Single Cell Analysis of MAPK Signaling Dynamics during Tissue Homeostasis

组织稳态过程中 MAPK 信号动力学的单细胞分析

• 批准号: 10669592
• 负责人: Sergi Regot
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-07 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10669592
• 关键词: Address; Apoptosis; Biological Assay; Biosensor; Cell Culture Techniques; Cell Cycle; Cell Fate Control; Cells; Cellular Stress; Development; Embryo; Environment; Equilibrium; Event; Generations; Goals; Growth Factor; Homeostasis; Individual; Laboratories; MAPK8 gene; Measures; Methodology; Methods; Microscopy; Mitogen-Activated Protein Kinases; Modeling; Molecular; Organoids; Outcome; Phenotype; Phosphorylation; Phosphotransferases; Population; Pre-implantation Embryo Development; Proliferating; Research; Resolution; Role; Signal Transduction; Signaling Molecule; Specific qualifier value; Stimulus; System; Tissues; Work; cell behavior; cell growth; cell type; clinically relevant; cytokine; experience; nucleocytoplasmic transport; p38 Mitogen Activated Protein Kinase; response; senescence; sensor; single cell analysis; temporal measurement; tumorigenesis

Project Summary The long term goal of our research is to understand how MAPK signaling dynamics controls and coordinates cell fate during tissue homeostasis and development. Mitogen Activated Protein Kinases (MAPKs) are clinically relevant signaling molecules that orchestrate cellular responses to a diverse array of stimuli. There are three major MAPK signaling cascades (ERK, p38 and JNK) that control opposing cellular decisions such as survival/apoptosis or proliferation/senescence. Even though these opposed functional roles have been well- characterized, a wide variety of stimuli (i.e. cytokines, cellular stresses or growth factors) have been shown to activate all branches of this highly interconnected network. In addition, whether cells finally apoptose, senesce or enter cell cycle is a highly heterogeneous outcome, even in isogenic cells experiencing the same environment. Our current understanding of how the MAPK network controls cell fate is incomplete because: (i) a lack of integrated methods to quantify the dynamics of the network as a whole and (ii) the use of cell population assays that average unsynchronized single cell behaviors. To address this need, my laboratory has pioneered a new generation of biosensors that allow simultaneous quantification of multiple kinase activities in thousands of live single cells. These biosensors convert phosphorylation into a nucleocytoplasmic shuttling event that can be easily measured by fluorescent microscopy. Our unique methodology features the high temporal resolution, sensor multiplexing capabilities, and single cell resolution essential for studying signaling network dynamics in single cells of a multicellular system. Our central hypothesis is that the signaling equilibrium between MAPKs is critical to regulate single cell outcomes (i.e. proliferation, quiescence, senescence, apoptosis). However, the crosstalk dynamics between individual MAP kinases has not been systematically studied. This is, in part, because MAPK studies use a wide variety of experimental conditions, cell types and genetically altered cells. In this project we will dissect MAPK signaling dynamics at the molecular, cellular and multicellular levels: In Project 1 we will systematically interrogate the crosstalk dynamics between MAP kinases and the phenotypic consequences of this crosstalk. In Project 2 we will use cell culture and primary organoid models to understand the role of MAPK signaling in maintaining tissue homeostasis during early oncogenesis. In Project 3 we will study how MAPK signaling dynamics robustly specifies the mammalian embryo during preimplantation development.

项目概要 我们研究的长期目标是了解 MAPK 信号动力学如何控制和协调 组织稳态和发育过程中的细胞命运。丝裂原激活蛋白激酶（MAPK）在临床上 协调细胞对各种刺激做出反应的相关信号分子。有三个 主要的 MAPK 信号级联（ERK、p38 和 JNK）控制相反的细胞决策，例如 存活/凋亡或增殖/衰老。尽管这些对立的职能角色已经很好地 根据特征，多种刺激（即细胞因子、细胞应激或生长因子）已被证明可以 激活这个高度互联网络的所有分支。另外，细胞最终是否会凋亡、衰老？ 或进入细胞周期是一个高度异质的结果，即使在经历相同的同基因细胞中 环境。我们目前对 MAPK 网络如何控制细胞命运的理解并不完整，因为：(i) 缺乏量化整个网络动态的综合方法以及（ii）细胞的使用 对不同步的单细胞行为进行平均的群体分析。 为了满足这一需求，我的实验室率先推出了新一代生物传感器，可以同时进行 定量数千个活单细胞中的多种激酶活性。这些生物传感器将 磷酸化成核细胞质穿梭事件，可以通过荧光轻松测量 显微镜。我们独特的方法具有高时间分辨率、传感器复用功能、 和单细胞分辨率对于研究多细胞的单细胞信号网络动力学至关重要 系统。 我们的中心假设是 MAPK 之间的信号平衡对于调节单细胞至关重要 结果（即增殖、静止、衰老、凋亡）。然而，之间的串扰动态 单个 MAP 激酶尚未得到系统研究。这在一定程度上是因为 MAPK 研究使用了广泛的 各种实验条件、细胞类型和基因改变的细胞。在这个项目中我们将剖析 MAPK 分子、细胞和多细胞水平的信号动力学：在项目 1 中，我们将系统地 询问 MAP 激酶之间的串扰动态以及该串扰的表型后果。 在项目 2 中，我们将使用细胞培养和原代类器官模型来了解 MAPK 信号传导在 在早期肿瘤发生过程中维持组织稳态。在项目 3 中，我们将研究 MAPK 信号如何 动力学有力地指定了植入前发育过程中的哺乳动物胚胎。