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。基质力学特性的感知在细胞分化、再生和疾病的许多方面起着至关重要的作用。基体机械传感之所以不为人所知，部分原因是其中涉及的许多步骤发生在纳米分子尺度上。我们最近表明，亚微米柱在早期由120纳米的局部收缩而移位。这在较大（bb10 - 1mm）的柱子上没有观察到。由于这些早期的纳米位移在细胞中发生了改变，而细胞无法对基质刚度的变化做出反应，因此我们建议细胞使用产生120纳米位移所需的力作为基质刚度的度量，正如其他人所认为的那样。要了解这个过程是如何发生的，将需要亚微米制造和定量细胞生物学技术的结合，这两位研究人员可以带来对这个问题的承担。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，这些设备将测试基质面积、间距和刚度变化的影响，甚至在单个细胞上。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。这些工具将使我们能够分析影响粘附成熟的机械因素，并了解完全扩散的细胞是否会继续局部收缩。使用间充质干细胞，我们将确定在硬和软小柱分化之前收缩的时间过程。这项资助将使我们能够确定干细胞分化中涉及的机械化学步骤的基础，这将使我们能够针对特定步骤来操纵分化或疾病过程。

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.