Elucidating the Biophysical Mechanisms of Notch Activation

描述（由申请人提供）：Notch受体协调细胞间的通讯，细胞生长，并决定细胞命运。Notch信号的功能障碍与包括癌症在内的一系列疾病有关，这激发了人们对了解其激活的分子机制的兴趣。本研究的中心假设是Notch配体与受体相互作用、机械转导和空间排列的物理方面积极地为信号调节提供了分子机制。本建议的长期目标是全面了解空间-机械输入如何对生化信号传导过程施加调节控制。我们寻求对细胞外环境如何影响细胞内化学信号传导的基本理解。为了实现这一目标，我们提出了一种高度多学科，混合物理和生物学的方法，旨在解构Notch受体切割如何对聚类，力学和空间排列敏感。除非引入一种新的方法来操纵和研究单个活细胞中的Notch，否则这些问题无法解决。我们将采用基于表面的Notch激活来概括其固有的二维几何形状，目的是解决关于空间和时间输入以及随机噪声在触发信号通路中的作用的长期问题。初步数据表明，合成脂膜平台为激活哺乳动物细胞系中的Notch通路提供了一种生理上更准确的方法。一种新开发的荧光力传感器将允许直接测量机械应变和蛋白酶活性之间的关系。基于显微镜的单细胞转录程序分析将用于测量配体诱导的激活。这些实验将对该通路进行定量描述，并可能有助于理解Notch分子调控在人类癌症中的作用。

DESCRIPTION (provided by applicant): The Notch receptors coordinate cell to cell communication, cell growth, and determine cell fates. Malfunctions in Notch signaling have been linked to a range of diseases including cancer, which has spurred interest in understanding its molecular mechanisms of activation. The central hypothesis of the proposed work is that physical aspects of the Notch ligand-receptor interaction, mechanotransduction, and spatial arrangement, actively provide a molecular mechanism for signal regulation. The long-term goal of this proposal is to develop a complete understanding of how spatio-mechanical inputs can exert regulatory control over biochemical signaling processes. We seek a fundamental understanding of how the extracellular environment influences a cell's intracellular chemical signaling. To achieve this goal, we propose a highly multidisciplinary, hybrid physical and biological approach aimed at deconstructing how the Notch receptor cleavage is sensitive to clustering, mechanics, and spatial arrangement. These questions cannot be addressed unless a new approach is introduced to manipulate and to investigate Notch in individual living cells. We will employ surface-based activation of Notch to recapitulate its innate two-dimensional geometry with the goal of addressing long-standing questions regarding the role of spatial and temporal inputs and stochastic noise in triggering the signaling pathway. Preliminary data indicates that the synthetic lipid membrane platform provides for a more physiologically accurate approach to activate the Notch pathway in mammalian cell lines. A newly developed fluorescence force sensor will allow the direct measurement of the association between mechanical strain and protease activity. Microscopy-based single cell analysis of the transcriptional program will be used to measure ligand-induced activation. These experiments will yield a quantitative description of the pathway and may help in understanding the role of molecular Notch deregulations in human cancers. PUBLIC HEALTH RELEVANCE: The Notch pathway is universally employed between animal cells to control differentiation, growth, and development. Malfunctions in processing and relaying Notch signals are responsible for a range of human disorders and cancers. The goal of this proposal is to better understand Notch signaling by developing novel approaches to trigger the pathway and characterize its response functions in mammalian cells.