Cell-Matrix Interactions and Migration

细胞-基质相互作用和迁移

基本信息

批准号：
7967049
负责人：
Kenneth Yamada
金额：
$ 69.33万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/7967049
关键词：
Actins Actomyosin Address Adhesions Affect Belief Biochemical Cell Culture Techniques Cell physiology Cell-Matrix Junction Cells Cellular Morphology Cellular Structures Centrosome Chimera organism Chimeric Proteins Complex Cytoskeletal Modeling Cytoskeletal Proteins Cytoskeleton Development Disease Environment Extracellular Matrix Family Fibroblasts Fibronectins Fluorescence Recovery After Photobleaching Genes Goals Golgi Apparatus Guanosine Triphosphate Phosphohydrolases Health Human Development Image Imaging Techniques Immigration Integrins Knowledge Length Life Link Malignant Neoplasms Mediating Microscopy Microtubules Molecular Molecular and Cellular Biology Myosin ATPase Myosin Type II Nonmuscle Myosin Type IIA Physiological Play Procedures Process Protein Dynamics Protein Isoforms Publishing Regulation Role Signal Pathway Signal Transduction Structure Subcellular structure Surface System Testing Time Tissue Engineering Tissues Total Internal Reflection Fluorescent Wound Healing base cell motility cellular imaging directional cell embryonic stem cell extracellular in vivo inhibitor/antagonist interest migration molecular dynamics mutant novel strategies prevent programs receptor response rho rho GTP-Binding Proteins surface coating tool two-dimensional

项目摘要

Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute in complex fashion to cell migration and signaling. We are addressing the following questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell video or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated extracellular and intracellular molecules in the mechanisms and topographical regulation of cell migration. Cell and tissue dynamics occur in three-dimensional (3D) environments in vivo. We have established the importance of one-dimensional (1D) migration for understanding 3D migration. Using a newly developed procedure termed micro photoablation, we demonstrated that cell migration through aligned fibrillar 3D cell-derived matrices is readily mimicked by simple 1.5 micron-wide micropatterned lines in a process we term 1D migration. We showed that many aspects of 3D fibrillar cell migration, including spindle cell morphology, migration velocity (both increased in 1D and 3D compared to 2D), cytoskeletal organization (both actin- and microtubule-based), centrosome and Golgi orientation, and responses to contractile inhibitors are reproduced much more effectively by this system with 1D fibrillar topography versus the traditional two-dimensional surfaces used for cell culture. A particularly interesting finding involves differences in the cellular responses to inhibitors of contractility during cell migration under 1D, 2D, and 3D conditions. Treatment of fibroblasts with inhibitors of actomyosin contractility on 2D fibronectin-coated surfaces leads to a nominal increase in cell migration velocity and cell spreading, whereas the same treatment of cells plated on 1D (fibronectin-coated) or 3D fibrillar cell-derived matrix substantially inhibits migration greater than two-fold. Based on these observations, together with our finding that adhesions to the underlying substratum in 1D form a unique, lengthy adhesion structure unlike that found on 2D surfaces, we are testing whether changing the topography and physical structure of cell-ECM adhesions affects the basic morphological and biochemical mechanisms proposed to mediate cell migration. In order to quantify the dynamics of proteins potentially comprising a clutch-like mechanism implicated in cell-matrix interactions, we are currently analyzing fluorescence recovery after photobleaching (FRAP) of GFP-linked fusion proteins together with other live-cell imaging techniques (Spinning Disk and TIRF microscopy) to track the dynamics of the proteins involved in the postulated molecular clutch to determine how 1D ECM enhances cell migration. More generally, we feel that studying cells migrating in 1D will provide a powerful new tool for analyzing the molecular mechanisms of cell migration, because the components of the molecular machinery are arrayed linearly along the length of a steadily migrating cell that remains oriented in a single direction. Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. We and others recently published studies on the roles of the major myosin II genes, myosin IIA and IIB. We found that myosin IIA plays central roles in fibroblast and embryonic stem cell contractility, actin cytoskeletal organization, and organization of cell-matrix adhesions. Unexpectedly and in contradiction to the belief that myosin II molecules are essential for cell migration, we showed that myosin IIA is not required, and in fact it serves as a brake on migration in 2D cell culture. We also found strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. For technical reasons, it was necessary in our original study to use standard 2D culture systems for visualizing cytoskeletal dynamics and Rho GTPase functions. We had also previously established a key role for Rac in helping to regulate directional cell migration in such 2D settings. Two new projects are testing whether these principles of myosin II crosstalk and Rho family GTPase functions in migration are correct in 1D, 2D, and 3D cell migration systems. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosin and microtubules. All of these processes will need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.

整联蛋白，细胞外基质分子和细胞骨架蛋白以复杂的方式有助于细胞迁移和信号传导。我们正在解决以下问题： 1。哪些亚细胞结构和信号通路对于有效的细胞迁移很重要？ 2。整联蛋白，细胞外基质和细胞骨架的功能如何整合，它们之间的调节串扰如何协调以产生细胞迁移？我们正在使用各种细胞和分子生物学方法来解决这些问题，包括生化分析，荧光嵌合体以及活细胞视频或共焦延时显微镜。我们已经生成了各种荧光分子嵌合体和细胞骨架蛋白的突变体，这是一项长期程序的一部分，以分析其在整联蛋白介导的过程中的功能。我们一直专注于整合素的功能以及相关的细胞外和细胞内分子在细胞迁移的机理和地形调节中。细胞和组织动力学发生在体内的三维（3D）环境中。我们已经确定了一维（1D）迁移对理解3D迁移的重要性。使用新开发的过程称为微光启动，我们证明了细胞通过对齐的纤维3D细胞衍生的矩阵迁移，很容易通过简单的1.5微米宽的微图案模仿，在一个过程中，我们术语1D迁移。我们表明，3D原纤维细胞迁移的许多方面，包括纺锤细胞的形态，迁移速度（与2D相比，迁移速度增加（在1D和3D中增加），细胞骨架组织（基于肌动蛋白和微管），中心体和Golgi方向以及对收缩抑制剂的响应是更有有效的。用于细胞培养的二维表面。一个特别有趣的发现涉及在1D，2D和3D条件下细胞迁移期间对收缩力抑制剂的细胞反应差异。用肌动蛋白收缩抑制剂在2D纤连蛋白涂层表面上的抑制剂治疗成纤维细胞会导致细胞迁移速度和细胞扩散的名义增加，而在1D（纤维蛋白涂层）上镀的细胞相同的治疗方法（纤维蛋白涂层）或3D纤维细胞细胞细胞衍生的基质均高于两种迁移。基于这些观察结果，以及我们发现，与在2D表面上发现的一维底层的粘附形式形成了独特的，冗长的粘附结构，我们正在测试改变细胞ECM粘附的地形和物理结构是否会影响基本的形态学和生物化学机制，以介绍细胞迁移。为了量化蛋白质的动力学，可能包含与细胞 - 玛质相互作用有关的离合器样机制，我们目前正在分析GFP连接融合蛋白光漂白（FRAP）后的荧光恢复（FRAP），以及其他活细胞成像技术（旋转磁盘和TIRF Microsocy）与Outtul of Dynermits Clotems Clotems Clotems Clotim Clotim compter Motil有关。增强细胞迁移。更普遍地，我们认为研究细胞以1D为单位将为分析细胞迁移的分子机制提供强大的新工具，因为分子机械的成分沿着稳定迁移的细胞的长度线性阵列，该长度保持在单个方向上。非肌肉细胞肌醇和肌动蛋白被认为在细胞迁移以及许多发育和伤口修复过程中起着至关重要的作用，但是主要肌球蛋白IIA基因的作用尚不清楚。我们和其他人最近发表了有关主要肌球蛋白II基因，肌球蛋白IIA和IIB的作用的研究。我们发现，肌球蛋白IIA在成纤维细胞和胚胎干细胞收缩力，肌动蛋白细胞骨架组织以及细胞 - 矩阵粘附的组织中起着核心作用。出乎意料的和矛盾的是，肌球蛋白II分子对于细胞迁移至关重要，我们表明肌球蛋白IIA不需要，实际上它是2D细胞培养中迁移的刹车。我们还发现肌球蛋白IIA和微管动力学之间的强烈交叉调节，这些动力学调节了RAC定位和细胞迁移。出于技术原因，在我们的原始研究中，有必要使用标准的2D培养系统可视化细胞骨架动力学和Rho GTPase功能。我们还以前还确立了RAC在帮助调节这种2D设置中定向细胞迁移的关键作用。两个新项目正在测试肌球蛋白II串扰和RHO家族GTPase在迁移中的这些原理是否正确，在1D，2D和3D细胞迁移系统中是正确的。这些正在进行的关于整联蛋白以及细胞内和细胞外分子在细胞迁移中的功能的研究，我们有能力对早期细胞突起的活细胞分子动力学以及细胞内肌球蛋白和微管的形象。所有这些过程都需要实时并在更生理的1D和3D矩阵环境中并行分析，以便能够理解体内细胞迁移的机制。这种合并的知识应提供新颖的方法来理解，预防或改善细胞在异常发育和癌症中使用的迁移过程。对细胞如何移动和与基质环境相互作用的准确理解也将促进组织工程研究。