Elucidating the Biophysical Mechanisms of Notch Activation

阐明Notch激活的生物物理机制

基本信息

批准号：
8833297
负责人：
Khalid S Salaita
金额：
$ 29.1万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8833297
关键词：
Address Animals Benchmarking Biochemical Biological Biophysical Process Cell Communication Cell Line Cell Nucleus Cells Chemicals Coculture Techniques Complex Data Differentiation and Growth Disease Edetic Acid Endocytosis Endosomes Environment Eukaryotic Cell Extracellular Domain Fluorescence Fluorescence Resonance Energy Transfer Gene Activation Geometry Goals Growth and Development function Human Hybrids Individual Intercellular Junctions Life Ligand Binding Ligands Link Liquid substance Magnetism Malignant Neoplasms Mammalian Cell Maps Measurement Measures Mechanics Mediating Membrane Membrane Lipids Methods Microscopy Modeling Molecular Noise Pathway interactions Peptide Hydrolases Process Receptor Activation Regulation Research Resolution Role Signal Pathway Signal Transduction Site Source Supporting Cell Surface Testing Work base cell growth extracellular inhibitor/antagonist interdisciplinary approach interest multidisciplinary notch protein novel strategies programs receptor receptor binding research study response secretase sensor signal processing single cell analysis stoichiometry transcription factor transmission process two-dimensional

项目摘要

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.

描述（由申请人提供）：Notch受体将细胞与细胞通信，细胞生长和确定细胞命运坐在一起。 Notch信号传导中的故障与包括癌症在内的一系列疾病有关，这些疾病刺激了人们对其分子激活机制的兴趣。所提出的工作的中心假设是Notch配体 - 受体相互作用，机械传输和空间排列的物理方面积极地提供了一种用于信号调节的分子机制。该提案的长期目标是对空间机械输入如何对生化信号过程产生调节性控制。我们寻求对细胞外环境如何影响细胞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的混合物理和生物学方法，旨在解构Notch受体裂解如何对聚类，力学和空间排列敏感。除非引入一种新方法来操纵和调查个别活细胞中的缺口，否则无法解决这些问题。我们将利用基于表面的Notch激活来概括其先天的二维几何形状，目的是解决有关空间和时间输入和随机噪声在触发信号传导途径中的作用的长期问题。初步数据表明，合成脂质膜平台提供了一种更精确的方法，可以激活哺乳动物细胞系中的Notch途径。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联。基于显微镜的转录程序的单细胞分析将用于测量配体诱导的激活。这些实验将对途径产生定量描述，并可能有助于理解分子缺口在人类癌中的作用。