Single Cell Analysis of MAPK Signaling Dynamics during Tissue Homeostasis

项目摘要 我们研究的长期目标是了解MAPK信号动力学是如何控制和协调的 组织动态平衡和发育过程中的细胞命运。丝裂原活化蛋白激酶(MAPKs)在临床上 相关的信号分子，协调细胞对各种刺激的反应。一共有三个 主要的MAPK信号级联(ERK、p38和JNK)控制着相反的细胞决定，如 存活/凋亡或增殖/衰老。尽管这些对立的职能角色一直很好- 各种刺激(即细胞因子、细胞应激或生长因子)已被证明具有 激活这个高度互联网络的所有分支机构。此外，细胞是否最终凋零、衰老 或进入细胞周期是一种高度异质性的结果，即使在同基因细胞中也是如此 环境。我们目前对MAPK网络如何控制细胞命运的理解是不完整的，因为：(I) 缺乏量化整个网络动态的综合方法和(2)单元的使用 群体分析平均不同步的单细胞行为。 为了满足这一需求，我的实验室开创了新一代生物传感器，它可以同时 对数千个活的单细胞中的多个激酶活性进行量化。这些生物传感器可以将 磷酸化为核质穿梭事件，可通过荧光轻松测量 显微镜。我们独特的方法具有高时间分辨率、传感器多路传输功能、 以及研究多小区的单个小区中的信令网络动力学所必需的单小区分辨率 系统。 我们的中心假设是，MAPK之间的信号平衡对调节单细胞至关重要 结果(即增殖、静止、衰老、凋亡)。然而，相互之间的串扰动态 单个MAP激酶还没有得到系统的研究。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、细胞类型和转基因细胞。在本项目中，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这种串扰的表型后果。 在项目2中，我们将使用细胞培养和初级器官模型来了解MAPK信号转导在 在肿瘤发生的早期维持组织的动态平衡。在项目3中，我们将研究MAPK信号如何 Dynamic有力地确定了哺乳动物胚胎在植入前发育过程中的情况。

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.