Regulation of the Micromechanical Properties of Cells by Intermediate Filaments

中间丝对细胞微机械性能的调节

基本信息

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
项目类别：
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

项目摘要

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的独特物理特性，包括高柔韧性与极高的抗性能力的结合，导致较大的应变范围内应变流变学，可以与更硬但更脆的，更脆的，更脆的肌动蛋白网络，尤其是在细胞皮质上，尤其是在细胞皮质上，以高度局部和快速可调节的方式控制细胞机制（1,2）。图1显示了丝蛋白网络（4 mg/ml）如何在小菌株下僵硬地交联，但仍处于小于20％的菌株下破裂。波形蛋白网络继续在大型菌株下施加压力并抵抗破裂。合成水凝胶（如聚丙烯酰胺）仅表现出线性弹性。 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将张力施加到细丝链上时，这些网络会变硬。由动力蛋白和驱动蛋白电动机介导的VIF和微管之间的活性界面（3）可以产生内部预压力，从而使VIF网络和集成的细胞骨架变硬。大多数对皮质细胞僵硬度的研究都强调了肌动蛋白网络的贡献，因为在低菌株下，体外交联肌动蛋白形成最僵硬的网络，并且由于肌动蛋白最集中在局灶性粘附位点和细胞表面上，在该探针上，探针（如光学和磁性操纵珠），原子力显微镜，原子质显微镜，和型玻璃玻璃纤维。即使在旨在测试细胞表面或片状膜片附近的刚度的测量中，latrunculin和corchicine对肌动蛋白和微管的解聚也不会降低细胞的弹性模量，而不是因素。来自波形蛋白无效小鼠的3个细胞比野生型动物的相应细胞柔软40％通过磁性扭曲变进学测量（4）。与VIF的应变效果一致，刚度 WT和VIM“”细胞之间的差异随变形的增加而增加（5）。这种对全球细胞的影响刚度与最近的多尺度模拟研究一致（6）。氧化的LDL的发现增加了人类内皮细胞的刚度与VIF网络的重组（7）一致，这表明VIF的局部和时间变化有助于人类疾病的僵硬相关变化。 VIF对细胞僵硬的贡献不仅仅是纯VIF网络的流变学产生的，因为在细胞VIF中，VIF与肌动蛋白丝和微管相互互动，并且在细胞骨架的拥挤背景下，空间和生物化学相互作用都对机械响应有助于。 VIF和F-肌动蛋白形成的复合网络的协同流变响应如图2所示。此处，由于纯化的F-肌动蛋白丝类型的相等重量浓度（1 mg/ml），绘制了增加菌株引起的应力。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（8）强得多，而应力 - 应变图的向上曲率表明，IF网络的变形特征的增加（1）。在较高的F-肌动蛋白和波形蛋白（通常超过10 mg/ml）的细胞浓度下，局部僵硬，尤其是在大的情况下。