Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface

重建和解构膜-细胞质界面的细胞内信号传导

• 批准号: 10449754
• 负责人: Yuan-Chi Huang
• 金额: $ 10万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449754
• 关键词: Adaptor Signaling Protein; Award; Benchmarking; Biochemical; Biological Assay; Biological Models; Biophysics; Buffers; Cell Extracts; Cell Nucleus; Cell membrane; Cell model; Cells; Collaborations; Comparative Study; Complement; Complex; Coupled; Coupling; Cues; Cytoplasmic Protein; Cytosol; Data; Deposition; Development; Diffusion; Disease; Effectiveness; Egg Preservation; Environment; Epidermal Growth Factor Receptor; Event; Faculty; Feedback; Fluorescence Microscopy; Geometry; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Image; Individual; Kinetics; Lateral; Light; Liquid substance; MAP Kinase Gene; Malignant Neoplasms; Maps; Membrane; Membrane Proteins; Mentors; Mentorship; Methods; Microscopy; Molecular; Nuclear; Pathway interactions; Pharmaceutical Preparations; Physical condensation; Principal Investigator; Problem Solving; Property; Proteins; Protocols documentation; Reaction; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Research; Role; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Speed; System; Systems Biology; Testing; Therapeutic; Time; Training; Universities; Xenopus; Xenopus laevis; base; cancer cell; cellular imaging; collaborative environment; experience; extracellular; fluidity; imaging modality; inhibitor; innovation; member; membrane model; membrane reconstitution; molecular modeling; mutant; novel; preservation; programs; real-time images; reconstitution; single molecule; spatiotemporal; therapeutic target

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.

项目摘要 在细胞信号转导，物理机制和信号蛋白如何在A中的动力学路径 网络传输信息仍然知之甚少。长期目标是构建分子模型 定量地描述了从受体触发到下游激活的细胞内信号传导，均在 健康和疾病。该提议的目的是推进一种新型的重组方法 整合模型膜和细胞提取物，以研究受体酪氨酸中的膜 - 胞质耦合 激酶（RTK）信号通路。中心假设是膜的相关调节和动力学 信号转导取决于胞质分子和环境，通常未捕获 通过常规的膜重建。该提议的基本原理是，这种方法提供了 独特的实验优势，可以补充活细胞研究，以开发针对的定量描述 早期信号转导。动力学瓶颈和反馈的识别可以提供可行的治疗性 目标。中心假设将通过四个特定目的来提出：1）优化稳健的膜 - 杂质溶胶 重建方案，2）比较细胞质与膜中分子的第一键率，3） 重新构建和表征RTK-RAS-MAPK途径的时间调节，4）剖析 RTK-RAS-MAPK信号传导的时空耦合和动力学路径。膜 - 胞质 重构代表了一种概念和技术创新的方法来询问细胞内信号 在膜 - 胞质界面。初步数据支持此重构的生化可行性 方法。结合高级荧光显微镜，该平台可以控制和 实时信号事件的表征，直至单分子水平。这项研究的意义 程序是RTK信号通路的机械和动态框架的开发，该框架的开发 充当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 信号转导的组织原理，并改变我们对疾病和治疗学的看法。 Yuan-Chi Huang博士（William Y. C. Huang）是该项目的主要研究者。黄博士的目标是 成为细胞信号转导生物物理学的领先专家。黄博士有广泛的研究 开发基于成像的膜测定的经验，这些测定将复杂的信号反应映射到可量化 重构系统。该奖项使Huang博士能够整合一种其他成像方法，Lattice Light- 薄板显微镜，以解决胞质动力学，并在单细胞成像中获取实验训练。 Huang博士受到系统生物学领导者James Ferrell博士的指导，并得到了强大的支持 合作团队，史蒂文·拳击手，克里斯托弗·加西亚博士和乔安娜·韦索卡博士。他们都是教师 斯坦福大学的成员。这种安排表明了非常合作的环境 斯坦福大学（Stanford University），并强调了指导与协作的可行性和有效性。