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的独特物理性质，包括高柔韧性与非常高的抗断裂性的组合，导致在大应变范围内的应变硬化流变学，其可以与更硬但更脆的交联肌动蛋白网络联合收割机结合，特别是在细胞皮层，以高度局部化和快速可调的方式控制细胞力学（1，2）。图1显示了肌动蛋白网络（4 mg/ml）在小应变下如何被细丝蛋白交联，但在应变小于20%时仍然断裂。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表现出线性弹性。VIF网络的非线性弹性意味着当张力通过外部或内部产生的应力施加到细丝股线时，这些网络会弯曲。由动力蛋白和驱动蛋白马达（3）介导的VIF和微管之间的活性界面可以产生内部预应力，使VIF网络和整合的细胞骨架变硬。大多数皮质细胞刚度的研究强调了肌动蛋白网络的贡献，因为在低应变，在体外交联肌动蛋白形成最硬的网络，因为肌动蛋白是最集中的焦点粘附位点和细胞表面的探针，如光学和磁性操纵珠，原子力显微镜的提示，和校准的玻璃纤维连接。即使在设计用于测试细胞表面或板状伪足附近的刚度的测量中，肌动蛋白和微管被latrunculin和秋水仙素解聚也不会使细胞的弹性模量降低超过一个因子。 为3.来自波形蛋白缺失小鼠的细胞比来自野生型动物的相应细胞软40%， 通过磁扭转流变仪（4）测量。与VIF的应变刚化效应一致，刚度 WT和VIM“细胞之间的差异随着变形的增加而增加（5）。这种对全局细胞的影响 刚度与最近的多尺度模拟研究一致（6）。氧化LDL增加人内皮细胞硬度的发现与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硬度相关的变化。 VIF对细胞刚度的贡献不需要简单地从纯VIF网络的流变学产生，因为在细胞中VIF与肌动蛋白丝和微管交错，并且在细胞骨架的拥挤环境中，空间和生化相互作用都有助于机械响应。由VIF和F-肌动蛋白两者形成的复合网络的协同流变响应的实例示于图2中。在此，对于等重量浓度（1 mg/ml）的纯化的F-肌动蛋白丝类型，绘制由增加的应变引起的应力。在这些相对较低的浓度下，F-肌动蛋白和波形蛋白形成弱网络，但它们的组合比其部分的总和强得多（8），应力-应变图的向上弯曲说明了IF网络随着变形特性的增加而变硬（1）。在较高的细胞浓度的F-肌动蛋白和波形蛋白（一般超过10毫克/毫升）的局部硬化，特别是在大。