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: 6511906
• 负责人: JOSEF A KAS
• 金额: $ 10.36万
• 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 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511906
• 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丝 然后通过辅助蛋白排列到肌动蛋白细胞骨架中 （交流）。 AC 的破坏与重要的细胞有关 软化，在细胞迁移的情况下，也会导致细胞损失 动力。 AC 在细胞机械强度中的确切作用 还没有完全理解。 AC大致可分为两部分 叠加弹性元件：均匀的蛋白质网络 主要位于质膜和应力纤维下方的细丝 跨越细胞内部。 该提案将重点关注 AC 的同质皮质部分。 拟议的研究需要两个时间 新方法：（1）体外测量将研究如何 肌动蛋白网络的弹性响应是在微观水平上产生的 网络中单个肌动蛋白丝的测量和（2）体内测量 皮质 AC 的具体弹性将决定 肌动蛋白网络实现全细胞弹性。 同时测量 F-肌动蛋白的弹性特性 单个成分的溶液/凝胶和荧光显微镜 剪切下的肌动蛋白丝将提供详细的表征 肌动蛋白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 并导致对 AC 强度的定量预测 其组成的功能。 这些知识将改善 了解细胞运动。 将特别关注如何 瞬时交联剂和分子马达调节强度 这些网络。 初步结果表明，失活的肌球蛋白 II 作为交联剂，而活性肌球蛋白则液化 F-肌动蛋白网络 在没有其他交联蛋白的情况下。 的依赖性 将研究机械强度对长丝刚度的影响 调节肌动蛋白网络强度的额外可能性。 准确测量大的细胞弹性，统计 大量的细胞，已经开发出一种光学工具来非 在两束激光之间破坏性地拉伸单个细胞。 悬浮细胞的测量，显示外周 AC，但没有 应力纤维，将使 PI 能够量化肌动蛋白的贡献 网络到细胞弹性。 这些网络对网络的贡献 电池的整体机械强度将通过测量来确定 具有不同 AC 的细胞的弹性。 这也可以确定 同质肌动蛋白网络体外研究的相关性 细胞的细胞骨架。 主要关注点是在多大程度上 致癌载体引起的细胞恶性转化 - 这是 与 AC 的变化相关 - 可以通过细胞弹性进行监测 测量。