Analyses of 120nm Local Contractions Linked to Rigidity Sensing

描述（由申请人提供）： 为了使细胞正确分化，它必须感知其微环境的机械和化学特性。基质机械性质的传感起着关键作用，并影响细胞分化、再生和疾病的许多方面。矩阵机械传感不被理解的部分原因是，许多涉及的步骤发生在纳米的分子尺度。我们最近表明，亚微米柱位移在早期的局部收缩120 nm。这在较大（>1 mm）柱的情况下未观察到。由于这些早期纳米位移在无法对基质刚性变化做出反应的细胞中发生改变，我们建议细胞使用产生120纳米基质位移所需的力作为基质刚性的衡量标准，正如其他人也认为的那样。要了解这一过程是如何发生的，需要将亚微米制造和定量细胞生物学技术结合起来，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制造设备在第二和纳米水平上定义局部收缩的步骤，该设备将测试矩阵面积，间距和刚度变化的影响，甚至对单个细胞的影响。通过对该过程步骤的描述，我们将分析每个步骤中涉及哪些分子复合物。这些相同的工具将使我们能够分析影响粘附成熟的机械因素，并了解局部收缩是否在完全展开的细胞中继续。使用间充质干细胞，我们将确定的时间过程中的收缩之前的分化，以应对刚性和软的小支柱。这项资助将使我们能够确定干细胞分化中所涉及的机械化学步骤的基础，这将使我们能够针对分化或疾病过程的具体步骤进行操作。

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.