Inferring the Physics of mRNA Trafficking in Neuronal Systems

信使RNA(Messenger RNAs，mRNAs)的主动转运机制是神经元网络发育和功能的核心。荧光成像通过直接记录活细胞中mRNA的位置和拷贝数，是解决mRNA转运的物理基础的有力手段。然而，解决这些机制需要定量的、基于物理学的方法，对核糖体-mRNA关联、拷贝数以及需要局部翻译的突触和细胞骨架位置的招募进行建模。本项目将活细胞成像与随机分子运输和拷贝数变化的基于物理学的建模和推断相结合，以表征神经突触发育的分子基础，这是生命系统中大脑发育和功能的核心。PI提出的教育倡议包括本科生和研究生课程的改进，包括麻省理工学院生物工程系、物理系和生物系联合教授的基于讨论的生命系统物理学研讨会课程。此外，PI还积极开发和维护免费的网络服务器，这些服务器在全球范围内分发由他的团队开发的基于物理的推理程序。在1月份的活动间歇期间，国际学生联合会通过在生物系组织的年度讲习班上授课，参与了对代表不足的少数群体的外联活动。PI的教育和研究活动通过麻省理工学院的本科生研究机会项目以及来自外国的国际学生的宿主访问被翻译给本科生。本项目的目的是了解受神经细胞主动运输机制调控的信使RNA的翻译动力学。神经元由高度伸长的轴突和树突组成，这些突起将数百到数千个细胞体从发生转录的细胞核延伸出去。因此，神经元发育和学习的可塑性要求突触和细胞骨架蛋白在这些延长的过程中局部合成，而这些过程不能仅通过被动的mRNA运输机制来实现。为了实现这一功能，mRNAs被分子马达主动运送到突触位置，以使当地的蛋白质生产成为可能。在这个项目中，PI将应用单分子活细胞成像，以及基于物理学的信使核糖核酸和核糖体运输的建模，以了解神经元信使核糖核酸运输的分子基础。PI将开发随机建模和推理程序，以推断mRNAs和核糖体的关联动力学，以及它们与丝状肌动蛋白网络、微管和突触蛋白的物理关联，以确定调节mRNA翻译的细胞标志。荧光涨落分析用于推断核糖核蛋白复合体中mRNAs和核糖体的拷贝数，这些拷贝数通过使用可交换DNA探针的固定神经元样本的多路超分辨率荧光成像进行交叉验证。这项工作将有助于解决突触形成和周转中mRNA招募和运输的物理基础，突触是神经元发育和可塑性的核心。该项目由物理部的生命系统物理学计划和分子和细胞生物科学部的细胞动力学和功能簇共同支持。

Active transport mechanisms of messenger RNAs (mRNAs) are core to neuronal network development and function. Fluorescence imaging is a powerful approach to resolving the physical basis of mRNA transport using direct reporters of mRNA location and copy number in live cells. However, resolving these mechanisms requires quantitative, physics-based approaches that model ribosome-mRNA associations, copy numbers, and recruitment to synaptic and cytoskeletal sites where local translation is needed. The present project integrates live-cell imaging with physics-based modeling and inference of stochastic molecular transport and copy number variations to characterize the molecular basis of neuronal synapse development that is core to brain development and function in living systems. Educational initiatives advanced by the PI include undergraduate and graduate curriculum enhancements including a discussion based seminar course on the physics of living systems that is taught jointly between the Departments of Biological Engineering, Physics, and Biology at MIT. The PI is additionally active in developing and maintaining free web servers that distribute worldwide physics-based inference procedures developed by his group. The PI participates in outreach to under-represented minorities through teaching in an annual workshop organized by the Department of Biology over the inter-activity period in January. Educational and research activities of the PI are translated to undergraduate students through MIT's Undergraduate Research Opportunities Program, as well as through host visitations of international students from foreign countries.The objective of this project is to understand the translational dynamics of messenger RNAs that are regulated by active transport mechanisms in neuronal cells. Neurons consist of highly elongated axonal and dendritic processes that extend hundreds to thousands of cell bodies away from the nucleus, where transcription occurs. Consequently, neuronal plasticity in development and learning requires synaptic and cytoskeletal proteins to be synthesized locally within these extended processes that cannot be reached by passive mRNA transport mechanisms alone. To achieve this function, mRNAs are actively trafficked by molecular motors to synaptic sites to enable local protein production. In this project the PI will apply single-molecule live-cell imaging together with physics-based modeling of mRNA and ribosomal transport to understand the molecular basis of neuronal mRNA transport. The PI will develop stochastic modeling and inference procedures to infer the association dynamics of mRNAs and ribosomes, as well as their physical association with filamentous actin networks, microtubules, and synaptic proteins to identify cellular landmarks that regulate mRNA translation. Fluorescence fluctuation analysis is performed to infer copy numbers of mRNAs and ribosomes in ribonucleoprotein complexes, which are cross-validated using multiplexed super-resolution fluorescence imaging in fixed neuronal samples with exchangeable DNA probes. This work will help resolve the physical basis of mRNA recruitment and trafficking in the formation and turnover of synapses that are central to neuronal development and plasticity.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences.