Mechanical Regulation of Cell Adhesion

细胞粘附的机械调节

• 批准号: 8939797
• 负责人: Clare Michal Waterman
• 金额: $ 143.45万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939797
• 关键词: Actins; Actomyosin; Adhesions; Agreement; Atomic Force Microscopy; Back; Behavior; Binding; Biological Assay; Biological Process; Calpain; Cell Adhesion; Cell membrane; Cell physiology; Cells; Cellular biology; Chemotaxis; Complex; Cues; Cytoskeleton; Differentiation and Growth; Distal; Endocytosis; Environment; Eukaryotic Cell; Exhibits; Extracellular Matrix; Focal Adhesions; Guanine Nucleotide Exchange Factors; Integrins; Life; Light; Link; Magnetism; Mechanics; Mediating; Methods; Microscopy; Modeling; Molecular; Monomeric GTP-Binding Proteins; Motion; Myosin ATPase; Myosin Type II; National Heart, Lung, and Blood Institute; Neoplasm Metastasis; Nonmuscle Myosin Type IIA; Optics; PTK2 gene; Paper; Phosphorylation; Physiological; Positioning Attribute; Process; Property; Proteins; Proteome; Proteomics; Publications; Publishing; Recruitment Activity; Regulation; Relaxation; Research Institute; Resolution; Role; Signal Transduction; Small Interfering RNA; Stress; Stress Fibers; Tail; Testing; Time; Traction; Transfection; Translating; Tyrosine; Variant; Vinculin; Work; angiogenesis; cell behavior; cell motility; cellular imaging; mechanical behavior; migration; mimetics; mutant; neurogenesis; paxillin; polyacrylamide; protein complex; receptor; residence; response; rho; screening

Project 1: Rac1 induces PCK-dependent myosinIIA phosphorylation to regulate association with focal adhesions and cell migration. Pasapera AM1, Fischer RS1,Plotnikov SV1,Egelhoff T2, Waterman CM1. Cell Biology and Physiology Center, NHLBI, NIH1;Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic.2 Cell migration requires coordinated assembly of focal adhesions and contraction in the actomyosin cytoskeleton. The small GTPase Rac1 is critical to cell migration through its known functions in regulation of focal adhesion and actin cytoskeletal assembly dynamics, but its role in regulation of myosin II is not known. Myosin II dynamically assembles into minifilaments at the leading edge of migrating cells, and PKC-mediated phosphorylation in Ser 1916 in the non-helical tail is one of the main regulators. We hypothesized that Rac1 may regulate myosin II minifilament assembly dynamics during cell migration via downstream regulation of PKC and Ser 1916 phosphorylation. To test this, we analyzed the effects of Rac1 activation on the phosphorylation and dynamics of myosin IIA in U2OS cells. We found that transfection of active Rac1 (Rac1V12) induced PKC- and integrin-dependent myosin IIA phosphoryation on Ser 1916. Live cell imaging of GFP-myosin IIA revealed that Rac1 activation promotes rapid assembly, motion, and turnover of myosin IIA minifilaments, as well as perpendicular orientation to the leading edge, resulting in its accumulation specifically in focal adhesions. To determine the role of Ser 1916 phosphorylation on myosin IIA dynamics and localization, we expressed phospho-mimetic (S1916D) and non-phosphorylatable mutants (S1916A) of myosin IIA. This showed that phosphorylation is critical to the Rac1-induced rapid assembly and turnover of myosin IIA minifilaments as well as to the focal adhesion association of myosin IIA. Thus, Rac1 acts as a master regulator of cell migration by coordinating actin assembly-mediated protrusion, adhesion, and actomyosin contraction dynamics. This work is being prepared for publication Project 2: PROTEOMIC ANALYSIS OF FOCAL ADHESION MATURATION Proteomic Analysis of Myosin II-mediated Focal Adhesion Maturation Reveals a Role for -Pix in Relaxation-mediated Rac1 Activation J. Kuo, X. Han, C.T Hsiao, J. Yates, C. M. Waterman Focal adhesions (FAs) undergo contraction-mediated maturation wherein they grow and change composition to differentially transduce signals from the extracellular matrix to modulate cell migration, growth and differentiation. To determine how FA protein composition is globally modulated by myosinII contraction, we developed a proteomics approach to isolate native FAs, identify their protein composition, and compare specific protein abundance in FAs from cells with and without myosinII inhibition. We reproducibly identified 905 FA-associated proteins, half (402) of which changed in FA abundance in response to perturbation of myosinII activity, thus defining the myosinII-responsive FA proteome. FA abundance of 75% of proteins in the myosinII-responsive FA proteome were enhanced by contractility, including those involved in Rho-mediated FA maturation, stress fiber formation, and endocytosis- and calpain-dependent FA disassembly. Surprisingly, 25% of the myosinII responsive FA proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in FA by myosinII inhibition, establishing for the first time negative regulation of FA protein recruitment by contractility. We focused on the role of the Rac guanine nucleotide exchange factor, -PIX, documenting its depletion from FA during myosin-mediated FA maturation and its role in negative regulation of FA maturation to promote rapid FA turnover, lamellipodial protrusion and fast cell migration. A mthods paper describing our method was published this year. Project 3: Analysis of traction stress variation across single focal adhesions. Sergey V. Plotnikov, Benedikt Sabass, Clare M. Waterman The ability of eukaryotic cells to sense mechanical properties of the extracellular matrix (ECM) and to exhibit durotaxis (directed migration toward stiffer environments) is thought to underly many biological processes including angiogenesis, neurogenesis and cancer metastasis. ECM stiffness sensing is achieved by integrin-mediated focal adhesions (FA), protein assemblies that couple contractile actomyosin bundles to the plasma membrane and transmit force generated by the cytoskeleton to the ECM. Although it has been shown that both structural and signaling components of FA are crucial to translate mechanical cues into cell behavior, the molecular mechanism of mechanosensing remains unknown. Here we demonstrate that a molecular clutch, a mechanical link between the actin cytoskeleton and ECM-engaged integrins, acts as a mechanosensor in FAs, and the strength of the clutch determines range of ECM stiffness cells are able to sense to mediate durotaxis. Using high-resolution traction force microscopy on polyacrylamide ECMs of varying stiffnesses, we found that the mechanical behavior of the integrin-actin interface at FA exhibited ECM stiffness-dependent switching between a load-and-fail compliance sensing regime and a frictional slippage regime as described in the clutch oscillation model (Chan and Odde, 2008). In the load and fail regime, the position of peak traction within the FA resided on average at the distal FA tip, but oscillated over time towards the FA center and back to the tip. As ECM rigidity was increased, the traction peak did not oscillate and remained in the FA center, signifying the frictional slippage regime. We found that perturbing the gradient of paxillin phosphorylation across FA by expressing Y31/118E- or Y31/118F-paxillin mutants or by inhibiting FAK weakened the molecular clutch and switched FAs from load-and-fail compliance sensing to frictional slippage regime on compliant ECMs. In agreement with the clutch oscillation model, the load-and-fail regime could be rescued by further decreasing either substrate stiffness or myosin II contractility. Since paxillin phosphorylation on tyrosine residues 31 and 118 mediates vinculin recruitment into FAs, we demonstrated that vinculin and the paxillin-vinculin interaction are essential to strengthen the molecular clutch and to enable mechanosensing over a wide range of ECM compliances. We demonstrated the physiological importance of the load-and-fail compliance sensing regime by showing a requirement for this FA behavior in durotaxis, but not in chemotaxis in a boyden chamber assay or in random cell migration. This work resulted in 2 publications

项目1：RAC1诱导PCK依赖性肌动脉磷酸化，以调节与局灶性粘连和细胞迁移的关联。 Pasapera AM1，Fischer RS1，Plotnikov SV1，Egelhoff T2，Waterman CM1。 NHLBI，NIH1；细胞生物学和生理中心；克利夫兰诊所Lerner Research Institute NC-10细胞生物学系。2 细胞迁移需要协调组装局灶性粘连和肌动蛋白细胞骨架中的收缩。小型GTPase Rac1对于细胞迁移至关重要，通过其在调节局灶性粘附和肌动蛋白细胞骨架装配动力学方面的已知功能，但尚不清楚其在调节肌球蛋白II中的作用。 肌球蛋白II在迁移细胞的前缘动态组装成微型刚毛，而在非螺旋尾部Ser 1916中PKC介导的磷酸化是主要调节剂之一。 我们假设RAC1可以通过PKC和SER 1916磷酸化来调节细胞迁移过程中肌球蛋白II微型组装动力学。为了测试这一点，我们分析了Rac1激活对U2OS细胞中肌球蛋白IIA的磷酸化和动力学的影响。 我们发现，活性Rac1（Rac1v12）的转染诱导的PKC和整联蛋白依赖性肌球蛋白IIA IIA IIA磷酸化在Ser1916中。GFP-肌球蛋白IIA的活细胞成像，Rac1激活揭示了肌动蛋白IIA Minifilaments的快速组装，运动和失误，例如以及垂直于前缘的垂直方向，导致其在局灶性粘连中的积累。为了确定SER 1916磷酸化在肌球蛋白IIA动力学和定位中的作用，我们表达了肌球蛋白IIA的磷酸模拟（S1916D）和非磷酸磷酸突变突变体（S1916A）。 这表明磷酸化对于Rac1诱导的快速组装和肌球蛋白IIA微型纤维的快速组装以及肌球蛋白IIA的焦点粘附关联至关重要。因此，Rac1通过协调肌动蛋白组装介导的突出，粘附和肌动蛋白收缩动力学来充当细胞迁移的主要调节剂。 这项工作正在准备出版 项目2：局部粘附成熟的蛋白质组学分析 肌球蛋白II介导的焦点粘附成熟的蛋白质组学分析揭示了-pix在弛豫介导的Rac1激活中的作用 J. Kuo，X。Han，C.T HSIAO，J.Yates，C.M。Waterman 局灶性粘连（FAS）经历收缩介导的成熟，在其中它们生长，并且 将组成从差异传递信号从细胞外基质转换为调节 细胞迁移，生长和分化。确定FA蛋白成分在全球范围内如何 通过肌性收缩调节，我们开发了一种蛋白质组学方法来分离天然FAS， 鉴定其蛋白质组成，并比较细胞中Fas中的特定蛋白质丰度 有和没有肌科抑制作用。我们可重复地鉴定出905个FA相关蛋白，一半 （402）响应肌动症活性的响应而变化了FA的丰度 定义肌科反应FA蛋白质组。 FA在该蛋白质中的FA丰度为75％ 肌醇反应的FA蛋白质组通过收缩性增强了，包括参与的蛋白质组 Rho介导的FA成熟，应力纤维形成以及内吞和钙蛋白酶依赖性 FA拆卸。出人意料的是，肌动蛋白的25％反应FA蛋白质组，包括蛋白 参与RAC介导的层状突起，通过肌动脉抑制在FA中富集， 首次通过收缩对FA蛋白募集的负面调节。我们 专注于RAC鸟嘌呤核苷酸交换因子的作用-pix，记录了其 在肌球蛋白介导的FA成熟过程中FA的耗竭及其在负调节中的作用 FA成熟以促进快速的FA更新，层状叶片突出和快速细胞迁移。 一份描述我们方法的文章已于今年发表。 项目3：分析单个局灶性粘附之间的牵引力变化。 Sergey V. Plotnikov，Benedikt Sabass，Clare M. Waterman 人们认为，真核细胞感知细胞外基质（ECM）和表现出杜罗可去的机械性能（直接迁移到更硬环境）的能力被认为是基本的许多生物学过程，包括血管生成，神经发生，神经发生和癌症转移。 ECM刚度传感是通过整联蛋白介导的局灶性粘附（FA），蛋白质组件实现的，将收缩性肌动蛋白捆绑到质膜上，并通过细胞骨架产生的力传递到ECM。尽管已经表明，FA的结构和信号传导成分对于将机械提示转化为细胞行为至关重要，但机械感应的分子机制仍然未知。在这里，我们证明了分子离合器，即肌动蛋白细胞骨架和ECM引进的整联蛋白之间的机械连接，是FAS中的机械传感器，并且离合器的强度决定了ECM刚度细胞的范围能够介导Durotaxis。 使用高分辨率的牵引力显微镜在不同刚度的聚丙烯酰胺ECM上使用，我们发现FA上整联蛋白 - 肌动蛋白界面的机械行为表现出ECM刚度依赖性依赖性依赖性的切换，在负载和耐受的依从性感应方面和摩擦式滑移方案之间显示在离合器振荡模型中描述（Chan and Odde，2008）。 在负载和失败状态下，FA内的峰值牵引力的位置平均在远端FA尖端驻留，但随着时间的推移朝向FA中心并回到尖端。随着ECM刚度的提高，牵引峰不会振荡并留在FA中心，这表示摩擦滑移状态。我们发现，通过表达Y31/118E-或Y31/118F - 帕西林突变体或抑制FAK来削弱paxillin磷酸化的梯度，或者通过抑制FAK削弱了分子离合器，并从负载和脱落的合规性转移到符合良好的ECMS上的FAR和FAB依从性依从性ecms的FAS中。 。与离合器振荡模型一致，可以通过进一步降低底物刚度或肌球蛋白II的收缩性来挽救负载和失败状态。由于酪氨酸残基上的帕西林磷酸化31和118将Vinculin的募集介导为FAS，我们证明了vinculin和paxillin- vinculin的相互作用对于增强分子离合器和启用机械启动在广泛的ECM Appliances中是必不可少的。我们通过表明对Durotaxis的FA行为的要求，但在Boyden室分析中或随机细胞迁移中的趋化性中，我们证明了负载和失败依从性感应制度的生理重要性。 这项工作导致了2个出版物