Physical Approaches for Probing the Mechanical Properties of Intermediate Filamen

细胞的机械特性是所有细胞行为的根本基础；细胞必须支撑力，必须施加力，并且必须对力做出反应(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它在较大有机体中的主要功能；如果不能承受环境的力量，细胞将根本无法发挥作用。细胞的机械性能在很大程度上由细胞骨架、肌动蛋白、微管和中间丝(IF)内的三个丝状网络决定(1)。虽然肌动蛋白网络和微管已经被研究得相当好，但IF网络的情况并非如此，它的研究明显落后于其他网络(6，7-9)。事实上，已经有人提出IF网络在决定几乎所有脊椎动物细胞的机械特性和机械转导中是必不可少的。 然而，几乎没有直接证据支持这一拟议的IF功能。细胞内的IF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，当它们被拉伸时，实际上变得更加坚硬(6)。 然而，细胞内的环境是高度异质和复杂的，这使得确定这些网络的潜在力学性质变得极其困难。IF是非常动态的，并且不断地被重塑和重新组装，推测是由沿着细胞中的微管或肌动蛋白细丝运行的马达以某种方式驱动的，这些马达必须引导VIF的组装。据推测，还存在调节和控制VIF特性的相关蛋白质，并提供细胞内和VIF网络本身内的网络与周围网络的交叉连接(11-16)。 然而，VIF在细胞内的行为的复杂性和丰富性，尽管控制了大部分功能，也使得阐明基本属性变得更加困难；此外，它排除了以允许确定网络基本设计原则的方式测量机械属性的可能性。因此，本计划项目这一部分的首要目标是在更可控的环境中测量VIF的性能，从而使我们能够阐明它们在建立和调节细胞机械性能方面的作用(17)。 这里提出的工作将从详细研究波形蛋白中间丝(VIF)网络的性质开始，VIF可以在细菌中表达，使我们能够产生足够的数量来将蛋白质重组成网络，并对这些网络的机械性能进行详细的测量。这些测量将使用传统的整体流变学进行(18)。此外，我们将开发几种基于多粒子跟踪的新分析方法，即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作用力的影响。这些示踪剂颗粒的运动将用微观流变学的形式来解释，以测量网络的弹性和粘性。我们将研究生理浓度的多价阳离子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那里获得波形蛋白突变的结构，并将使用这些结构在细菌中表达突变(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。为了补充对重组网络的这些研究，我们还将形成“幽灵”，在那里，大多数细胞蛋白被洗涤剂冲走，几乎保留完整的IF网络(24)。通过用探测粒子播种这些网络，我们将测量它们的弹性性质，并与重组网络的弹性性质进行比较。这将为这些VIF网络对细胞弹性的贡献提供一个直接的探测。重要的是，这些也将使我们能够直接测量网络对剪切的响应；细胞将在准备重影之前被剪切，使我们能够探索由于剪切而对VIF网络的结构和机制的修改。此外，我们将把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的运动，施加磁场或光钳(8，25)。这些研究将与本项目资助的其他研究相结合，以阐明VIF网络弹性的基本设计原则。

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.