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

基本信息

批准号：
10640274
负责人：
Yuan-Chi Huang
金额：
$ 10万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10640274
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 Cytoplasm Cytoplasmic Protein Cytosol Data Deposition Development Diffusion Disease Effectiveness 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 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 Visualization Xenopus Xenopus laevis cancer cell cellular imaging collaborative environment egg experience extracellular fluidity imaging modality inhibitor innovation member membrane model membrane reconstitution molecular modeling mutant novel preservation programs protein purification real-time images reconstitution single molecule spatiotemporal therapeutic target transmission process

项目摘要

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中，信号蛋白如何发挥作用的物理机制和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描述了从受体触发到下游激活的细胞内信号传导，两者都在健康和疾病。该提案的目的是提出一种新型的重组方法整合模型膜和细胞提取物来研究受体酪氨酸中的膜-细胞质偶联激酶 (RTK) 信号通路。中心假设是膜的相关调节和动力学信号转导依赖于细胞质分子和环境，通常不被捕获通过传统的膜重构。该提案的基本原理是，这种方法提供了独特的实验优势补充了活细胞研究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目标。中心假设将通过四个具体目标来实现：1）优化稳健的膜-细胞质重构方案，2) 比较细胞质与膜中分子的首次相遇率，3) 重建并表征 RTK-Ras-MAPK 通路的时间调节，以及 4) 剖析 RTK-Ras-MAPK 信号的时空耦合和动态路径。膜-细胞质重构代表了一种在概念和技术上创新的研究细胞内信号传导的方法在膜-细胞质界面。初步数据支持这种重组的生化可行性方法。与先进的荧光显微镜相结合，该平台可以实现控制和实时信号事件的表征，直至单分子水平。本研究的意义该计划是开发 RTK 信号通路的机械和动力学框架，充当研究其他信号通路的范例。这些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的看法。黄元奇博士（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的领先专家。黄博士研究广泛具有开发基于成像的膜测定的经验，该测定可将复杂的信号反应映射到可量化的重构的系统。该奖项使黄博士能够整合一种额外的成像方法，晶格光- 片状显微镜，以解决细胞溶质动力学问题，并获得单细胞成像的实验训练。黄博士得到了系统生物学领域的领军人物 James Ferrell 博士的指导，并得到了强大的支持。合作团队包括 Steven Boxer 博士、Christopher Garcia 博士和 Joanna Wysocka 博士。全部都是教员斯坦福大学成员。这样的安排展现了非凡的协作环境斯坦福大学的教授，并强调了指导和合作的可行性和有效性。