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.

项目总结/摘要 生物分子凝聚物（BMC）定义了小的无膜细胞区室， 蛋白质和核酸来产生细胞功能。光漂白后的荧光恢复（FRAP） 目前，选择体外和体内表征BMC并推导平均扩散的方法， 捕获这些冷凝物中的分子。虽然FRAP是一个很好的方法来检索这些重要的 它极大地限制了我们对活细胞中BMC纳米级组织和动力学的理解， 排除了对纳米级BMC的分析（低于光的衍射~200 nm）。单分子成像 超分辨率显微镜允许直接成像的分子被困在BMC与更大的 在体外和活神经元中的时空分辨率，并有可能揭示纳米级BMC和 它们在衰老和阿尔茨海默病（AD）背景下的演变。然而，目前没有 生物信息学工具在单分子水平上专门分析BMC。我们开发了一种新的工具， 称为纳米尺度时空索引聚类（NASTIC），（沃利斯等人，bioRxiv，2021，参考文献7）， 这为蛋白质经历液-液相的动力学提供了前所未有的见解 在大的和纳米级的BMC中，在活的神经元中的分离/转变。我们将Tau和突触核蛋白鉴定为 候选人的能力，产生突触BMC在海马神经元。NASTIC分析显示， Tau分子确实可以在活的海马神经元中形成小的纳米簇，其中它们表现出非常低的 对1，6-己二醇的迁移率和敏感性，1，6-己二醇是一种脂肪醇，用于抑制弱分子相互作用， 介导BMC。NASTIC将成为我们的核心能力，为理解 因为它提供了前所未有的机会来研究神经元中的BMC的时空行为， 这些凝聚物中的分子。NASTIC将进一步发展，以涵盖双色单分子 分析.这将有助于评估这些纳米级BMC与它们的时空关系。 二维和三维的细胞环境。我们将验证我们的双色NASTIC实现与成对对偶 Tau野生型和AD突变体P301 L和α-突触核蛋白的成像。这将使我们能够破译共同集群 动态和检查一个nanoBMC对另一个的潜在分层依赖性， 了解突触中纳米级BMC的产生。我们预计P301 L突变将 很大程度上增加了Tau nanoBMCs的大小和寿命，并改变了它们与其他突触的关系。 分子。 这笔赠款的结果将是开发和验证一个分析管道， 探索活海马神经元中Tau和α-突触核蛋白纳米BMC的时空关系。 因此，我们的项目将产生数据和急需的技术， 了解BMC在神经元功能和AD中的作用。