Physical Approaches for Probing the Mechanical Properties of Intermediate Filamen

探测中间丝机械性能的物理方法

• 批准号: 8142485
• 负责人: DAVID A WEITZ
• 金额: $ 29.55万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142485
• 关键词: Actins; Agitation; Bacteria; Behavior; Biological Assay; Cations; Cells; Characteristics; Complement; Complex; Controlled Environment; Cytoskeleton; Detergents; Elasticity; Endothelial Cells; Environment; Exhibits; Fibroblasts; Genetic; Goals; In Vitro; Intermediate Filaments; Investigation; Left; Life; Link; Magnetism; Measurement; Measures; Mechanics; Microfilaments; Microfluidics; Microtubules; Modification; Molecular Motors; Motion; Motor; Nature; Organism; Phosphorylation; Physiological; Post-Translational Protein Processing; Program Research Project Grants; Property; Proteins; Regulation; Rheology; Role; Running; Serum; Site; Stretching; Structure; Techniques; Testing; Tracer; Vimentin; Work; base; crosslink; design; laser tweezer; magnetic beads; magnetic field; mutant; particle; programs; reconstitution; research study; response; shear stress

The mechanical properties of cells fundamentally underlie all cellular behavior; the cell must support forces, must exert forces and must respond to forces (1-5). Moreover, while the genetic response within the cell ultimately provides the control mechanism, it is the mechanical response of the cell that dictates its primary function within a larger organism; without being able to withstand the forces of its environment, the cell would not be able to function at all. The mechanical properties of a cell are determined to a large extent by the three filamentous networks within the cytoskeleton, actin, microtubules and intermediate filaments (IF) (1). While actin networks and microtubules have been rather well studied, this is not the case for IF networks, whose study has significantly lagged that of the others (6, 7-9). Indeed, it has been proposed that networks of IF are essential in determining the mechanical properties of and mechanotransduction in virtually all vertebrate cells. However, there is little direct evidence supporting this proposed IF function. The IF networks within a cell are thought to be able to withstand very large strain; this can often be well in excess of 100% (6, 7, 10) In addition, the IF networks exhibit pronounced strain stiffening, effectively becoming much stiffer as they are stretched (6). However, the intracellular environment is highly heterogeneous and complex, making determination of the underlying mechanical properties of these networks extremely difficult. The IFs are remarkably dynamic, and are constantly being remodeled and reassembled, presumably driven in some fashion by the motors that run along either microtubules or actin filaments in the cell and these must guide the assembly of the VIF. There are also, presumably, associated proteins which regulate and control the VIF properties, and which provide crosslinking of the network to the surrounding networks within the cell, and within the VIF network itself (11-16). However, the complexity and richness of the behavior of the VIF within the cell, while controlling much of the function, also makes elucidating the fundamental properties much more difficult; moreover, it precludes measurement of the mechanical properties in a fashion that would allow determination of the underlying design principles of the network. The overarching goal of this section of the Program Project is therefore to measure the properties of VIF in a more controlled environment, thereby enabling us to elucidate their roles in establishing and regulating the mechanical properties of cells (17). The work proposed here will begin with a detailed study of the properties of networks of vimentin intermediate filament (VIF), which can be expressed in bacteria to enable us to produce sufficient quantifies to reconstitute the protein into networks and to make detailed measurements of the mechanical properties of these networks. These measurements will be performed using traditional bulk rheology (18). In addition, we will develop several new assays based on multi-particle tracking, measurements of the motion of small tracer particles embedded within the network and subject either to thermal agitation or to externally applied forces controlled by a magnetic field. The motion of these tracer particles will be interpreted using the formalism of microrheology to measure the elastic and viscous properties of the network. We will investigate the role of physiological concentrations of multivalent cations in regulating the network (6). In addition, we will work with the Goldman lab to investigate the role of phosphorylation in regulating VIF network elasticity (19, 20). We will also obtain constructs for vimentin mutants from our collaborator Harald Herrmann, and will use these to express the mutants in bacteria (21-23). This will enable us to elucidate fundamental design principles for the elasticity of these VIF networks. To complement these investigations of reconstituted networks, we will also form 'ghosts', where most of the cell proteins are washed away with detergent, leaving nearly the full IF network intact (24). By seeding these networks with probe particles, we will measure their elastic properties and compare to those of the reconstituted networks. This will provide a direct probe of the contribution of these VIF networks to cell elasticity. Importantly, these will also enable us to directly measure the response of the networks to shear; cells will be sheared prior to preparing the ghosts, allowing us to probe modifications in the structure and mechanics of the VIF networks due to the shear. We will, in addition, extend these particle tracking measurements to living cells: We will inject the cells with tracer particles and measure the motion of these particles due to both the internal molecular motors within the cell and to external forces, applied either with a magnetic field or with optical tweezers (8, 25 ). These studies will link with the others of this project program grant to elucidate the fundamental design principles of the elasticity of VIF networks.

细胞的机械特性从根本上构成了所有细胞行为的基础。细胞必须支持力、必须施加力并且必须对力作出反应 (1-5)。此外，虽然细胞内的遗传反应最终提供了控制机制，但细胞的机械反应决定了其在更大的有机体中的主要功能。如果不能承受环境的力量，细胞就根本无法发挥作用。细胞的机械特性在很大程度上由细胞骨架内的三个丝状网络、肌动蛋白、微管和中间丝 (IF) 决定 (1)。虽然肌动蛋白网络和微管已得到相当深入的研究，但 IF 网络的情况并非如此，其研究明显落后于其他网络 (6, 7-9)。事实上，有人提出，IF 网络对于确定几乎所有脊椎动物细胞的机械特性和机械转导至关重要。 然而，几乎没有直接证据支持这个提议的 IF 函数。细胞内的中频网络被认为能够承受非常大的应变；这通常可以远远超过 100% (6, 7, 10) 此外，IF 网络表现出明显的应变硬化，在拉伸时实际上变得更加坚硬 (6)。 然而，细胞内环境高度异质且复杂，使得确定这些网络的潜在机械特性变得极其困难。 IF 非常动态，并且不断地被重塑和重新组装，大概是由沿着细胞中的微管或肌动蛋白丝运行的马达以某种方式驱动，并且这些马达必须引导 VIF 的组装。据推测，还存在调节和控制 VIF 特性的相关蛋白，并提供网络与细胞内周围网络以及 VIF 网络本身的交联 (11-16)。 然而，细胞内 VIF 行为的复杂性和丰富性在控制大部分功能的同时，也使得阐明其基本特性变得更加困难。此外，它无法以允许确定网络基本设计原则的方式测量机械特性。因此，该计划项目这一部分的总体目标是在更受控的环境中测量 VIF 的特性，从而使我们能够阐明它们在建立和调节细胞机械特性中的作用 (17)。 这里提出的工作将从详细研究波形蛋白中间丝 (VIF) 网络的特性开始，该网络可以在细菌中表达，使我们能够产生足够的定量以将蛋白质重建为网络，并对机械强度进行详细测量。这些网络的属性。这些测量将使用传统的本体流变学进行 (18)。此外，我们将开发几种基于多粒子跟踪的新测定方法，测量嵌入网络中的小示踪粒子的运动，并受到热搅拌或由磁场控制的外部施加力的影响。这些示踪粒子的运动将使用微流变学的形式来解释，以测量网络的弹性和粘性特性。我们将研究多价阳离子的生理浓度在调节网络中的作用 (6)。此外，我们将与高盛实验室合作，研究磷酸化在调节 VIF 网络弹性中的作用 (19, 20)。我们还将从我们的合作者 Harald Herrmann 处获得波形蛋白突变体的构建体，并使用它们在细菌中表达突变体 (21-23)。这将使我们能够阐明这些 VIF 网络弹性的基本设计原则。为了补充这些重建网络的研究，我们还将形成“幽灵”，其中大部分细胞蛋白被洗涤剂洗掉，几乎完整的 IF 网络完好无损 (24)。通过在这些网络中植入探针粒子，我们将测量它们的弹性特性并与重构网络的弹性特性进行比较。这将直接探讨这些 VIF 网络对细胞弹​​性的贡献。重要的是，这些还将使我们能够直接测量网络对剪切的响应；在准备幽灵之前，细胞将被剪切，使我们能够探测由于剪切而导致的 VIF 网络结构和力学的变化。此外，我们还将这些粒子跟踪测量扩展到活细胞：我们将向细胞注入示踪粒子，并测量这些粒子由于细胞内的内部分子马达和外力（通过磁力施加）而产生的运动。现场或使用光镊 (8, 25)。这些研究将与该项目计划资助的其他研究联系起来，以阐明 VIF 网络弹性的基本设计原则。