Inferring the Physics of mRNA Trafficking in Neuronal Systems

推断神经系统中 mRNA 运输的物理原理

• 批准号: 1707999
• 负责人: Mark Bathe
• 金额: $ 72万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1707999&HistoricalAwards=false
• 关键词: Inferring; Physics; mRNA; Trafficking; Neuronal

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.

信使RNA（mRNAs）的主动转运机制是神经网络发育和功能的核心。荧光成像是使用活细胞中mRNA位置和拷贝数的直接报告者来解析mRNA转运的物理基础的有力方法。然而，解决这些机制需要定量的，基于物理学的方法，模型核糖体-mRNA协会，拷贝数，并招聘到突触和细胞骨架网站需要本地翻译。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的建模和推断相结合，以表征神经元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。由PI推进的教育举措包括本科生和研究生课程的增强，包括基于讨论的研讨会课程，该课程在麻省理工学院生物工程，物理学和生物学系之间联合授课。PI还积极开发和维护免费的Web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。PI通过在1月份的活动期间由生物系组织的年度研讨会上授课，参与对代表性不足的少数民族的外联活动。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计划以及来自外国的国际学生的接待访问转化为本科生。本项目的目的是了解神经细胞中由主动运输机制调节的信使RNA的翻译动力学。神经元由高度伸长的轴突和树突状突起组成，这些突起延伸数百至数千个远离细胞核的细胞体，在细胞核中发生转录。因此，神经元在发育和学习中的可塑性需要突触和细胞骨架蛋白在这些扩展过程中局部合成，而这些扩展过程不能仅通过被动mRNA转运机制来实现。为了实现这一功能，mRNA被分子马达主动运输到突触位点，以使局部蛋白质产生。在本项目中，PI将应用单分子活细胞成像以及基于物理的mRNA和核糖体转运建模，以了解神经元mRNA转运的分子基础。PI将开发随机建模和推理程序，以推断mRNA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。进行荧光波动分析以推断核糖核蛋白复合物中mRNA和核糖体的拷贝数，其使用具有可交换DNA探针的固定神经元样品中的多路复用超分辨率荧光成像进行交叉验证。这项工作将有助于解决mRNA募集和运输的形成和神经元发育和可塑性的核心突触的营业额的物理基础。该项目由物理学系的生命系统物理学计划和分子与细胞生物科学系的细胞动力学和功能集群共同支持。

项目成果

Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes

• DOI: 10.1038/s41467-019-12372-6
• 发表时间: 2019-09-26
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Guo, Syuan-Ming;Veneziano, Remi;Bathe, Mark
• 通讯作者: Bathe, Mark

DoGNet: A deep architecture for synapse detection in multiplexed fluorescence images

• DOI: 10.1371/journal.pcbi.1007012
• 发表时间: 2019-05-01
• 期刊: PLOS COMPUTATIONAL BIOLOGY
• 影响因子: 4.3
• 作者: Kulikov, Victor;Guo, Syuan-Ming;Lempitsky, Victor
• 通讯作者: Lempitsky, Victor

Molecular Diversity of Glutamatergic and GABAergic Synapses from Multiplexed Fluorescence Imaging

• DOI: 10.1523/eneuro.0286-20.2020
• 发表时间: 2021-01-01
• 期刊: ENEURO
• 影响因子: 3.4
• 作者: Danielson, Eric;de Arce, Karen Perez;Bathe, Mark
• 通讯作者: Bathe, Mark