NanoPlan - Deciphering the molecular construction rules of integrin-based cell adhesions.

细胞粘附到细胞外基质（ECM）是许多发育和病理生理过程的基础。它是由整合素受体介导的，整合素受体结合广泛的细胞外配体，并在称为局灶黏附（FAs）的大分子结构中与肌动蛋白细胞骨架连接。FAs是高度复杂的组件，包含数百种不同的蛋白质，在细胞粘附过程中处理化学和机械信号；然而，控制FA组织和机械化学信号传导的分子过程仍然不完全清楚。此外，哺乳动物表达24种不同的整合素受体，具有特定的配体结合和信号特性，但介导整合素受体特异性的潜在机制尚不清楚。更好地理解整合素功能的主要障碍是缺乏适当的技术，无法对FAs进行系统的研究，具有足够的时间和空间分辨率。因此，我们法德联盟的成员已经开发出能够进行这种分析的技术。我们已经建立了独特的细胞系统来研究不同的整合素受体及其生理表达水平的细胞内调节因子，我们已经开发了超分辨率显微镜方法来研究单个FAs蛋白的动态纳米级组织。此外，我们已经开发了质谱方法来研究FAs的分子组成和工程生物传感器，以皮牛顿灵敏度量化细胞中的整合素力转导。我们建议结合我们的工具来研究整合素及其相关分子如何调节FAs的纳米级组织，以整合素亚型特异性的方式调节细胞粘附过程中的化学和机械信号。在该项目的第一部分，将使用单蛋白跟踪和超分辨率显微镜来确定基因修饰细胞中不同整合素亚基的动态。我们将结合超分辨率显微镜和纳米图谱来控制整合素亚型特异性配体的横向间距，并应用分子张力传感器来关联FA纳米级组织与整合素力转导。在第二部分，我们将分析整合素调节剂在FA纳米级组织、分子动力学和力转导中的作用。最后，我们将评估外部施加的力是如何被不同的整合素受体及其相关分子处理的。这些实验将利用定制的细胞拉伸器，允许超分辨率显微镜以及活细胞的分子力测量。总之，所提出的实验应该为FAs的分子结构规则提供前所未有的见解。预期的结果应该是有价值的细胞粘附社区和普遍感兴趣的细胞生物学和生物物理社区。

Cell adhesion to the extracellular matrix (ECM) is fundamental to many developmental and pathophysiological processes. It is mediated by integrin receptors that bind to a wide range of extracellular ligands and intracellularly connect to the actin cytoskeleton in macromolecular structures called focal adhesions (FAs). FAs are highly complex assemblies containing hundreds of different proteins processing chemical as well as mechanical signals during cell adhesion; however, the molecular processes that govern FA organization and mechano-chemical signaling remain incompletely understood. Furthermore, mammals express 24 distinct integrin receptors with specific ligand binding and signaling properties but the underlying mechanisms mediating integrin receptor specificity are still unclear. The major obstacle for a better understanding of integrin function has been the lack of suitable techniques that allow a systematic investigation of FAs with sufficient temporal and spatial resolution. We, the members of this French-German Consortium, have therefore developed techniques that will allow such an analysis. We have established unique cell systems to study distinct integrin receptors and their intracellular regulators at physiological expression levels and we have developed super-resolution microscopy methods to study the dynamic nanoscale organization of individual FAs proteins. In addition, we have developed mass-spectrometry approaches to study the molecular composition of FAs and engineered biosensors to quantify integrin force transduction with piconewton sensitivity in cells. We propose to combine our tools to investigate how integrins and their associated molecules regulate the nanoscale organization of FAs to modulate chemical and mechanical signaling during cell adhesion in an integrin subtype specific fashion.In the first part of the project, single-protein tracking and super-resolution microscopy will be used to determine the dynamics of distinct integrin subunits in genetically modified cells. We will combine super-resolution microscopy with nano-patterning to control the lateral spacing of integrin subtype specific ligands and apply molecular tension sensors to correlate FA nanoscale organization with integrin force transduction. In the second part, we will analyze the role of integrin regulators on FA nanoscale organization, molecular dynamics and force transduction. Finally, we will evaluate how externally applied forces are processed by distinct integrin receptors and their associated molecules. These experiments will utilize custom-built cell stretchers that allow super-resolution microscopy as well as molecular force measurements in living cells. Together, the proposed experiments should provide unprecedented insights into the molecular construction rules of FAs. The expected results should be valuable to the cell adhesion community and be of general interest to the cell biological and biophysical community.