Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface

项目摘要 在细胞信号转导中，细胞内信号转导蛋白的物理机制和动力学路径 人们对网络传输信息的了解仍然很少。长期目标是构建一个分子模型 这定量地描述了从受体触发到下游激活的细胞内信号，两者都是在 在健康和疾病方面。这项提议目的是提出一种新型的重建方法 结合模型膜和细胞提取物研究酪氨酸受体的膜-胞浆偶联 激酶(RTK)信号通路。中心假说是膜的相关调节和动力学 信号转导依赖于胞浆分子和环境，而这些分子和环境通常不被捕获 通过常规的膜重建。这项提议背后的理由是，这种方法提供了一种 独特的实验优势，补充了活细胞研究在开发定量描述 早期信号转导。识别运动瓶颈和反馈可提供可行的治疗方法 目标。中心假设将通过四个具体目标来实现：1)优化健壮的膜-细胞质 重组方案，2)比较细胞质和膜中分子的初见率，3) 重组和表征RTK-RAS-MAPK通路的时间调节，以及4)解剖 RTK-RAS-MAPK信号的时空耦合和动态路径。膜-胞浆 重构代表了一种在概念上和技术上创新的方法来询问细胞内信号 在膜-细胞质交界处。初步数据支持这一重组的生化可行性 接近。与先进的荧光显微镜相结合，该平台能够控制和 实时信号事件的特征，下至单分子水平。这项研究的意义 该计划是RTK信号通路的一个机械性和动力学框架的发展，它 作为研究其他信号通路的范例。这样的努力可能会广泛影响我们的理解 信号转导的组织原则，并转变我们对疾病和治疗的看法。 黄元池博士(William Y.C.Huang)是该项目的首席研究员。黄博士的目标是 成为细胞信号转导生物物理学方面的领先专家。黄博士有广泛的研究 开发基于成像的膜分析方法，将复杂的信号反应映射到可量化的 重组的系统。该奖项使黄博士能够将另一种成像方法--晶格光-- 薄片显微镜，以解决细胞液动力学，以及获得单细胞成像的实验培训。 黄博士由系统生物学领域的领军人物詹姆斯·费雷尔博士指导，并得到了一位强大的 合作小组，Steven Boxer博士，Christopher Garcia博士和Joanna Wyocka博士。他们都是教职员工 斯坦福大学的成员。这样的安排表明了特殊的合作环境 并强调了导师关系和协作的可行性和有效性。

PROJECT ABSTRACT In cellular signal transduction, the physical mechanism and the dynamical path of how signaling proteins in a network transmit information remains poorly understood. The long-term goal is to construct a molecular model that quantitatively describes intracellular signaling from receptor triggering to downstream activation, both in health and in diseases. The objective of this proposal is to advance a novel type of reconstitution approach integrating model membranes and cell extracts to study the membrane-cytosol coupling in the receptor tyrosine kinase (RTK) signaling pathway. The central hypothesis is that the relevant regulation and kinetics of membrane signal transduction is dependent on the cytosolic molecules and environment, which are generally not captured by conventional membrane reconstitution. The rationale underlying this proposal is that such approach offers a unique experimental advantage that complements live-cell studies in developing a quantitative description of early signal transduction. Identification of kinetic bottleneck and feedbacks could provide viable therapeutic targets. The central hypothesis will be pursued by four specific aims: 1) Optimize a robust membrane-cytosol reconstitution protocol, 2) Compare the first-encounter rate of molecules in the cytosol versus membrane, 3) Reconstitute and characterize the temporal regulation of the RTK-Ras-MAPK pathway, and 4) Dissect the spatiotemporal coupling and dynamical path of the RTK-Ras-MAPK signaling. The membrane-cytosol reconstitution represents a conceptually and technically innovative approach to interrogate intracellular signaling at the membrane-cytosol interface. Preliminary data support the biochemical feasibility of this reconstitution approach. In combination with advanced fluorescence microscopy, this platform enables control and characterization of real-time signaling events, down to the single-molecule level. The significance of this research program is the development of a mechanistic and dynamical framework of the RTK signaling pathway, which acts as a paradigm for studying other signaling pathways. Such efforts could broadly impact our understanding of the organizing principles of signal transduction, and transform our view on diseases and therapeutics. Dr. Yuan-Chi Huang (William Y. C. Huang) is the principal investigator of this project. Dr. Huang's goal is to become a leading expert in the biophysics of cellular signal transduction. Dr. Huang has extensive research experience developing imaging-based membrane assays that map complex signaling reactions to quantifiable reconstituted systems. This award enables Dr. Huang to integrate an additional imaging method, lattice light- sheet microscopy, to resolve cytosolic dynamics, as well as acquire experimental training in single-cell imaging. Dr. Huang is mentored by a leader in systems biology, Dr. James Ferrell, and is further supported by a strong collaboration team, Dr. Steven Boxer, Dr. Christopher Garcia, and Dr. Joanna Wysocka. All of them are faculty members at Stanford University. Such arrangement demonstrates the exceptionally collaborative environment of Stanford University, and highlights the feasibility and effectiveness of the mentorship and collaboration.