Single molecule biomolecular condensate analysis in neurons

神经元中的单分子生物分子凝聚物分析

• 批准号: 10583437
• 负责人: Frederic Andre Meunier
• 金额: $ 14.59万
• 依托单位: UNIVERSITY OF QUEENSLAND
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583437
• 关键词: 3-Dimensional; Actins; Aging; Alcohol consumption; Algorithms; Alzheimer' s Disease; Amyloid; Astrocytes; Axon; Behavior; Binding; Biology; Cell physiology; Cells; Cellular biology; Cluster Analysis; Code; Color; Critical Pathways; Data; Dependence; Detection; Development; Diffusion; Disease; Drops; Environment; Evolution; Exhibits; Fluorescence Recovery After Photobleaching; Generations; Genes; Grant; Health; Hippocampus; Image; In Vitro; Individual; Label; Liquid substance; Measures; Mediating; Membrane; Methodology; Methods; Molecular; Mutation; Names; Nerve; Neurons; Nucleic Acids; Organelles; Outcome; Periodicals; Phase; Physical condensation; Protein Dynamics; Proteins; Pythons; Recurrence; Research; Resolution; Solid; Structure; Synapses; Technology; Time; Trees; Validation; Video Games; alpha synuclein; bioinformatics tool; density; diffraction of light; experimental analysis; graphical user interface; in vivo; indexing; insight; light microscopy; molecular imaging; mutant; nanocluster; nanoscale; neglect; novel; open source; particle; segregation; single molecule; sound; spatiotemporal; superresolution imaging; superresolution microscopy; synuclein; tau Proteins; tau mutation; tool; ultra high resolution

PROJECT SUMMARY/ABSTRACT Biomolecular condensates (BMCs) define small membraneless cellular compartments that segregate specific proteins and nucleic acids to generate cellular functions. Fluorescence recovery after photobleaching (FRAP) is currently the method of choice to characterize BMCs in vitro and in vivo and derive the average diffusion and trapping of molecules trapped in these condensates. Although FRAP is a good way to retrieve these important metrics, it greatly limits our understanding of BMCs nanoscale organization and dynamics in live cells and precludes the analysis of nanoscale BMCs (below the diffraction of light ~200 nm). Single-molecule imaging super-resolution microscopy allows direct imaging of molecules trapped in BMCs with much greater spatiotemporal resolution both in vitro and in live neurons, and has the potential to reveal nanoscale BMCs and their evolution in the context of ageing and Alzheimer’s disease (AD). However, there are currently no bioinformatic tools to specifically analyze BMCs at the single-molecule level. We have developed a novel tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), (Wallis et al., bioRxiv, 2021, ref. 7), which offers unprecedented insights into the dynamics of proteins undergoing liquid-liquid phase separation/transition in large and nanoscale BMCs, in live neurons. We identified Tau and synuclein as candidates for their ability to generate synaptic BMCs in hippocampal neurons. NASTIC analysis reveals that Tau molecules can indeed form small nanoclusters in live hippocampal neurons in which they exhibit very low mobility and sensitivity to 1,6-hexanediol, an aliphatic alcohol used to inhibit weak molecular interactions that mediate BMCs. NASTIC will be at the core of our ability to make a significant contribution to the understanding of BMCs in neurons because it offers unprecedented opportunities to examine the spatiotemporal behaviour of molecules in these condensates. NASTIC will be developed further to encompass two-color single-molecule analysis. This will be instrumental to assess the spatiotemporal relationship of these nanoscale BMCs with their cellular environment in 2 and 3D. We will validate our two-color NASTIC implementation with the pairwise dual imaging of Tau wildtype and AD mutant P301L, and a-synuclein. This will allow us to decipher the co-clustering dynamics and examine potential hierarchical dependency of one nanoBMC upon the other allowing refined understanding of the generation of nanoscale BMCs in synapses. We anticipate that the P301L mutation will largely increase the size and lifetime of the Tau nanoBMCs and alter their relationship with other synaptic molecules. The outcome of this grant will be the development and validation of an analytical pipeline that will enable exploration of the spatiotemporal relationship of Tau and a-synuclein nanoBMCs in live hippocampal neurons. Our project will therefore generate data and a much-needed technology opening new avenues for our understanding of BMCs in neuronal function and AD.

项目摘要/摘要 生物分子冷凝物（BMC）定义了小型无膜细胞室，该室分离特异性 蛋白质和核酸产生细胞功能。光漂白后的荧光恢复（FRAP）为 目前，选择在体外和体内表征BMC的首选方法，并得出平均扩散和 被困在这些冷凝物中的分子的捕获。尽管FRAP是检索这些重要的好方法 指标，它极大地限制了我们对BMCS纳米级组织的理解和活细胞中的动态 排除了纳米级BMC的分析（低于200 nm的光的衍射）。单分子成像 超分辨率显微镜可以直接成像被困在BMC中的分子 时空分辨率在体外和活性神经元中都有可能揭示纳米级BMC和 它们在衰老和阿尔茨海默氏病（AD）中的演变。但是，目前没有 生物信息学工具可在单分子水平上专门分析BMC。我们已经开发了一个新颖的工具， 命名纳米时空索引聚类（Nastic），（Wallis等，Biorxiv，2021，参考文献7）， 它为经历液态液相的蛋白质动态提供了前所未有的见解 活神经元中的大和纳米级BMC中的分离/过渡。我们将tau和突触核素确定为 候选者在海马神经元中产生突触BMC的能力。分析表明 tau分子确实可以在活的海马神经元中形成小纳米簇，其中它们表现出很低 迁移率和对1,6-己二二醇的敏感性，一种用于抑制弱分子相互作用的脂肪醇 中介BMC。 Nastic将是我们为理解做出重大贡献的能力的核心 BMC在神经元中的作用，因为它提供了前所未有的机会来检查的时空行为 这些冷凝物中的分子。将进一步开发出来以包含两色单分子 分析。这将有助于评估这些纳米级BMC与它们的空间时间关系 2和3D中的细胞环境。我们将使用成对双重的验证我们的两色nastic实现 Tau WildType和Ad突变体P301L和A-核蛋白的成像。这将使我们能够破译共同集群 动力学和检查一种纳米BMC对另一种允许精制的潜在分层依赖性 了解突触中纳米级BMC的产生。我们预计P301L突变将 很大程度上增加了tau nanobmcs的大小和寿命，并改变了与其他突触的关系 分子。 这笔赠款的结果将是对分析管道的开发和验证，这将使 在活海马神经元中tau和a-核蛋白纳米BMC的时空关系探索。 因此，我们的项目将生成数据和急需的技术为我们的 对神经元功能和AD中BMC的了解。