球蛋白II 作为交联剂，而活性肌球蛋白液化F-肌动蛋白网络 在没有其他交联蛋白的情况下。对人类的依赖 对纤维硬度的机械强度将作为一种 调节肌动蛋白网络强度的另一种可能性。 为了准确测量大型细胞的弹性，统计 大量的细胞，一种光学工具已经被开发成非 在两束激光之间破坏性地拉伸单个细胞。 悬浮液中细胞的测量，显示外围AC，但没有 应激纤维，将允许PI量化肌动蛋白的贡献 网络到细胞弹性。这些网络对 电池的整体机械强度将通过测量 不同AC的细胞弹性。这也让我们能够下定决心 均相肌动蛋白网络的体外研究对 细胞的细胞骨架。一个主要的焦点将是在多大程度上 通过致癌载体使细胞恶性转化-这是 可通过细胞弹性监测与AC-的变化相关 测量。