Cell Signaling and Cell Decisions

项目总结/摘要 人类细胞整合外部和内部信号输入，以做出改变的调节决策 功能输出。细胞的两个这样的决定--迁移和迁移的决定以及进入 增殖状态-是多细胞发育和组织维持的中心。监管 细胞如何开始迁移或增殖的反馈和核心开关机制还不清楚。 由于显著的细胞间变异性和缺乏同步性， 调节基序不能仅通过生物化学和遗传方法获得。然而近日 开发的活性报告和快速扰动策略使得研究复杂的 空间和时间信号反馈架构和决策过程中活的单细胞， 这种方法可以揭示反馈机制并规避技术批量测定问题。我们的工作 试图通过使用这些活细胞来理解人类细胞中细胞决策过程的原理， 方法来监测，干扰和自动分析相关的信令过程，并最终得出 如何做出具体决策的量化模型。 我们提出的工作有两个主题：在我们的第一个主题中，我们确定细胞如何启动和建立 细胞极性以及已经极化的细胞在定向迁移和趋化过程中如何引导它们的前沿。我们 开发了自动荧光显微镜方法来监测和干扰关键的Rho家族 小GTP酶和相关的第二信使，并制定了方法来量化不同的变化， 肌动蛋白结构这些方法将使我们能够了解细胞的核心调控机制， 极化和细胞迁移过程中的转向。在我们的第二个主题中，我们试图了解细胞如何决定 通过研究竞争的分子机制， 通过确定细胞周期不可逆点的分子机制， 进入，并通过探索顺序信号事件如何防止相同DNA的重新复制，以确保 DNA只能复制一次我们已经开发了一些活细胞周期活性报告，微扰 策略和自动化的单细胞分析方法，这将有助于我们了解和建模的监管 控制人类细胞周期进入的机制。 我们的两个主题将导致人类决策过程的逻辑的新概念，并将提供 详细的分子和机制模型解释了细胞如何整合信号输入开始迁移， 进入细胞周期。最后，癌症中细胞增殖和迁移的普遍失调， 在退行性疾病、免疫疾病和其他疾病中，这些过程经常失调， 对细胞迁移和增殖的复杂调控结构的分子理解可能导致 治疗多种疾病的新治疗策略。

PROJECT SUMMARY/ABSTRACT Human cells integrate external and internal signaling inputs to make regulatory decisions that change functional outputs. Two such decisions of cells – the decision to polarize and migrate and the decision to enter the proliferative state - are central to multicellular development and tissue maintenance. The regulatory feedbacks and the core switch mechanisms of how cells start to migrate or proliferate are not yet understood. Due to significant cell-to-cell variability and lack of synchronization, an understanding of the underlying regulatory motifs cannot be achieved by biochemical and genetic approaches alone. However, recently developed activity reporters and rapid perturbation strategies have made it possible to investigate complex spatial and temporal signaling feedback architectures and decision processes in living single cells, an approach that can reveal feedback mechanisms and circumvent the technical bulk assay issues. Our work seeks to understand the principles of cellular decision processes in human cells by employing these live-cell methods to monitor, perturb and automatically analyze the relevant signaling processes and ultimately derive quantitative models of how specific decisions are made. Our proposed work has Two Themes: In our first theme, we determine how cells initiate and establish cell polarity and how already polarized cells steer their front during directed migration and chemotaxis. We have developed automated fluorescence microscopy methods to monitor and perturb the critical Rho family small GTPases and relevant second messengers, and developed methods to quantify changes in different actin structures. These approaches will allow us to understand the core regulatory mechanisms for cell polarization and cell steering during migration. In our second theme, we seek to understand how cells decide to transition from a quiescent to a proliferative state by investigating molecular mechanisms of competition between stress and mitogens, by determining the molecular mechanism of the point-of-no return for cell cycle entry, and by exploring how sequential signaling events prevent the re-replication of the same DNA to ensure that DNA is only replicated once. We have developed a number of live cell cycle activity reporters, perturbation strategies and automated single-cell analysis methods that will help us understand and model the regulatory mechanisms controlling human cell cycle entry. Both our themes will lead to new concepts of the logics of human decision processes and will provide detailed molecular and mechanistic models explaining how cells integrate signaling inputs to start to migrate or enter the cell cycle. Finally, the universal dysregulation of cell proliferation and migration in cancer, and the frequent dysregulation of these processes in degenerative, immune and other diseases, argues that a molecular understanding of the complex regulatory architecture of cell migration and proliferation may lead to new therapeutic strategies for the treatment of a broad range of diseases.