Regulation of the Micromechanical Properties of Cells by Intermediate Filaments

中间丝对细胞微机械性能的调节

• 批准号: 8142486
• 负责人: Paul A Janmey
• 金额: $ 27.23万
• 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/8142486
• 关键词: Actins; Affect; Animals; Antibodies; Atomic Force Microscopy; Biochemical; Biological Assay; Cell Culture Techniques; Cell Shape; Cell Volumes; Cell surface; Cells; Cellular Morphology; Characteristics; Chemicals; Colchicine; Complement; Crowding; Cytoskeleton; Dominant-Negative Mutation; Dynein ATPase; Elasticity; Elements; Endothelial Cells; Exhibits; F-Actin; Fibroblasts; Filament; Focal Adhesions; Frequencies; Gel; Generations; Genetic Transcription; Human; Hydrogels; Image; Imagery; In Vitro; Intermediate Filaments; Kinesin; Knockout Mice; Lateral; Lead; Life; Liquid substance; Magnetism; Measurement; Measures; Mechanical Stress; Mechanics; Mediating; Microfilaments; Microtubules; Modification; Motor; Mutation; Null Lymphocytes; Phosphorylation; Phosphorylation Site; Property; Proteins; Regulation; Resistance; Retroviral Vector; Reverse Transcriptase Polymerase Chain Reaction; Rheology; Role; Rupture; Scanning Probe Microscopes; Side; Signal Transduction; Site; Stress; Stretching; Sum; Testing; Traction; Vertebrates; Vimentin; Weight; cantilever; cell cortex; cell motility; cell type; crosslink; depolymerization; design; fiberglass; filamin; flexibility; fluorophore; human disease; in vivo; instrumentation; novel; oxidized low density lipoprotein; physical property; polyacrylamide; programs; response; shear stress; simulation; soft tissue; submicron; viscoelasticity

The unique physical properties of VIF, including the combination of high flexibility with very high resistance to breakage, lead to strain-stiffening rheology over a large strain range that can combine with the stiffer, but more brittle, crosslinked actin network, especially at the cell cortex, to control cell mechanics in a highly localized and rapidly tunable manner (1,2). Figure 1 shows how actin networks (4 mg/ml) crosslinked by filamin stiffen at small strains, but still break at strains less than 20%. Vimentin networks continue to strain-stiffen at large strains and resist breakage. Synthetic hydrogels like polyacrylamide exhibit only linear elasticity. The nonlinear elasticity of VIF networks means that these networks stiffen when tension is applied to the filament strands either by externally or internally generated stresses. The active interface between VIF and microtubules mediated by both dynein and kinesin motors (3) can generate internal pre-stress that stiffens VIF networks and the integrated cytoskeleton. Most studies of cortical cell stiffness have emphasized the contribution of actin networks, because, at low strains, in vitro crosslinked actin forms the stiffest networks and because actin is most concentrated at focal adhesion sites and the cell surface where probes such as optically and magnetically manipulated beads, atomic force microscope tips, and calibrated glass fibers attach. Even in measurements designed to test the stiffness near the cell surface or lamellipodium, depolymerization of actin and microtubules by latrunculin and colchicine does not decrease the cell's elastic modulus more than a factor of 3. Cells from vimentin null mice are 40% softer than corresponding cells from wild type animals when measured by magnetic twisting rheometry (4). Consistent with the strain-stiffening effect of VIF, stiffness differences between wt and vim''' cells increase with increasing deformation (5). This effect on global cell stiffness is consistent with a recent multi-scale simulation study (6). The finding that oxidized LDL increases human endothelial cell stiffness coincident with a reorganization of the VIF network (7) suggests that local and temporal changes in VIF contribute to the stiffness-related changes in human disease. The contribution of VIF to cell stiffness need not result simply from the rheology of pure VIF networks since in the cell VIFs interdigitate with actin filaments and microtubules and, in the crowded context of the cytoskeleton, both steric and biochemical interactions contribute to the mechanical response. An example of the synergistic rheological response of composite networks formed by both VIF and F-actin is shown in Figure 2. Here the stress resulting from increasing strain is plotted for equal weight concentrations (1 mg/ml) of purified F-actin filament types. At these relatively low concentrations F-actin and vimentin form weak networks, but their combination is much stronger than the sum of its parts (8), and the upward curvature of the stress-strain plot illustrates the stiffening with increasing deformation characteristic of IF networks (1). At the higher cellular concentrations of F-actin and vimentin (generally more than 10 mg/ml) the local stiffening, especially at large.

VIF的独特物理性能，包括高柔韧性和非常高的抗断裂能力，导致在大应变范围内的应变硬化流变学，可以与更坚硬但更脆弱的交联型肌动蛋白网络结合，以高度局部化和快速可调的方式控制细胞力学(1，2)。图1显示了细丝蛋白交联的肌动蛋白网络(4 mg/ml)如何在小的应变下变硬，但在低于20%的应变下仍然断裂。波形蛋白网络在大应变下继续应变硬化，并抵抗断裂。像聚丙烯酰胺这样的合成水凝胶只显示出线状弹性。VIF网络的非线性弹性意味着当通过外部或内部产生的应力对纤维股施加张力时，这些网络会变得坚硬。VIF和微管之间的活跃界面由动力蛋白和动蛋白马达(3)共同介导，可以产生内部预应力，使VIF网络和完整的细胞骨架变得坚硬。大多数关于皮质细胞硬度的研究都强调了肌动蛋白网络的作用，因为在低应变下，体外交联的肌动蛋白形成了最坚硬的网络，而且因为肌动蛋白最集中在焦点黏附位置和细胞表面，探针如光学和磁力操纵的珠子、原子力显微镜尖端和校准的玻璃纤维。即使在设计用于测试细胞表面或片层基质附近的硬度的测量中，用长春新素和秋水仙素解聚肌动蛋白和微管也不会使细胞的弹性模数降低超过一个因素。 波形蛋白缺失小鼠的细胞比野生型动物的相应细胞柔软40% 用磁扭转流变仪测量(4)。与VIF的应变硬化效应一致，刚度 Wt和vim‘’细胞之间的差异随着变形的增加而增大(5)。这对全局小区的影响 刚度与最近的多尺度模拟研究一致(6)。氧化的低密度脂蛋白增加了人内皮细胞的僵硬，这一发现与VIF网络的重组一致(7)，表明VIF的局部和时间变化有助于人类疾病中与僵硬相关的变化。 VIF对细胞硬度的贡献不一定简单地来自于纯VIF网络的流变学，因为在细胞中，VIF与肌动蛋白细丝和微管交错，在拥挤的细胞骨架环境中，空间和生化相互作用都有助于机械反应。图2显示了VIF和F-肌动蛋白形成的复合网络的协同流变响应的一个例子。在这里，对于相同重量浓度(1毫克/毫升)的纯化的F-肌动蛋白细丝类型，绘制了由于应变增加而产生的应力。在这些相对较低的浓度下，F-肌动蛋白和波形蛋白形成了弱网络，但它们的结合比其各部分之和强得多(8)，应力-应变曲线的向上曲率说明了IF网络的硬化和变形增加的特征(1)。当F-肌动蛋白和波形蛋白的细胞浓度较高时(一般大于10 mg/ml)，局部僵硬，尤其是广泛的。