Analyses of 120nm Local Contractions Linked to Rigidity Sensing

描述(申请人提供)：为了让细胞正确分化，它必须感知其微环境的机械和化学特性。对基质力学性质的感知起着至关重要的作用，并影响细胞分化、再生和疾病的许多方面。基质机械传感之所以不被理解，部分原因是涉及的许多步骤都发生在纳米级的分子尺度上。我们最近已经证明，亚微米级的柱子在早期被局部收缩120纳米所移位。较大(&gt；1 mm)的柱子不会出现这种情况。由于这些早期的纳米位移在无法响应基质刚性变化的细胞中发生变化，我们建议细胞使用产生120 nm基质位移所需的力作为基质刚性的衡量标准，其他人也这么认为。要了解这一过程是如何发生的，需要结合亚微米制造和定量细胞生物学技术，这两位研究人员可以利用这些技术来解决这个问题。我们将使用新的纳米级设备在第二和纳米级别定义局部收缩的步骤，这些设备将测试基质面积、间距和刚性的变化甚至对单个细胞的影响。通过对过程中各个步骤的描述，我们将分析每个步骤涉及哪些分子络合物。这些工具将使我们能够分析影响黏附成熟的机械因素，并了解在充分展开的细胞中局部收缩是否继续。使用间充质干细胞，我们将确定在分化之前对坚硬和柔软的小柱做出反应的收缩的时间进程。这笔赠款将使我们能够确定干细胞分化所涉及的机械力化学步骤的基础，这将使定向操作分化或疾病过程的具体步骤成为可能。

DESCRIPTION (provided by applicant): For a cell to differentiate properly it must sense both the mechanical and chemical properties of its microenvironment. Sensing of the matrix mechanical properties plays a critical role and influences many aspects of cellular differentiation, regeneration and disease. Matrix mechanosensing is not understood in part because many of the steps involved occur at a molecular scale of nanometers. We have recently shown that submicrometer pillars are displaced at early times by a local contraction of 120 nm. This is not observed with larger (>1 mm) pillars. Because these early nanometer displacements are altered in cells that are unable to respond to matrix rigidity changes, we suggest that the cells use the force needed to produce a 120 nm displacement of matrix as a measure of matrix rigidity as others also believe. To understand how this process occurs will require a combination of submicrometer fabrication and quantitative cell biological techniques that the two investigators can bring to bear upon this problem. We will define the steps in the local contractions at a second and nanometer level using new nanofabricated devices that will test the effects of changes in matrix area, spacing and rigidity even on single cells. From a description of the steps in the process, we will analyze which molecular complexes are involved in each step. These same tools will enable us to analyze the mechanical factors that affect adhesion maturation and to know if local contractions continue in fully spread cells. Using mesenchymal stem cells, we will determine the time course of the contractions that precede differentiation in response to rigid and soft small pillars. This grant will enable us to determine the bases for the mechanochemical steps involved in stem cell differentiation that will enable targeting of the specific steps for manipulations of differentiation or disease processes.