Mechanical Regulation of Cell Adhesion

细胞粘附的机械调节

• 批准号: 9572281
• 负责人: Clare Michal Waterman
• 金额: $ 225.05万
• 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/9572281
• 关键词: ANGPTL2 gene; ATP phosphohydrolase; Actins; Actomyosin; Adhesions; Adhesives; Affect; Antigens; Apoptotic; Behavior; Binding; Biosensor; Bulla; Bundling; Calcium; Cell Adhesion; Cell Differentiation process; Cell Nucleus; Cell Survival; Cells; Chimera organism; Chimeric Proteins; Complex; Cues; Cytoskeleton; DNA Damage; DNA Double Strand Break; Data; Dendritic Cells; Development; Environment; Epithelial; Europe; Exclusion; Extracellular Matrix; Extravasation; F-Actin; Family; Focal Adhesions; G Actin; Gene Targeting; Growth; Head; Human; Immune response; Immune system; Inflammatory Response; Integrins; Intestines; Lead; Ligands; Light; Link; MYLK gene; Magnetism; Maintenance; Mechanics; Mediating; Melanoma Cell; Mesenchymal; Metastatic Neoplasm to the Lung; Microbe; Microfilaments; Microscopy; Mitogen-Activated Protein Kinases; Molecular; Morphogenesis; Morphology; Motor; Movement; Mus; Myosin ATPase; Myosin S-2; Myosin Type II; Nature; Neck; Neoplasm Metastasis; Nuclear; Nuclear Envelope; Oncogenic; Optics; Particulate; Pathway interactions; Perception; Phagocytes; Phagocytosis; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Plasticizers; Play; Positioning Attribute; Process; Property; Protein Tyrosine Kinase; Proteins; Proteomics; Publications; Publishing; Recruitment Activity; Regulation; Reporting; Rho-associated kinase; Role; Sentinel; Shapes; Signal Pathway; Signal Transduction; Small Interfering RNA; Structure; Surface; System; T-Lymphocyte; Tail; Testing; Tissues; Traction; Transcription Coactivator; Up-Regulation; Work; base; biophysical properties; cancer cell; cell motility; fluorescence imaging; live cell imaging; macrophage; mechanical force; mechanical properties; mechanotransduction; melanoma; migration; neoplastic cell; non-muscle myosin; overexpression; particle; pressure; prevent; protein complex; receptor; residence; sarcoma; screening; src-Family Kinases; symposium; tumor; uptake

Project 1: FMN2 makes perinuclear actin to protect nuclei during confined migration and promote metastasis. Colleen Skau Bob Fischer Hawa Thiam Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas, and show that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration, and thus promote cancer metastasis. This work was published in Cell Project 2:Mechanosensing by integrins regulates signaling and actin dynamics during phagocytosis Valentin Jaumouille Tissue resident phagocytes, such as macrophages and dendritic cells, act as sentinels of the immune system. They play a major role in the clearance of large particulate material, such as apoptotic cells and microbes. Depending on the nature of the particle they engulf, macrophages and dendritic cells will initiate an inflammatory response and present antigens to T lymphocytes. Phagocytosis depends on the reorganization of the actin cytoskeleton, driven by surface receptors. Although multiple signaling pathways have been identify, little is known about the molecular mechanisms underlying the formation of signaling complexes by the receptors, how actin reorganization is adjusted to the target biophysical properties, the mechanical forces involved in the uptake and whether they affect the downstream immune responses. We observed that engagement of 2 integrins by stiff particles lead to the assembly of a molecular platform that can act as a clutch to transduce mechanosensation. Whereas it has been previously established that 2-mediated phagocytosis of soft particles is independent of tyrosine kinases, we found that engulfment of stiff particles requires Src family kinases, Syk and the Arp2/3 complex. Live cell imaging reveals how target mechanical properties regulate actin dynamics in macrophages. This work was presented at two Gordon conferences and ASCB and several seminars in europe Project 3:Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells. Michelle Baird Bob Fischer The role of non-muscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, however whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A pulses occur in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or MLCK-mediated NM2 regulatory light chain phosphorylation, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. This work was published in Mol. Biol. Cell Project 4: YAP nuclear localization in the absence of cell-cell contact is mediated by a filamentous actin-dependent, myosin II- and phospho-YAP-independent pathway during ECM mechanosensing Arupratan Das YAP and TAZ transcriptional co-activators mediated up regulation of the target genes are responsible for development, tumor formation and cell differentiation. Nuclear exclusion of YAP through Hippo signaling mediated phosphorylation at S112 residue is well characterized. Actin being shown as common regulator of both Hippo signaling dependent as well as in Hippo independent regulation of YAP. Cellular perception of mechanical environment and regulation of YAP nuclear localization through actin is reported to determine differentiation fate. Here we showed that actomyosin contractility suppresses YAP phosphorylation at S112 residue however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. Essential player for YAP nuclear localization is F-actin, which even triggers pS112-YAP nuclear localization independent of myosin contractility. Such actin mediated regulation is also conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced F/G actin ratio leading to nuclear exclusion of both YAP and pS112-YAP. These data provide evidences for actomyosin contractility and phosphorylation independent regulation of YAP nuclear localization. In support to the physiological relevance, this study might help to explain reported observations where YAP dependent intestinal tissue growth persisted even in the activation of Hippo signaling and YAP phosphorylation at S112 residue This work was published in J. Biol. Chem. Project 5: Jeremy Logue and Richard Chadwick Abstract Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large leader bleb. Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement. This project resulted in 3 publications Project 6: Functional Behavior of Overexpressed Fusion Proteins in Melanoma Cells Under Confinement Mediating Leader Bleb Based Motility Greg Adams Cell movement is mediated by a remarkably plastic array of morphologies. Cancer cells migrating in confined 3D environments mimicking tissue microenvironment can switch between modes of migration depending on the degree of confinement and the availability of adhesive ligand. Under high confinement and low adhesion, cells undergo a mesenchymal-to-amoeboid switch and take on a highly stereotypical morphology with fast, persistent movement termed leader bleb-based migration (LBBM). The morphology is characterized by formation of a large ( 20.5 um) sausage-shaped bleb that points in the direction of movement (the leader bleb) separated from a smaller ( 12.6 um) sphe

项目1：FMN2使核周肌动蛋白在受关闭的迁移过程中保护核并促进转移。 Colleen Skau Bob Fischer Hawa Thiam 受限制的3D组织微环境中的细胞迁移对于正常生理功能和肿瘤细胞的传播至关重要。我们发现了一种细胞骨架结构，可防止在狭窄的微环境中迁移期间对核的损害。形成型肌动蛋白丝核定核FMN2与核周肌动蛋白/局灶性粘附（FA）系统相关联，该系统与先前表征的肌动蛋白/FA结构不同。该系统控制在2D表面迁移的细胞中的核形状和定位。在受限的3D微环境中，FMN2通过限制核包膜损伤和DNA双链断裂来促进细胞存活。我们发现FMN2在人黑色素瘤中被上调，并表明小鼠黑色素瘤细胞中FMN2的破坏会抑制其渗出和转移到肺部。我们的结果表明，FMN2在生成核周肌动蛋白/FA系统中的关键作用，该系统可保护细胞核和DNA免受损害，以促进受封闭迁移期间的细胞存活，从而促进癌症转移。 这项工作发表在细胞中 项目2：整联蛋白通过吞噬作用调节信号传导和肌动蛋白动力学的机械感应 Valentin Jaumouille 组织常驻的吞噬细胞，例如巨噬细胞和树突状细胞，充当免疫系统的哨兵。 它们在清除大颗粒物材料（例如凋亡细胞和微生物）中起着重要作用。 根据它们吞噬的粒子的性质，巨噬细胞和树突状细胞将引发炎症反应并呈现对T淋巴细胞的抗原。 吞噬作用取决于由表面受体驱动的肌动蛋白细胞骨架的重组。 尽管已经确定了多个信号通路，但对受体信号复合物形成的分子机制知之甚少，如何将肌动蛋白的重组调整到靶标生物物理特性，摄取中涉及的机械力以及它们是否影响下游免疫反应。 我们观察到，刚性颗粒将2种整合素的接合导致分子平台的组装，该平台可以充当传输机械敏的离合器。 尽管以前已经确定，软颗粒的2介导的吞噬作用与酪氨酸激酶无关，但我们发现僵硬的颗粒需要SRC家族激酶，SYK和ARP2/3复合物。 活细胞成像揭示了靶向机械性能如何调节巨噬细胞中的肌动蛋白动力学。 这项工作在欧洲的两个戈登会议和ASCB和几个研讨会上介绍 项目3：局部搏动性收缩是粘合细胞皮质细胞骨架中肌球蛋白2a运动的固有特性。 Michelle Baird Bob Fischer 已经表征了非肌肉肌球蛋白2（NM2）搏动动力学在产生发育形态发生所需的收缩力中的作用，但是这些脉动收缩是否是所有肌动蛋白网络的固有特性。 在这里，我们使用了活细胞荧光成像，以表明NM2A脉冲的瞬时组装出现在间质，上皮和肉瘤起源的单个粘附细胞的皮质细胞骨架中，独立于发育信号线索和细胞细胞或细胞-ECM相互作用。我们表明，皮质细胞骨架中的脉冲需要RHO相关激酶或MLCK介导的NM2调节轻链磷酸化，胞质钙和NM2 ATPase活性的增加。 令人惊讶的是，我们发现皮质细胞骨架脉冲特别需要NM2A的头部结构域，因为它们在NM2B或2b-head-2a-2a-tail嵌合体中都不发生。 因此，我们的结果表明，皮质细胞骨架中的脉冲收缩是NM2A电动机的内在特性，可以介导其在粘附细胞的皮质细胞骨架中张力张力中的作用。 这项工作发表在摩尔中。生物。细胞 项目4：在没有细胞细胞接触的情况下，YAP核定位是由丝状肌动蛋白依赖性，肌球蛋白II-和磷酸-YAP独立途径在ECM机械传感器期间介导的 Arupratan Das YAP和TAZ转录共激活因子介导的靶基因的调节是导致发育，肿瘤形成和细胞分化的原因。通过HIPPO信号传导在S112残基处介导的磷酸化的核排除是很好的特征。肌动蛋白作为河马信号依赖性以及河马独立调节YAP的常见调节剂。据报道，通过肌动蛋白对机械环境的细胞感知和对YAP核定位的调节，以确定分化的命运。在这里，我们表明肌动球蛋白的收缩力抑制了S112残基处的YAP磷酸化，但是，YAP磷酸化的收缩力损失和增加均不足以使其核排斥。 YAP核定位的重要参与者是F-肌动蛋白，甚至触发了独立于肌球蛋白收缩性的PS112-YAP核定位。这种肌动蛋白介导的调节在机械转导过程中也是保守的，因为底物的依从性增加了YAP磷酸化并降低了f/g肌动蛋白比，从而导致YAP和PS112-YAP的核排除。这些数据为肌动球蛋白的收缩力和磷酸化的独立调节提供了证据。为了支持生理相关性，这项研究可能有助于解释报道的观察结果，即即使在S112残基上激活HIPPO信号传导和YAP磷酸化的激活中，依赖于YAP的肠道组织的生长也持续存在 这项工作发表在J. Biol。化学 项目5：杰里米·洛格（Jeremy Logue）和理查德·查德威克（Richard Chadwick） 抽象的 在组织的范围内，癌细胞可以使用泡沫来迁移。 EPS8是一种肌动蛋白捆绑和封盖蛋白，其封闭活性被ERK抑制，ERK是一种由致癌信号传导激活的关键MAP激酶。 我们检验了以下假设：EPS8充当ERK效应子来调节肌动蛋白皮层力学，从而介导基于BLEB的癌细胞迁移。 限制在非粘附环境中的细胞朝着非常大的领导者爆炸的方向迁移。 EPS8捆绑活性促进皮层张力和细胞内压力驱动领导者的BLEB形成。 EPS8封盖和捆绑活动在拮抗领导者中组织肌动蛋白的作用，ERK介导了这种效果。 ERK生物传感器揭示了领导者泡沫中的浓缩激酶活性。 BLEB含量被狭窄的颈部捕获，该颈部将引导液与细胞体分开。 因此，ERK活性促进了EPS8捆绑的肌动蛋白，以增强皮质张力并在非粘附性限制下驱动癌细胞的基于BLEB的迁移。 该项目产生了3个出版物 项目6：在限制介导引导者BLEB运动的黑色素瘤细胞中过表达融合蛋白的功能行为 格雷格·亚当斯（Greg Adams） 细胞运动是由形态的塑性阵列介导的。 在模仿组织微环境的约束3D环境中迁移的癌细胞可以根据限制程度和粘合剂配体的可用性在迁移模式之间进行切换。 在高限制和低粘附力下，细胞会经历间充质到木质造成的开关，并进行高度刻板的形态，并具有快速，持续的运动称为领导者基于BLEB的迁移（LBBM）。形态的特征是形成了一个大的（20.5 um）香肠形状的泡沫，指向移动方向（领导者BLEB）与较小（12.6 um）SPHE分离