CAREER: Mechanisms Controlling Signaling Reactions on Membranes through Molecular Assembly at Multiple Length Scales

该奖项的全部或部分资金来自《2021年美国救援计划法案》(公法117-2)。细胞依靠复杂的生化反应来发出启动基本功能的信号。了解确保这些生化反应在正确的时间在正确的地点发生的控制措施是基本的。在许多情况下，信号是由蛋白质相互作用并在膜表面组装成功能复合体来执行的。然而，我们对这些过程的理解是有限的。Lee实验室使用生物化学和生物物理技术的组合来揭示分子组装是如何调节生化反应的。这项拟议的研究具有重大而广泛的影响，从提高我们对众多生物过程的基础知识到改进疾病的治疗策略。这项研究的组成部分是一个教育和更广泛的影响项目，为学生提供真实的科学学习体验，包括那些来自历史上代表性不足的少数群体的学生。计划的活动包括(1)开发智能手机光学显微镜，用于实施基于课程的本科生研究体验(CURE)，以解决现实世界的问题；(2)为本科生提供个性化的研究指导；以及(3)与区域科学教师合作，为圣地亚哥县及其他地区高需求学校的学生开发科学实验室材料和课程。美国救援计划的资金被用来支持这位职业生涯早期的研究员在他职业生涯的关键阶段。蛋白质组装，如二聚化、聚集和微观/宏观相分离，似乎在嘈杂的细胞环境中提高了敏感性和特异性。然而，关于这些过程如何改变蛋白质的结构和功能以产生所需的信号结果，仍有许多需要了解。该项目主要研究真核细胞中的主要信号蛋白--Raf蛋白。中心假设是Raf的激活受到膜表面不同的分子组装模式的调节。为了验证这一假设，Lee实验室将在支撑的脂质双层上重建Raf信号，并在多个长度尺度上操纵分子组装，从结构上定义明确的复合体形成到蛋白质的宏观液-液相分离(LLP)。单分子荧光显微镜和时间分辨荧光光谱将用于定量膜结合、分子扩散、酶活性和构象动力学。这项研究将为细胞质信号分子如何利用多尺度分子组装来定义膜表面生化反应的特异性和敏感性提供新的见解。由于蛋白质组装是信号转导中一个反复出现的主题，本项目中开发的实验方法和理论框架可以很容易地适用于其他膜信号反应。该奖项由MCB的分子生物物理和细胞动力学和功能计划联合资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). Cells rely on sophisticated biochemical reactions to signal the initiation of essential functions. Understanding the controls that ensure that these biochemical reactions occur in the right place at the right time is fundamental. In many cases, the signaling is carried out by proteins that interact and assemble into functional complexes at the membrane surface. However, our understanding of these processes is limited. The Lee lab uses a combination of biochemistry and biophysical techniques to unveil how molecular assembly modulates biochemical reactions. The proposed research has significant and broad implications, from advancing our fundamental knowledge of numerous biological processes to improving therapeutic strategies for disease. Integral to this research is an education and broader impacts program that provides authentic science learning experiences to students, including those from historically underrepresented minority groups. The planned activities include (1) developing smartphone optical microscopes for implementing course-based undergraduate research experiences (CUREs) that tackle real-world problems, (2) providing personalized research mentorships for undergraduate students, and (3) collaborating with area science teachers to develop science laboratory materials and curriculum for students in high-needs schools in San Diego County and beyond. American Rescue Plan funding is used to support this early career investigator at a critical stage in his career.Protein assembly, such as dimerization, clustering, and micro/macroscopic phase separation appears to enhance sensitivity and specificity in noisy cellular environments. However, much remains to be understood about how these processes alter the structure and function of proteins to produce the desired signaling outcome. This project focuses on Raf kinases, major signaling proteins in eukaryotic cells. The central hypothesis is that activation of Raf is regulated by different modes of molecular assembly at membrane surfaces. To test this hypothesis, the Lee lab will reconstitute Raf signaling on a supported lipid bilayer and manipulate molecular assembly at multiple length scales, from structurally well-defined complex formation to macroscopic liquid-liquid phase separation (LLPS) of proteins. Single-molecule fluorescence microscopy and time-resolved fluorescence spectroscopy will be used to quantify membrane binding, molecular diffusion, enzymatic activity, and conformational dynamics. The research will provide new insights into how cytosolic signaling molecules utilize multiscale molecular assembly to define specificity and sensitivity of biochemical reactions at the membrane surfaces. As protein assembly is a recurring and emerging theme in signal transduction, the experimental approach and theoretical framework developed in this project can be readily adapted to other membrane signaling reactions.This award was funding jointly by the Molecular Biophysics and Cellular Dynamics and Function Programs of MCB.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.