Nanostructure Devices for Measuring Cell Mechanics

描述(由申请人提供)：细胞环境是决定细胞行为和最终细胞表型的主要因素。最近的许多研究表明，环境的机械方面是决定细胞反应的重要因素。关键的力学因素是纳米级的间距、曲率和细胞外基质成分的刚性。我们已经组成了两个纳米制造实验室和一个细胞生物学实验室之间的合作，以探索这些因素是如何在分子水平上被细胞感知的。使用电子束写入系统，我们将能够创建具有一系列纳米级特征的细胞大小的阵列，这将使我们能够筛选触发细胞响应的关键距离和图案。一旦我们定义了细胞反应的关键距离，如细胞扩散，我们将筛选一些缺失关键运动性和信号蛋白的细胞系，以确定细胞扩散反应是否发生改变。特别感兴趣的是Talin，其具有间隔约55 nm的多个整合素结合位点。在膜曲率方面，我们观察到细胞对胶原和其他纤维的反应是通过伸展含有肌球蛋白II-B的片状脂膜，而肌球蛋白II-B-/-细胞以低于30%的效率收缩纤维。我们将制造具有不同曲率半径的纤维和二维表面特征，并将GFP-肌球蛋白II-B在片状脂膜中的组装作为定义引起响应的曲率范围的标准。细胞不仅跟踪纤维，而且通过接触引导跟踪表面特征，我们将探索确定细胞极化和跟踪的重要参数。最近的结果表明，特定的突变将极大地影响细胞极化和移动的能力，导致严重异常的形态，这将使我们能够了解接触指导中涉及的重要细胞参数。刚性检测对细胞的扩散和生长至关重要，我们已经定义了一些蛋白质，它们是检测刚性表面和柔软表面之间的差异所必需的。开发一种探测表面硬度变化对细胞表面相邻区域影响的装置，将使我们能够探索硬度传感的详细机制。了解细胞所感知的表面特征的大小和几何形状将使我们能够以一种定义的方式得出给定的细胞行为。了解所涉及的蛋白质将使我们能够使用靶向药物抑制剂以确定的方式改变形态。这些研究在组织工程和设计伤口愈合、癌症和各种疾病的潜在治疗方法方面有许多实际应用。

DESCRIPTION (provided by applicant): Cellular environment is a major factor in determining cell behavior and ultimately cell phenotype. Many recent studies have shown that the mechanical aspects of the environment are important factors in determining the cell response. The mechanical factors that appear critical are the nanometer level spacing, curvature, and rigidity of the extracellular matrix components. We have formed a collaboration between two nanofabrication labs and a cell biology lab to explore how these factors are sensed by cells at the molecular level. Using an e-beam writing system, we will be able to create cellular sized arrays with a range of nanometer level features that will enable us to screen for the critical distances and patterns that trigger cell responses. Once we define the critical distance for a cell response such as cell spreading, we will screen a number of cell lines that are missing critical motility and signaling proteins to determine if the cell spreading response is altered. Of particular interest is talin that has multiple integrin binding sites spaced over about 55 nm. In regard to membrane curvature, we have observed that cells respond to collagen and other fibers by extending lamellipodia with myosin II-B in them and myosin II-B-/- cells contract fibers at less than 30% efficiency. We will fabricate fibers and 2-D surface features with different radii of curvature and use the assembly of GFP-myosin II-B in lamellipodia as a criterion for defining the range of curvatures that elicit the response. Cells not only follow fibers but also follow surface features through contact guidance and we will explore the important parameters in determining cell polarization and tracking. Recent results indicate that specific mutations will dramatically affect the ability of cells to polarize and move, giving rise to grossly abnormal morphologies that will enable us to understand important cellular parameters involved in contact guidance. Rigidity sensing is critical for cell spreading and growth and we have defined a number of proteins that are needed to sense the difference between rigid and soft surfaces. The development a device for probing the effect of changes in surface rigidity on adjacent regions of the cell surface will enable us to probe the detailed mechanism of rigidity sensing. Knowing both the size and geometry of surface features that are sensed by the cells will enable us to elicit given cell behaviors in a defined way. Knowing the proteins involved will enable us to use targeted pharmacological inhibitors to alter morphology in defined ways. These studies have many practical applications in tissue engineering and in designing potential therapies for wound healing, cancer and a variety of disorders.